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trichrome staining

Charles T. Faulkner ctfaulkn at utkux.utcc.utk.edu
Fri Jul 18 13:33:25 EST 1997

>  June <dejmw at hotmail.com> 7/17/97  6:21 pm wrote: 

> Our parasit lab is experiencing problems with our trichrome stain.
> We recently changed manufacturers and that might be part of the problem.

	[snip, snip]

>  Does anyone know how to trouble shoot the trichrome??? I would really

	I have found that most of my problems with trichrome stem from
inadequate fixation. It seems that the PVA supplied in the transport vials
(ParaPak, Meridian Diagnostics) is quite variable in its ability to
adequately penetrate and fix the fecal material.  This opinion is based on
staining of approx 400 fecal samples...  some of which are known positives
based on ZNSO4 flotation w/ Lugol's iodine. Staining characteristics of
samples processed within a single lot of transport vials ranged from
perfect fixation (all colors represented) to useless for diagnostic
evaluation (only light green).  These results are probably not due to
variation in sample processing because I have followed the same basic
protocol, and they are not due to run to run variation in staining because
many of these samples are typically stained in the same tray with other
properly fixed samples.  Our trichrome is obtained from AJP Scientific,
Clifton NJ (201) 472-7200.

	Our protocol for troubleshooting the problem included:  systematic
variation of the basic Trichrome stain protocol described on pg 54 of
Price's "Procedure Manual for Diagnosis of Intestinal Parasities"  CRC
	stain from 8 min, 10 min, 12 min
        destaining in 90 or 95% acid alcohol: 1 dip, 5 sec, 10 sec
 	remove destain 5 dips in 95% or 100% ETOH

Each variant was run with a control slide of recently acquired E. coli
obtained from a baboon at the local zoo fixed with bulk purchased PVA
(Meridian Diagnostics).  Best results were obtained with staining at 10
min, destain 1 dip 95% acid alchol (1 drop glacial acetic per 10 ml 95%
ETOH), remove destain 5 dips in 100% ETOH.  The remainder of our procedure
is unmodified through the xylene step, except that we use the maximum
times given.  

    I have noticed that Meridian recently added a low viscosity
formulation PVA to their product line with the claim that it "allows for
better fixation due to faster penetration".  

	Other problems could stem from use of the ZN-PVA and CU-PVA which
substitutes zinc sulfate and copper sulfate for mercuric chloride.  I
think the staining characteristics of these "new" media were recently
reviewed by Garcia et al....(1996, Jour Clin Micro??).    

     *  Charles T. Faulkner, M.A.                   *
     *  Clinical Parasitology Service               *
     *  Dept. of Comparative Medicine               *
     *  2407 River Drive                            *
     *  Knoxville, TN 37996-4500                    *
     *  Voice: (423) 974-5645  Fax: (423) 974-5640  *
     *  E-Mail: ctfaulkner at utk.edu                  *

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