Thanks to all who responded to my post, I will certainly be trying out a
combination of the recommendations to see if I can 'clear' the problem. I
have included some of the ideas sent to me. Thanks again,
Institute of Aquaculture
University of Stirling
1. Could your racks be contaminated?
Do you wash and dry them thoroughly after each day? Are your slides
you put them in xylene?
Also try two changes of xylene 5 min each.
2. Are all your fluids at the same level? It sounds like possibly water
the top of the slides and is not rinsed off going through your
it goes into your xylene. This does sound like an issue of water
into your xylene. Do you use recycled alcohols and/or xylenes? If so
they are not distilled properly
3. Wherever the water is
coming from....be it atmospheric or just from not changing alcohols
enough...it is the water that causes Xylene to turn milky (and forms
droplets you describe as well).
4. Surely this has to be due to water carried into the
xylene because the last alcohol wasn't 100%. Two
possible causes suggest themselves:
1. Insufficient shaking off of solvents (alcohols)
during the steps of dehydration of the stained
2. Contamination of your supposedly absolute alcohol
with water. Test for this by mixing some with new
xylene. It shouldn't go milky. This fors the basis of
a very simple assay for water in alcohols:
Hall, J (2001) A simple, rapid method for measuring
the percentage of water in alcohols used for
dehydrating tissues. Biotech. Histochem. 76(1):41-42.
> The second and subsequent rack/s of
> slides can often clear perfectly.
Could it be that the xylene now contains enough alcohol
to prevent milkiness when more small amounts of water
are carried over into it? If this is the case, you
will probably see microscopic droplets of water in
the mounted preparations, even though they look
transparent to the unaided eye.
5. How humid is your air??Exposing the rack/slides to air, the alcohol
assume it IS a 100%) will pick up moisture and your Xylene will turn
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