[Histonet] annoying crystals on sections

E. Wayne Johnson ewj <@t> pigsqq.org
Mon Aug 20 18:20:06 CDT 2012

We are having problems with crystals precipitated on our slides which 
are H&E stains on tissues from pigs.
Tissues are fixed in buffered formalin.  We had trouble months ago with 
formalin pigment and we had resolved
that by using ammonia in EtOH or picric acid in EtOH.  Sometimes we 
receive fixed samples from the field that are
not buffered but presently all of our tissues are fixed in neutral 
phosphate buffered formalin.

We moved the Sakura autostainer to a different location under a fume 
hood on a
different floor of the building to get the solvent odor out of our work 
Immediately we began to see a tremendous degradation in slide quality 
due to what we initially thought was formalin pigment.
We have changed all of the solutions and all of the stains.  We find 
that if we use Milli-q water instead of tap water for
rinsing (done by hand in that case) we dont see the crystals, but the 
eosin staining quality is not acceptable after rinsing in the acidic
(ph ~5) Milli-Q water.

Our tap water is neutral to slightly alkaline and is very hard with calcium.

We do all sorts of tissues for diagnosis of pig diseases.  Sometimes the 
slides are quite acceptable but sometimes particularly
when looking at small intestine, the crystals are very annoying.  The 
crystals occur randomly on the slide except that there is a
tendency for them to be centered on nuclei particularly in intestinal 
epithelium.  The crystals are birefringent in polarized light but
seem to be generally clear not dark like the formalin pigment we had 
seen before.  Neither ammonia nor picric acid remove these,
and now if we use alcoholic ammonia to treat the slides, the slides come 
out too blue.  Our slides are cut at 4 to 5 microns.

Brain has the least problem, small intestine seems worst.

We have gone back and cut some blocks that previously stained 
beautifully with no pigment or precipitate problems
and those slides also now have the same problem, either crystals if 
washed with tap water, or poor eosin staining if rinsed with MilliQ water.

Our next step is to examine the slides microscopically at every step and 
try to find at which step the problem is occurring.

Any thoughts or similar experiences?

E. Wayne Johnson, DVM
Enruikang AgTech
MOA Feed Industry Centre

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