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Chemical dangers in Histology from personal experience

I feel like the dangers of histology chemicals, those that I know of, should be addressed. I work in a histology lab that is situated within many other different labs. It is the general belief within the histology world, that the chemicals (wet and dry) we deal with are harmful to breath, touch and be around. Maybe not in the short run (most of them) but definitely in the long haul. I know that the chemicals used in the other industries are by comparison are far worse. That may be, but the use in histology research by all labs seems to be unregulated for the most part. This is the opposite for clinical histology labs, they are highly regulated and controlled to a T.  In this country (US) everything to do with humans is highly regulated. For those researchers working in labs, safety may not always be a great concern. I realize, their focus is on something grander and may not be focusing on the day-to-day idiosyncrasies. This is all well and good for them, but their actions set the tone for others to follow. I call this monkey see monkey do. I know this happens because I did it.  In the past, I worked for a research lab that did not even have chemical, dissection, and safety hoods. Lab coats could not be found unless you bought your own. No one wore safety glasses, ever. There was no paid chemical waste removal. All chemicals went down the drain, regardless of what it was. The drain led into a septic system that was next to a river. Once I asked the owner if we could hire a company to remove our waste. He laughed and said it would cost too much money and if I wanted to get paid, I should never bring it up again. I did not agree with this practice but felt bad like my job was in jeopardy if I rocked the boat. Not a good situation for anyone there and anyone living in the neighborhood. I remember one day while I was there, one of the wet chemicals leaked through its container. I smelled something and got up to investigate. The smell was very strong and filled the entire building. My eyes watered and my nose began to clog. I found the offending liquid on the shelf leaking down to the next level. It was ammonium hydroxide (28-30%). The container was the wrong kind of plastic bottle and it leaked about 100 mills before I found it. Standing in front and on top of it I put gloves on and grabbed gauze to soak it up. After about 5 minutes, a co-worker came in and hauled me out of the room into the outdoors.  Out into the cold light and winter conditions, I still could not see the problem.  I was blinded by the habits of another that now were my own.  My throat and nasal passages had closed, eyes stinging, ears not hearing but still I protested.  Someone has to clean it up, I can’t leave it there, it’s dangerous.  5 1/2 years of negative conditioning had led me to a destructive path.  The owner sat less than 10 yards away but never came to see what was going on.  He could not smell it and had no idea that there was a spill.  We did not have a safety plan in place.  The fire department was never alerted, no report was ever written, but I suffered.  The physical damage was done in a matter of minutes.  In the weeks following, I came to realize I could no longer smell many substances that before the accident were easily identified.  In hindsight, I realize that I took on characteristics of my former boss who did not use safety guidelines in his company.  This took me years and a couple different lab settings to realize the gross negligence of safety.  This will never happen again.  Too bad I had to learn the hard way.  Today, safety is my number one concern for others and myself when working in a laboratory setting.

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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
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Internship Program

Histologistics and Worcester Polytechnic Institute (WPI) have created an internship program for area high school and college students. Are you interested in the fields of biology and chemistry but don’t know what direction you are heading. We can help! By doing an internship with us you will be exposed to a wide range of laboratory methodologies. Students who are accepted into this program will be working with all areas of histology and will include the Life Sciences and Bio Engineering department. The histology labs are located inside the Life Sciences and Bio Engineering department. This means students will get to see what projects graduate students are working on while they themselves are working on their projects.

Leica RM 2235 Microtome
Leica RM 2235 Microtome

This has already begun.  The second round of students are in their first week of the internship.  We now have 4 shared interns learning histology.  Two are new high school graduates and the other 2 are seeking bachelors degrees.  During August of 2013, we hope to share a total of 6 interns.  In mid July I will be interviewing students from 6 area high schools for the fall internships.  If you are interested or know of someone who is, contact me at hans@histologistics.com.

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