June:

Stephen Kayes skayes at USAMAIL.USOUTHAL.EDU
Thu Jul 17 17:29:13 EST 1997


June:  
Haven't done trichromes since grad. school but here is what I  found on my bookshelf:

I.  Trichrome:
 (1)  Introduction:  The trichrome staining procedure is quick and easy to perform.  It
gives very good results for routine purposes.  Overstaining and differentiation are not
necessary to bring out the morphologic details of the parasites.  Also, mordanting is
not necessary before staining.  However, for best results, destaining is recommended
because it gives better differentiation of cellular detail.  This stain gives good results
with smears fixed with either plain Schaudin's or PVA-fixative solution.  Both
procedures follow:

 (2)  Stain Characteristics
  (a)  The cytoplasm of properly fixed and stained organisms is blue-green, tinged with
purple.  Occasionally, Entamoeba coli cysts stain more purplish than cysts of other
species.  The nuclear chromatin, chromatoid bodies, and ingested red blood cells and
bacteria stain red or purplish red.  Other ingested particles, such as yeast or molds,
generally stain green, but variations in color of ingested particles do occur.
Background material usually stains green, thus contrasting with he protozoa.
 
 (b) Cysts that do not stain and those that stain predominantly red are usually
associated with incomplete fixation.  Organisms that stain pale green are associated
with degenerate forms, although understained or overstained organisms can also
appear green.

 (c)  Eggs and larvae stain red and contrast strongly with the green background.  In
contrast to smears stained with hematoxylin, trichrome-stained smears have a
transparency which enables embedded protozoa to be identified in somewhat thicker
preparations.

 (3)  Reagents:  The solutions used in the trichrome procedure are the same as those
used for the Heidenhain stain except for the stain and the destaining solution.  The
trichrome stain solution is stable and can be used repeatedly.  Replace the lost volume
by adding stock solution.  However, the stain will be weakened when a large number of
smears are stained in a short period of time.  Its strength can be restored by allowing it
to evaporate in the open air for 3 to 8 hours.  Simply leave the cover off the staining
dish overnight.  

At this point, I could give you the recipe for making your own stain and the
recommended procedure if you want or need  that.  Back then, people made their own
stains rather than bought them from commercial vendors.  However, the newer vended
products are often modified to be quicker and more efficient.  If you want this
information, I will be happy to send it.  The above was excerpted from the Air Force
Clinical Laboratory Procedures--Parasitology manual (AFM160-48).  

Good Luck with your staining.  I believe a well stained section is a true work of art.

Steve Kayes


>>> dejmw <dejmw at hotmail.com> 7/17/97  6:21 pm >>>
Our parasit lab is experiencing problems with our trichrome stain.
We recently changed manufacturers and that might be part of the problem.
I had some literature entitled "What's wrong with my Trichrome?", but,
unfortunately I am not able to find it.  It was excellent, only 3 pages
long but it gave great tips such as, "if the background is too pink
then you left it in the clearing agent too long".  I'm not sure if
that's correct because I no longer have the info.
 Does anyone know how to trouble shoot the trichrome??? I would really
appreciate any information, or info on where to find it.
Thanking you in advance.
  June





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