RE: [Histonet] H&E water contamination[Scanned]

From:Kemlo Rogerson

Plus xylene will tolerate low levels of water (very low) without going
cloudy; the contamination only is seen microscopically as a fine 'mist' of
droplets. Knew a Chief Technician that kept anhydrous copper sulphate in his
last alcohols to absorb any water carry over. 

If you try staining in very cold and damp conditions then a mist can form
from one alcohol to another. It's something to do with the eutectic point of
water or I may just be making that up. All I know is, even with fresh
alcohols, staining in cold damp conditions can result in water from the
atmosphere contamination the xylenes. The only way I could get round it was
to use a hair dryer to raise the temperature a bit, and hope I didn't catch
fire. I hasten to add I did that before CPA, etc., and not in my present

Those were the good old days of sharpening knives with diamond paste and a
bit of wood, and making up your own stains and ripening the haematoxylin in
the window......... I must be old. I AM old!!!!

Kemlo Rogerson
Cellular Pathology Manager
East Lancashire Hospitals NHS Trust
DD. 01254-294162
Mobile 0774-9754194

-----Original Message-----
From: Tony Henwood [] 
Sent: 28 November 2004 23:00
Subject: RE: [Histonet] H&E water contamination[Scanned]

A few ideas.

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


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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