Water in the Xylenes, a Serious Problem in Histology


Most histology labs still use xylene for processing and in the staining process.  There are alternatives to xylene like limonene, Naturalene, MasterClear, and others but they are more expensive.   Do you know the oil saying, oil and water do not mix?  Well xylene and water does not mix also.  The mixing of the two has many bad results in processing and staining.  If water is present in the xylenes during tissue processing then the tissues are more prone to “frying” become brittle.  Finding water in the xylenes is usually found by someone who looks at the slides after they are done staining.   This is very unfortunate because the slides must now be de-cover slipped in xylene, this contaminates more xylene.  Then they must be dehydrated in 2 changes of 100% alcohol and possibly re-stained.  If these were to go out to a client or pathologist then besides looking really unprofessional slides with water under the coverslip can bleed out or fade the stain.  Another way water is discovered in xylenes is by looking at the bottom of the xylene container or bottle.  Water looks like bubbles or oil in water.  I have experienced this phenomenon at all the histology labs I have worked for.  The cause 99 percent of the time has been human error.  The histologist of today must be dynamic and do multiple jobs at once while also doing them very well.  We are very busy and run around and do not always pay attention to every detail.  So contamination does happen occasionally.  There are many questions when this does happen.  Many of them are asked and published on a histology blog called histonet. http://lists.utsouthwestern.edu/mailman/mmsearch/histonet.  This website is for questions all about histology and everything else that pertains to it, a very useful tool.  Most of the archives are in the form of threads, so finding an entire conversation may be difficult.

One of the labs I manage now has been having issues with water repeatedly showing up on coverslipped slides over the course of 4 months.  In this lab the amount of people and type change every day.  Regulating who uses the lab for what purpose is impossible.  I have tried to train everyone who is or will be using the staining apparatus. I thought that slides being stained and then run to xylene for cover slipping might have been moved too fast from 100% alcohol to xylene, not giving enough time for the water to come off the slides.  I also thought that maybe the last 100% alcohol was not being changed on a daily basis and therefore getting contaminated the day before.  This would make sense, the last 100% alcohol would be 98-99% alcohol and then have water carry over into the xylenes.   I sent out emails to notify of everyone about these problems.  I tried using dri-rite in the xylenes, to alert people of possible water contamination.  One day I changed all the xylenes and alcohols in the staining apparatus.  Then I did an H&E stain.  Looking through the microscope at the finished slides, I saw water droplets.  I took pictures and sent them to everyone in disgust. Then I thought, could it be the 100% alcohol was contaminated?  I had never heard of this, but still could be possible.  I looked at the label on the 5 gallon cube of alcohol.  It said 200 proof alcohol.  Just below that in fine print, read the words assay content 98.9-99.98% alcohol with 0.1-.02% water.  Water in the alcohol?  How could this be?  Was it human error or a bad batch?  No it was correct, I looked it up on the website, the MSDS and other publications led me to believe it was known.  It’s 100% ethanol. There is no denaturing chemical to render it waterless. I feel like I was led to believe this was 100% alcohol.  I guess I should have looked more closely.

As you can see below the problem (water) looks like bubbles in the tissue.  This is a Grocott’s Methenamine Silver (GMS) stain for fungi.  The small round black circles are fungus.  In between the tissue where the white space is are perfectly round almost clear circles.  This is what water looks like under glass.  Before I found the alcohol water problem, I did a gram stain.  I looked at this stain after a week of being coverslipped, all the gram negative/positive bacteria had decolorized.

Water Under Glass

One thought on “Water in the Xylenes, a Serious Problem in Histology

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