Microtomist question

[Olympusman]

I have just finished restoring a classic Spencer 820 rotary microtome and have sent off one of my two knives of to a sharpening service (writeup to follow when the knife gets back).

I intend to embed my specimens in a combination of paraffin and beeswax for sectioning.

I have read in The Microtomists Vade Mecum (a page turner if there ever was one) about an adhesive made up of egg white and Sodium Salicylate to adhere the sections of the slide to allow dissolving the wax with Xlycol/Xylene.

While I cant'maging any pathology lab compunding this formula today, what out there is available?

I know many labs today use cryogenic tissue preparation, but I am not about to acquire something the size of a teletype machine (shows my age) just for casual sectioning.

Thanks a load.

Regards
Mike

[Pau]

Usually labs use pre treated slides to facilitate sections adhesion (at least some of them are actually based in egg albumina).

When I was a young student so many years ago I worked whith a Reichert sliding microtome and tissue samples I embed in paraffin and I don't recall problems using plan slides without any adhesive tratement.

For people working at a pro lab with automatic preparation systems sample adhesion will be a much more important factor than for amateur hand work

[Cyk-Cyk]

Hi,

I read somewhere if you scratch slide with soap, then put a slice on it and put for heating 40C for 8h it should keep slide enough to put it in Xylen and then staining.

I also find information that you can just use egg albumina , as well there are slide with fixative on it but there are quiet expensive.

[yvan_be]

That would probably be "Mayers egg albumen". It is availlable ready made but it's easy to prepare it yourself.

I wrote some notes on attaching sections onto slides, years ago, see: http://www.microscopy-uk.org.uk/mag/ind ... 07ind.html

[Olympusman]

I am familiar with Yvan Lindekens "three-part" series on microtomy. It would have been nice if he had actaully presented us with part three. Never did find out what happened to part three.

In "The Microtomists Vades Mecum" (1898) by Arthur Bolles Lee, Meyer's Albumen is covered on pages 215-217. The formula is 50cc egg white, 50cc glycerin and 1 gram Sodium salicylate.

I'll look for prepared Meyer's Albumen on the internet.

By the way, "The Microtomists Vade Mecum" is an amazing resource. Someone on this website brought it to my attention a few years ago when I had questions about silver staining. Apparently Lee had reviewed a vast collection of papers by microsocpists on what they had learened about preparing specimens for microscopy. It's a free download online. I printed the whole thing one rainy afternoon a few years ago.

Mike

[yvan_be]

I am familiar with Yvan Lindekens "three-part" series on microtomy. It would have been nice if he had actaully presented us with part three. Never did find out what happened to part three. [...]

You probably mean part 4. Part 3: http://www.microscopy-uk.org.uk/mag/ind ... para3.html

Part 4 never materialized due to... well, personal reasons. Something I regret very much. I don't want to go into detail about that. I'm sure you'll understand.
But hey: I still have some paraffin, dyes, chemicals, microtomes, samples, ... left, so who knows...?

I Agree: Bolles Lee is a goldmine for microscopists..

[Olympusman]

Found Mayer's Albumen at http://www.seedsit.com/mayereggalbumina ... 125ml.aspx and ordered a bottle.
Yvan, I was looking forward to the last installment, as it appeared that it would have discussed mounting the sections and dissolving the paraffin. I understand how personal things can upset our lives. I really enjoyed the first installments and thought they were very thorough.

Regards
Mike

[yvan_be]

Hi,

I read somewhere if you scratch slide with soap, then put a slice on it and put for heating 40C for 8h it should keep slide enough to put it in Xylen and then staining.

I also find information that you can just use egg albumina , as well there are slide with fixative on it but there are quiet expensive.

That's right. Attaching sections onto slides using distilled water only is the method of choice, but it's only doable using really *perfect* sections, showing no wrinckles or any other artefact.

That limits the use of the technique to only some homogenious zoological samples, showing no, or only minor variations in density, such as adipose tissue.

This technique solves the problem that some adhesives, especially those using gelatin, are more or less strongly, stained by some basic dyes such as safranin or the more potent haematoxylin formulations such as Heidenhain's iron haematoxylin.

Marice Langeron describes the technique in the several editions of "Précis de Microscopie". He called it: " ... la plus élégante, puisqu'elle ne laisse, entre la lame et les coupes, aucune trace de colle ...". ("the most elegant method, as it leaves no trace of adhesive between section and slide").

But he also warned for the technique being all but fullproof: "... Elle ne donne pas une sécurité absolue et ne convient tout à fait que pour les coupes d'organes parenchymateux sans cavités ni portions chitineuses ou sclérosées.". ("it doesn't give absolute certainty and anyway, it's only usable for parenchymatic organs, chowing no cavities nor chitinous or sclerotic areas").

[yvan_be]

Found Mayer's Albumen at http://www.seedsit.com/mayereggalbumina ... 125ml.aspx and ordered a bottle.
Yvan, I was looking forward to the last installment, as it appeared that it would have discussed mounting the sections and dissolving the paraffin. I understand how personal things can upset our lives. I really enjoyed the first installments and thought they were very thorough.

Regards
Mike

I'll see what I can do with the remaining notes of that part 4.

[discomorphella]

Mayer's albumin adhesive is typically made by simply mincing and mixing equal parts of egg albumin and glycerol, with approx. 1% thymol or phenol as a preservative. It takes a bit of stirring to get it all mixed, then just let it sit in the fridge and use the clear part after a day or 2. Nobody that I know use the salicylate formula anymore. We used Mayers with either phenol or thymol at NCI for a long time, and it works pretty well. More sophisticated adhesion methods have supplanted it for the most modern labs, e.g. poly-lysine or various silane derivatives, but Mayer's works great. Another good technique is gelatin subbing, where you dip the slides in an approximately 0.1% gelatin / 0.01% chrome alum solution and then let them dry. I use mayer's for my home use, its fine unless you are going to digest the sections with a proteolytic enzyme. Make sure you dry the slides thoroughly and then give them a 15 minute 56 C heat treatment to help coagulate the albumin and hold the sections on better. See Ruzin or Kiernan (Histological and Histochemical Methods, fifth edition: Theory and Practice Fifth Edition Edition) for these and other methods. The vade mecum is a bit dated, I would grab a copy of Kiernan 4th ed or the newest 5th edition as a good primary reference.

David

[yvan_be]

This is the techniques I used to stretch and attach paraffin sections, using Mayer's egg albumen:

- Smear an as thin as possible layer of egg albumen on some slides

- Divide the ribbon into individual sections using a sharp knife, such as a scalpel:


- Put a drop of freshly boiled and cooled down distilled water on the slides

- add a section, the shiny side of the section should face the slide:


- Put the slide on a hotplate at a temperature +/- 5°C lower than the melting point of the paraffin used:


- The section will stretch slowly. Add a drop more distilled water if nessecary

- Take the slide from the hotplate, once the section is stretched. Keep the section at it's place using a dissecting needle or whatever, whlie tilting the slide to drain as much as possible of the remaining water:


- Return the slide to the hotplate and let dry for some hours


Picture below shows the back (L) and the front side (R) of a slide to which a paraffin section (Dandelion flower bud T.S.) is attached. When viewing the back side of the slide, once it has completely dried (tilt it against a window or another light source), no air between section and slide, resulting in a mirror alike image, should be noticed:


I emphasized above the use of "freshly boiled and cooled down distilled water". The reason for that: older batches of distilled/deïonozed water usualy contain dissolved gasses, that can end up as "air bubbles" between sections and slide, as can be seen here:


Bolle's Lee, 7th edition (1913), free download: http://www.stainsfile.info/StainsFile/d ... ecum-7.pdf

Lots of information on all aspects of (human/zoological) tissue preparation here: http://www.leicabiosystems.com/patholog ... eparation/

[Raymond]

Yvan,

thanks for sharing your valuable experiences! A great help.

There is an old inquiry of yours still floating around the Internet, asking for manuals for a Leitz model 1300 base sledge microtome. As it happens, I just dragged one of these into my home lab. It probably dates from between 1959 and the mid-sixties. Did you find something useful back in the day? Do you still have it at hand? If so, I would really appreciate a (digital) copy.

I found some pdf's on the web, but these are more like sales brochures with little information on using and servicing this microtome.

Thanks and best regards,
Raymond

[yvan_be]

You're welcome, Raymond.
I'm glad I can be of help with the few things I know. Ah well, as they say: "it takes some wisdom for a man to know that he doesn't know all that much". A comforting thought...

Regarding manuals on the Leitz 1300: my inquiry didn't return a 1300 manual, but someone send me a copy of the Leitz 1400 users manual, which turned out to be helpfull, as both machines have things in common.
I also have a copy of the 1300 (brief) user manual, equipped to cut semi-thin sections. I'll send both through PM, later today.

You won't regret that microtome: it's a nice and simple, thus: reliable machine, capable of cutting paraffin sections as large as some 8 cm * 12 cm. I used mine only a few times. I sold it a few years ago and I kept the other base sledge microtome I had: a Jung Tetrander, as it had a much smaller footprint.

Regards,

Yvan.