[Histonet] Cryostat chatter problems (long!)
One additional area to clean on a knife holder (paraffin microtomy) is the
BACK of the holder. If there is any paraffin buildup, you can have
defective ribbons plus icky sticky problems. Protect your all edges on
knife holder, and do not use solvents to clean off paraffin - this removes
lubricants put there by manufacturer, use a rough towel, and correct
cleaning materials to remove paraffin. Ask your manufacturer what they
We have three 1850's, and dearly love them. Cutting fast is a huge No No
in our lab - we teach a slow, steady motion as knife passes through
tissue. If you hesitate going through a tissue, you get BIG chatter or
uneven section thickness. Remember, the tissue is NOT embedded in hard
paraffin, and has only itself as support with OCT, a softer embedding media
than the paraffin ONLY surrounding the tissue. Cutting fast on the
cryostat is a No No in our lab! How you hold the flywheel is important,
if you grab it like driving a "Harley" down the road,( too tightly!) - you
can impart chatter through flywheel mechanism, use a light hold to get rid
of the VAROOM grip! Relax, and cut slowly.
If the temperature setting is too cold or block is not firmly attached to
metal block holder with a generous amount of OCT, you can get
chatter. Bubbles in or underneath a frozen block is the enemy! -20C is a
general cutting temperature, but it will depend on the tissue. For fatty
tissue, colder, for liver, brain, and spleen we warm to -16C -to
-17C. Very dense tissue (connective tissue) can cause chatter - if really
cold, harder to cut. You must be willing to change temperatures for any
given tissue. Thick prefixed sucrose cryoprotected brain (50um) is cut at
-19C or so, can be warmer, slowly and with antiroll device - no problems.
Metal chucks for 1850's are little mushrooms with shallow circle
depressions. These are fine for smaller, less dense tissues. You can buy
better designed metal chuck in several sizes from Thermo Electron aka
Shandon, but you must cut the stem to have the same stem length as the
original holders in order to fit in microtome block holder. Thermo chucks
have deeper, waffle weave depressions, basically an old fashioned style
used for paraffin blocks years ago. The waffle weave holds blocks far
better and more firmly than the Leica holders. Achieving stability in
block and knife is important for cryostat work.
If you take the block directly from the Peltier or cooling bars in the
1850, the block will be 10 degrees colder than the desired cutting
temperature at the block and knife. Mount the block, and set it NEXT to
the cryostat (use a microcentrifuge rack, bright colors and plastic for
many blocks) and allow the block to equilibrate to you chosen cutting
temperature - it takes about 20 minutes. If you set the cryostat cutting
temperature to -20C, it will be approx that at knife and block, but not on
those bars. Know your temperature differentials inside the cryostat - test
with thermometer to locate warm and cold areas, record the temperatures for
any given readout setting, then use these areas to your advantage.
How you orient block in holder can make a difference. If you orient a
tissue so the broadest, widest part of sample is cut by the knife FIRST,
you get a lot of resistance and the knife will "spring" no matter if
everything else is tight. Create a path of least resistance. Example, if
block is a perfect square, place narrowest part of tissue pointed towards
blade edge so it is cut first. In other words, the face of the block looks
like a diamond and we cut the narrowest point first to get that less
resistant pathway. One can trim rectangular blocks so you achieve a
similar look, and proper orientation of tissue in block at embedding helps
Change the knife frequently and try OTHER brands of disposable blades and
try high profile. Low profile are thinner and narrower, and not as sturdy
overall as high profiles. We use high profile disposable AccuEdge blades
(Sakura Finetek, VWR has them) and do not get chatter - when we change to
low profile AccuEdge and cutting denser tissue, chatter tends to return -
so we stay with high, since they are just as sharp but stable. We use low
profile occasionally but only for small, less dense tissues.
Make sure the levers one pulls out when cleaning any part of holders and
even with paraffin microtomes are reoiled so a dry metal surface is not
tightened down on another dry metal surface. My Leica rep indicated that
this is a source of poor tightening, and lubricated surfaces allows for
properly tight fits and not the "dry metal on dry metal" syndrome.
Whew, what a lecture!! Good luck
At 05:34 PM 8/19/2004, you wrote:
>I just wanted to say thanks to everybody who gave me suggestions/advice
>for transferring paraffin ribbons to the water bath. Some hints that
>proved particularly helpful were 1) Keeping the blocks cold--virtually
>eliminates wrinkles 2) Simply making sure that the plate of the knife
>holder and the knife itself are very, very clean. This helps in getting
>smooth, unwrinkled sections off of the knife 3) Keeping the pair of
>forceps used to remove the sections ice cold and keeping them very, very
>clean. This helps keep the paraffin from sticking to the forceps, thus
>allowing that first section to be kept if necessary.
>My sectioning is going much, much better, so thank you all very much.
>Now, if someone could solve my chatter problem on the cryostat.....
>It has all the service techs, including a specialist who flew in from
>California to look at it today, baffled. They're saying it might be the
>bearings, but they just don't know. Basically, the chatter occurs when
>cutting at a fast speed, and hardly at all when cutting slowly. We've
>tightened just about every nut, bolt, and lever on the cryostat, tried
>different blades, adjusted the clearance angle, and we're running out of
>ideas. It's a Leica CM1850. Any ideas are greatly appreciated.
> >>> "Sarah Jones" 08/01/04 2:11 PM >>>
> I use a wooden applicator stick that I angle cut, at the tip, with a
>single edge razor blade, making a fine point. I dip the tip of the
>stick into my waterbath before touching the ribbon and the water makes
>the ribbon adhere to the stick. I use them for both the beginning and
>the end of the ribbon. When doing serial sections, it's difficult to
>save every section if you need more than one ribbon. At the best of
>times, I'll usually lose one section at the beginning and one at the
>end. If I have trouble getting the ribbon started, I may lose even more
>than that. If researchers are doing 3D reconstruction, I just keep a
>record of where sections were lost and how many sections were lost. I'm
>doing that right now with some laser induced retinal lesions in the eye
>of the rat.
> If you need thin sections (3-5 microns), the blocks need to be cold.
>If the block isn't cold, the ribbon will be compressed. I always cut
>thin sections off of an ice tray. I use the dissecting pans (without
>wax) available from Fisher for my ice trays. If you are doing thicker
>sections, you can cut at room temperature.
> I once had a disposable knife holder where the sections would stick
>to the metal. I ended up putting a piece of wide, clear, plastic
>packaging tape over it and that kept the ribbon from sticking to it.
>You might try Paraguard too, a spray used for keeping paraffin from
>sticking to surfaces. It has a nasty smell that makes me nauseous so I
>don't use it myself. Also, make sure there isn't any water on the face
>of the block or on the knife before you start a ribbon.
>Sarah Jones HT(ASCP)
>Dept. of Vet. Anatomy & Public Health
>Texas A&M University
>College Station, TX 77843-4458
> >>> "Jacqueline Miller"
>7/30/2004 2:16:00 PM >>>
>I would like to know about the techniques that histotechs use to cut
>paraffin sections--especially how the ribbon is transferred from
>microtome to water bath while preserving the first and last sections.
>I've been sectioning paraffin blocks for almost 4 years now. However,
>I've never needed to cut serial sections and the samples were always
>large. So, I never worried about losing the first section of each
>ribbon (and sometimes the last). I would like to know what techniques
>the histologists out there use to transfer the ribbon from the
>to the water bath. I've used metal forceps and my fingers (gloved and
>not gloved) to grab the first section (but the section sticks to the
>forceps), and I'm using the wooden part of a cotton swab to lift off
>last section, which is working fairly well.
>I'm also using a new microtome, Leica RM2235, and having some trouble
>with the first section coming off the knife just wrinkling up. And,
>the surface below the knife is such that even if I grab the edge of
>section with a paintbrush, it won't budge. Is there anything I can do
>to correct these problems? Also, which is preferred, to cut the
>chilled or to leave them RT. I used to cut blocks chilled all the
>but here it's preferred that I cut them RT unless I have a problem.
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Veterinary Molecular Biology
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