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Microtomy school

 
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Olympusman



Joined: 15 Jan 2012
Posts: 3569

PostPosted: Fri Apr 13, 2018 8:33 am    Post subject: Microtomy school Reply with quote

I had the opportunity the other day to visit the histology department at a medical center to learn what I could about microtomy. Absolutely fascinating. Mainly I learned a great deal about the importance of temperature at various stages of tissue preparation and embedding. Got to watch a cryomicrotome in action. Also learned about dehydrating tissue samples. Instead of progressively stronger steps in alcohol the technique they use is
10% buffered Formalin
70% Formalin 30% alcohol
Pure alcohol
Pure alcohol
Pure alcohol
Xylene

I also learned about prepared ionized slides which are used with silver stained tissues (brain, bone). Silver stained tissues don't adhere to normal slide and the tissue washes off during staining.
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Michael Reese Much FRMS EMS Bethlehem, Pennsylvania, USA
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ChrisR
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Joined: 14 Mar 2009
Posts: 7609
Location: Near London, UK

PostPosted: Fri Apr 13, 2018 2:15 pm    Post subject: Reply with quote

I learned enough about it to be fascinated and, inevitably, accumulate some hardware. I doubt I have enough life left to actually get round to practicing, though!
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Chris R
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Olympusman



Joined: 15 Jan 2012
Posts: 3569

PostPosted: Sat Apr 14, 2018 1:09 pm    Post subject: Microtomy Reply with quote

Got out my binder of notes I accumulated over the years to review technique.
Purchased 10% buffered formalin and Potassium hydroxide. Also purchased a digital kitchen thermometer to establish heating of Paraplast and water bath atop a gravy warmer and a coffee cup warmer.
This is getting complicated for a hobby microscopist.
More hardware with a fixed amount of counter space in the lab.

Mike
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Michael Reese Much FRMS EMS Bethlehem, Pennsylvania, USA
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ChrisR
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Joined: 14 Mar 2009
Posts: 7609
Location: Near London, UK

PostPosted: Sun Apr 15, 2018 2:45 pm    Post subject: Reply with quote

It's also expensive. Minimum qty seems to be about $100.

I saw a Leitz microtome and bought it, expecting imagination to be fired. Still waiting.
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Chris R
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Olympusman



Joined: 15 Jan 2012
Posts: 3569

PostPosted: Mon Apr 16, 2018 2:01 pm    Post subject: Buffered formalin Reply with quote

The histology lab I visited had a 2.5 gallon cubitainer of formalin. I purchased two pair of jars that appear to hold about 40 or 50 ml on Amazon from TissuePro Technology www.tissueprotech.com

As for the KOH, I now have a lifetime supply. I found it is very effective at dissolving residue accumulated in beakers when I decapsulate microchips.

Mike
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Michael Reese Much FRMS EMS Bethlehem, Pennsylvania, USA
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discomorphella



Joined: 01 Oct 2006
Posts: 599
Location: NW USA

PostPosted: Sat Apr 21, 2018 9:05 am    Post subject: Reply with quote

Hi Mike,

One thing you may want to invest in is a small crock pot and a simple proportional temperature controller. You can find used YSI models on ebay. That makes a really nice paraffin oven, and avoids the problem of getting water into your paraffin. Also, a gallon of Histoclear2 is a much better option than xylene, its nontoxic, and you can use it with regular 99% usp grade isopropyl alcohol, also comparatively nontoxic, and cheaply available. A very good dehydrating/embedding schedule that works for all manner of samples is
wash in tap water to remove formalin
50% IPA
70% IPA
90% IPA
95%
99%
99%
1:1 IPA / HC2
HC2
HC2
HC2 saturated with paraplast
PARA1
PARA2
PARA3
embed

This works very well for all tissues, botanical as well as animal histological samples. More rugged samples can skip the 50% IPA step and the 90% IPA step and the IPA/HC2 and HC2/para as well.
The main advantage for this technic is enhanced safety for you. Xylene is toxic, and can contain ethyl benzene which is worse. HC2 is far safer. You can run this whole protocol on your benchtop without a hood. Just get yourself some 120 ml jars (or use old spice jars) and a few tissue cassettes or make some out of screen and you're good to go. Also, the celloidin coating technique is a great way to keep your sections from falling off, if regular albumin won't work. The silanized (typically surface treated with APTES) are not as effective for long soaking in high pH solutions.
Get a good box of real, chemo-tested nitrile gloves, like Cardinal Health for example, and a good pair of safety glasses.

David
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