Re: [Histonet] H&E water contamination

 Another source of water contamination in H&E sections can also occur in 
atmospheric conditions of high humidity (even in the UK), if there is the 
slightest delay in mounting the slides after their removal from xylene. 
This water contamination manifests itself as small collections of water 
micro droplets that seem to orientate themselves over nuclei, and look 
like a pigment lying over some of the nuclei. This artifact is also 
exhibits birefringence.  It took me a while to track down and prove the 
source of this contamination. In the end I took slides out of xylene and 
breathed on them several times prior to mounting, and these slides showed 
the this same nuclear water artifact. The artifact is obviously caused by 
water vapour condensing on the slide prior to mounting but I cannot say 
what level of atmospheric humidity was required for this to manifest 
itself because I did not measure the humidity levels.
Histopathology Group
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Message: 1
Date: Mon, 29 Nov 2004 10:00:02 +1100
From: Tony Henwood 
Subject: RE: [Histonet] H&E water contamination
To: "''" <>,
Message-ID: <>
Content-Type: text/plain

A few ideas.

Check your xylene for water.
Keep the alcohol and xylene containers covered when not in use. Alcohol 
absorb water from the air (possibly xylene as well - though of this I am 


Tony Henwood JP, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager & Senior Scientist
The Children's Hospital at Westmead,
Locked Bag 4001, Westmead, 2145, AUSTRALIA.
Tel: 612 9845 3306
Fax: 612 9845 3318

-----Original Message-----
From: [] 
Sent: Saturday, 27 November 2004 9:12 AM
Subject: [Histonet] H&E water contamination

Hi histonet
I have been performing H&E stains on sliced parraffin embedded mouse
hippocampal slices. I am consistently getting water contamination in my
slides and have been unable to rectify the problem. My procedure for
staining is: Xylene I for 7.5 minutes; Xylene II for 7.5 minutes; 100%
ethanol I for 2 minutes; 100% ethanol II for 2 minutes; 95% ethanol I and
70% ethanol I for 2 minutes each; Water I for a second; then "H" for 20
minutes; Water II for 10 minutes; "E" for about 13 seconds, Water III for
about 1 minute; 70% ethanol II for 50 seconds; 95% ethanol II for 5
minutes; 100% ethanol III and IV for 5 minutes each and then Xylene III
and IV for 7.5 minutes each.
I beleive the "70% ethanol II" is the key step for removing water after
the staining but increasing the amount of time spent in the solution
hasn't worked yet (though I have yet to leave it in for longer then a
Any tips would be greatly appreciated,

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