Staining, Histochemistry and Histotechnology
(Frequently Asked Questions)
Dr. John A. Kiernan
Department of Anatomy
and Cell Biology
The University of Western Ontario
Test for water in used absolute alcohol
How can I determine whether used "absolute" alcohol is still OK for the last stage of dehydrating specimens or slides?
Some people add anhydrous copper sulphate to the alcohols used for processing tissues. It changes colour (white to blue) in the presence of water, but this does not tell you if there is only a tiny trace of water or enough to make the alcohol immiscible with xylene.
You may be interested in a simple method I developed for this purpose. My job is evaluating histology equipment for the Medical Devices Agency, (an agency of the Department of Health), and I was interested in trying to establish "carry-over" in processing and staining instruments. I started off by adding known dilutions of alcohol, drop by drop, to different amounts of xylene, my basic thinking being that water turns xylene milky, and if one adds enough of the diluted alcohol, the mixture eventually becomes clear again. From this I developed the following method:
A measured 5 ml of xylene (the 5 ml is important) is placed in a 50 ml glass beaker and placed on a black background. Using a 1 ml plastic pasteur/transfer/dropping pipette, add the alcohol for analysis, drop by drop and keep count of the number of drops, until you can just detect a faint turbidity in the xylene. Carry on adding the alcohol to the xylene until the turbidity just clears, again taking note of how many drops were needed.
Using known dilutions of alcohol, I was able to set up and standardise the method and obtain reproduceable results consistently. The method was not sensitive enough to detect the water in 99% or 98% alcohol.
97% = 5 drops to turn xylene milky, 10 drops to clear the mixture
96% = 4 drops to turn xylene milky, 14 drops to clear the mixture
95% = 3 drops to turn xylene milky, 34 drops to clear the mixture
94% = 3 drops to turn xylene milky, 74 drops to clear the mixture
93% = 3 drops to turn xylene milky, 83 drops to clear the mixture
92% = 3 drops to turn xylene milky, 98 drops to clear the mixture
91% = 3 drops to turn xylene milky, 140 drops to clear the mixture
90% = 3 drops to turn xylene milky, 204 drops to clear the mixture
You would have to initially set up your own range of standard dilutions with the particular alcohol used in your laboratory for the sake of accuracy. The 1 ml plastic pasteur/transfer/dropping
pipettes, they can even be called pastettes, should be held vertically to standardise the size of the drops, and I tried to use the same brand each time.
This is a simple method, and quick to do, although I should think the method would give the Biochemists the shudders. It could help to prolong the life expectancy of the alcohols used in processors.