Re: FW: H & E Staining problems

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Dear Laurie,
Annoying isn't it! 
We too have had this problem in the past and it seems to boil down to water 
(or possibly excess alcohol) in the last xylene/antemedia bottle prior to 
One of our (past!) solvent suppliers used to leave the the empty xylene 
containers uncapped, open to the weather outside prior to filling with 
xylene, so that small amounts of rain water were included in the xylene. It 
was such a small amount of water that the Lab Aids changing the machine 
didn't see it and emptied the water into the processor too. 

Bitter experience has taught us that as little a 1ml of H2O in 2.25 litres of 
xylene will ruin a machine loaded with biopsies because the tissue is 
hydrophillic at all points in the processing schedule and preferentially 
absorbs water. When water coated biopsies go into hot wax something awful 
happens and the results are as we know - dreadful. In the UK this artefact is 
also known as "Pinks Disease" because of the peculiar eosin staining. 
On another occasion the inlet valves on the processor were drawing Formalin 
into the last xylene and exactly the same thing happened. We proved the 
presence of water in the xylene by drawing up xylene from the base of the 
processing bottle using a 10 ml glass pipette. There were small "air bubbles" 
in the xylene which were in fact Formalin, and we also saw damp streaks on 
the glass too. We proved the point by mixing the machine xylene and a sample 
of known pure xylene (from BDH, who can be relied upon to produce good 
reagents) with 1ml of Schiffs reagent. If the machine xylene goes pink and 
the BDH xylene doesn't, then you have formalin in your xylene and a fault 
with the machine.

Before we found this out we looked at everything, fixation, the time GI bxs 
were left "standing" prior to fixation, drying on times post microtomy, 
drying on temps, staining, water, everything. By the time we'd finished 
looking, the artefact went away because either we'd used a batch of xylene 
which came in containers left open in a dry period ( we do get them in the 
UK!) or we'd changed the xylenes on the machine and there was no Formalin in 
it (yet!). Very frustrating.

May I suggest that you check the xylenes/antemedia thoroughly?

If you let me know whether water contamination is present I would appreciate 
it. I'm trying to build up an idea of how common this cause of the artefact 


Glyn Woodward

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