Embedding in glycerin soap - recipes and experiences?

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MicroBob
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Embedding in glycerin soap - recipes and experiences?

#1 Post by MicroBob » Sun Nov 18, 2018 7:32 am

Hi together,
embedding in paraffine for making sections requires a comprehensive setup and is somewhat unwieldy for occasional use.
I read about embedding in glycerin soap, a material that only needs water as a solvent.
AO writes that Flemming introduced it in 1873 and that it "has been used sporadical since."
It probably can't compete with paraffine embedding for the dedicated user, but it may be useful for the hobbyist.

So far I haven't found much information about this method - do you have information about it or have you even used it?

Bob

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Re: Embedding in glycerin soap - recipes and experiences?

#2 Post by MichaelG. » Sun Nov 18, 2018 8:04 am

Good question, Bob ... You have captured my interest.

I have found one reference this morning:in a short article by Charles Sedgwick Minot, about the Sledge Microtome, published 1877
https://archive.org/details/jstor-2447828
When the object contains fatty tissue which it is wished to examine more closely, it may be imbedded in the so-called transparent glycerine soap in the way that has already been in use for several years.
If I find anything more detailed, I will update this post.

MichaelG.

.
Updates:
1. See pp86-87 here: http://bhl-china.org/bhldatas/pdfs/j/jo ... 01baus.pdf
From which, for convenient reference:
... The following bibliography contains only those articles referred to in this paper:

BIBLIOGRAPHY. Flemming, '73: Eine Einbettungs methode. Arch. f. mikr. Anat. 9:123-125.

Kadyi, H., '79: Seife als Einbettungs-masse bein Anfertigen Mikdoskopischen Schnitte. Zool. Anz. 37: 476-479.

Lee, A. B., '93: The Microtomists' Vade-Mecum, 3rd. Edit. 18S-191.

Pfitzer, E., '87: Ueber eine Einbettungs-Methode fuer Entwickelungsge-schichtliche Untersuchungen, Ber. d. Deut. bot. Gesell, 6: LXV-LXVIII.

Poll, A., '89: Note die Microtecnica, Malpighia 3:77-84. Also see for the same, Journal Royal Microscopical Society. 835-836, 1889.

Salensky, W. '77: Ueber die Knospung der Salpen Morph. Jahrb. 3:549-602. pi. 28-30.

PI Mead Wilcox.

Lab. of Veg. Physiology, Harvard Uni-versity, Cambridge, Mass, Feb. 8, 1898.
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Re: Embedding in glycerin soap - recipes and experiences?

#3 Post by MicroBob » Sun Nov 18, 2018 11:45 am

Hi Michael,
thank you very much for your research work! I will look into these sources and will continue to write here what I will find.
I bought this soap: https://www.ebay.de/itm/Glycerinseife-R ... :rk:2:pf:1
But so far I haven't got a proper recipe together and haven't tried it out.

Bob

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Re: Embedding in glycerin soap - recipes and experiences?

#4 Post by mrsonchus » Sun Nov 18, 2018 12:29 pm

MicroBob wrote:Hi together,
embedding in paraffine for making sections requires a comprehensive setup and is somewhat unwieldy for occasional use.
I read about embedding in glycerin soap, a material that only needs water as a solvent.
AO writes that Flemming introduced it in 1873 and that it "has been used sporadical since."
It probably can't compete with paraffine embedding for the dedicated user, but it may be useful for the hobbyist.

So far I haven't found much information about this method - do you have information about it or have you even used it?

Bob
Hi Bob - an interesting question....

Well, paraffin 'embedding' is as you say is very involved in that the tissue must first be thoroughly prepared. This is not without reason - the 'embedding' of the tissue is the final stage that follows infiltration - the stage that ensures that the tissue is thoroughly infiltrated right down to the molecular level (i.e. totally without any un-infiltrated areas/spots) with the wax.
This ensures that during the sectioning, sometimes down to 1µ (with an 'ordinary' rotary microtome), the tissue is totally supported.

For un-processed tissue such as live tissue just collected (perhaps a soft stem for example) simple embedding without infiltration is possible, to a limit of course, usually to about 20µ minimum. For this type of simple embedding the main criterion for choosing a supporting (embedding) medium would probably be a match in the hardness (here defined as resistance to the intended cutting blade) of the tissue and embedding medium.

This approach may be implicitly seen when for example 'bunching' tissues together before hand sectioning something such as pine needles (in fact I very recently used this technique to hand-section some truly tiny and delicate moss leaves). The supporting medium in this case, of equal hardness, will simply be the multiple needles surrounding whichever needle gives the best section.

This would seem to be the same principle for embedding in glycerine-soap.... The same result (or better or worse of course) may be achieved by embedding your tissue in wax (without infiltration) if the tissue is say a hard stem such as a rose. For a softer tissue such as a soft leaf from a tomato plant. bunching or embedding in gelatin may be used - I've used simple gelatin embedding for hand-sectioning of seeds which are very difficult to hold for hand sectioning, but not so when inside a nice block of gelatin. The gelatin may also be hardened to varying degrees by dipping briefly, complete with embedded tissue, in formalin....

There is of course also, for the harder tissue usually but not always, the use of the old-faithful carrot! For a slightly softer tissue there is also the option of 'pith tissue' (available online as a cleaning aid I believe) which is used the same way as carrot to hold the tissue.
There's also a good method for pretty thin sectioning of untreated wood with a sliding (not a sledge - sliding microtomes give the oblique cutting edge so useful for hard tissue) microtome which simply involves taking a small (say 2mm-3mm cube) piece and covering it with a resinous glue or similar before sectioning, after the glue has hardened.

The major factor for choice of embedding (as distinct from and without-prior infiltration) material would I think be the matching of hardness/blade resistance during sectioning....

In that respect I suspect that the soap may work quite well - but may be inclined to alter the morphology of the tissue if it is for example plant tissue.

An interesting question; I'd be interested to see it tried. If you do have a go perhaps you may let us know how it goes?

Hope this hasn't waffled-on too much, but this question did catch my eye!

John B.
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Re: Embedding in glycerin soap - recipes and experiences?

#5 Post by MicroBob » Sun Nov 18, 2018 12:53 pm

Hi John,
thank you for your reply! I'm just at the very beginning of looking into the topic so every detail helps.
Wouldn't it be possible to impregnate with soap too? My idea was that with water in the cells and soap and water around there will be reached an equilibrium of soap content and the cells would be filled with soap instead of paraffine. But this may of cause be too naive.

Bob

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Re: Embedding in glycerin soap - recipes and experiences?

#6 Post by MichaelBrock » Sun Nov 18, 2018 1:01 pm

I don't know anything about the use of wax or soap for sectioning but just about every homemade soap these days is glycerin based and the process is simple:

https://www.google.com/search?ei=mWHxW6 ... 8qOAYrI628

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Re: Embedding in glycerin soap - recipes and experiences?

#7 Post by mrsonchus » Sun Nov 18, 2018 1:14 pm

MicroBob wrote:Hi John,
thank you for your reply! I'm just at the very beginning of looking into the topic so every detail helps.
Wouldn't it be possible to impregnate with soap too? My idea was that with water in the cells and soap and water around there will be reached an equilibrium of soap content and the cells would be filled with soap instead of paraffine. But this may of cause be too naive.

Bob
Hi,
For plant tissue the soap would probably infiltrate to some degree, but I wouldn't like to imagine what the soap's presence may do to the cells' morphology....
The purpose of course is to use whatever embedding and/or infiltration medium as a tissue support-matrix as an aid to good sectioning of such unprepared tissue. I don't personally see what the use of the glycerine soap 'brings to the table' for this...

To start with I'd use simple bunching - take for example a dozen or so soft stems and simply draw a sharp blade across them perhaps using your fingernail held vertically as an edge....

Having said that I'd be interested to see what the soap method can do.

Give it a try, an interesting question for sure.

John B.
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Re: Embedding in glycerin soap - recipes and experiences?

#8 Post by desertrat » Sun Nov 18, 2018 6:56 pm

The sources listed above mention the 3rd edition of the Microtomist's Vade Mecum by Arthur Bolles Lee. This can be downloaded as a .pdf below:

https://www.biodiversitylibrary.org/ite ... 6/mode/1up

Between the Biodiversity Heritage Library, Google Books, and the Internet Archive, every edition of the above work can be downloaded for free except the last edition, the 11th, from 1950, which is probably still under copyright. The available editions are the 1st from 1885 through the 10th from 1937.

Only the early editions have instructions for soap embedding.

These works have a great deal of information on preparing zoological specimens for making permanent microscope slides. The 10th and 11th editions have sections on botanical preparation also.

Lee collaborated with other authors to produce similar works in French and German. He worked with Prof. Henneguy to write editions in French, and with Dr. Paul Mayer to write editions in German. Dr. Mayer was for many years a senior researcher at the marine biology laboratory in Naples, Italy, the SZN.

The microtechnique handbook in German:

https://archive.org/details/grundzgeder ... og/page/n5

The microtechnique handbook in French:

https://gallica.bnf.fr/ark:/12148/bpt6k ... /f11.image

Lee started his career in microtechnique as a technician at the marine biology laboratory in Nice, France, the so-called "Russian Laboratory".

Edit to add: Looks like the 11th edition from 1950 has recently become available for download from the Internet Archive:

https://archive.org/details/in.ernet.dli.2015.271798
Last edited by desertrat on Sun Nov 18, 2018 7:36 pm, edited 1 time in total.
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Re: Embedding in glycerin soap - recipes and experiences?

#9 Post by MicroBob » Sun Nov 18, 2018 7:18 pm

Hi John,
my idea was to get beyond what is possible with carrot-support in the hand microtome without goint the whole way to paraffine embedding. When I make a cross cut through a little blossom, it will fall apart. My hope was that soap embedding might make something like that possible. So a kind of carrot-all-around method that allows to get the cross section in one piece onto a slide.

The problem seems to be that there are few people that are looking for simplified methods like this. The professinals don't see the "proper"way as so problematic, and most amateurs stay away completly.

Yesterday we had our group meeting. I showed a new method to make radiolaria slides I have developed. Our members enjoyed it a lot and the marine zoology professor who held a great lecture afterwards would have been happy to have had this method in his active days. So it even happens that the needs of amateurs and professionals meet.

Do you think it is possible to get a proper cross section of a small blossom with soap embedding?

Bob

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Re: Embedding in glycerin soap - recipes and experiences?

#10 Post by mrsonchus » Sun Nov 18, 2018 7:32 pm

MicroBob wrote:Hi John,


Do you think it is possible to get a proper cross section of a small blossom with soap embedding?

Bob
Hi Bob, yes, I see exactly what you mean - the gulf between the full-on 'paraffin method' and the humble carrot does, without an alternative to 'fit between them' as it were, seem daunting.

Hmm, I've no experience whatsoever with the soap-embedding method, but I would certainly say that it's worth a try.
How do you propose to handle the section immediately after cutting and for mounting, with the parts from such a section being discontinuous? Is your intention to leave the soap in-situ around the section?
I'd really like to see this method tried, if you are to have a go I hope you'll keep us updated as it's an interesting idea for certain.

What's your next move with this old chap?

John B.
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Re: Embedding in glycerin soap - recipes and experiences?

#11 Post by MicroBob » Sun Nov 18, 2018 7:46 pm

Hi Rick,
thank you very much for the links to these books. Generally I'm quite happy with my internet capabilities, but in the scientific area I feel somtimes a bit lost.
I will have a look through them and see what use they are. I have an old M.Schanze, Leipzig microtome from about 1870 that would fit nicely to these old recipes.

I would like to reconstruct and develop this method to be useful for group meetings, e.g. one meeting embedding and one meeting cutting or so. Wait and see whether this will work.

@John:
I found a hint to this method a while ago and it got me thinking. My (probably naive) idea was to cut a section, glue the section to the cover slip, remove the soap with water, remove water with i-prop and cover with Euparal. Usually I'm not looking for 100%, I'm satisfied with 80% for 20% of the effort. :roll:

To keep amateur microscopy going, there is a need to single out and develop further methods that are really easy and quick to use. Of cause I will post what I will find, I'm looking for a benefit for all, not just for me.

Bob
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Re: Embedding in glycerin soap - recipes and experiences?

#12 Post by mrsonchus » Sun Nov 18, 2018 7:59 pm

Hi Bob, sounds good to me. That's basically the way the full paraffin method goe, the sections often 'stick themselves' to the slide, but certain methods are useful to make difficult cases stick. The method is called 'subbing' - and a very good mixture is a chrome-alum one which I have used myself on occasion.

Here's some info I gleaned in 2015 that may be helpful. If the tissue can be stuck well the removal of the soap and subsequent application of mountant should be pretty straightforward.
Chr_gelatin_subbing.JPG
Chr_gelatin_subbing.JPG (85.88 KiB) Viewed 5421 times
It really would be a great method to introduce to a group as you suggest, well worth a try Bob.

John B.
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Re: Embedding in glycerin soap - recipes and experiences?

#13 Post by MicroBob » Sun Nov 18, 2018 8:50 pm

Hi John,
thank you for the glue recipe - this sounds very promising!

I have trouble understanding the units though: "gm" is probably gram, but what is "gin" in (5 gin gelantin) ?

Bob

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Re: Embedding in glycerin soap - recipes and experiences?

#14 Post by mrsonchus » Sun Nov 18, 2018 9:27 pm

MicroBob wrote:Hi John,
thank you for the glue recipe - this sounds very promising!

I have trouble understanding the units though: "gm" is probably gram, but what is "gin" in (5 gin gelantin) ?

Bob
Hi Bob - I suspect that "gin" is a badly-scanned OCR version of 'gm', as the correct ratio of gelatin to alum is 10:1.

See this link to a great site I use often --> stainsfile

This mix is very strong indeed for the thinly-sectioned tissue, and may work for the thicker version.

John B.
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Re: Embedding in glycerin soap - recipes and experiences?

#15 Post by MicroBob » Tue Nov 20, 2018 7:46 pm

In the meantime I got this additional recipe:

1. Pflanzenprobe fixieren
2. Pflanzenprobe in Alkohol 30 % ca. 3 Std.
3.Probe in Glycerin + Glycerinseife 1:1 60 Grad C 8 Std.

4.Probe in Glycerinseife 100 % 60 Grad C 8 Std.
5. Probe im Block gießen, 60 Grad C nicht überschreiten !!!

Hans-Jürgen was not too excited about the method. E. Mead Wilcox (In Michaels linked document) reports that he has better success with glycerin and alcohol as a solvent. This seems to be the most promising recipe so far, even if it is from 1898. Right now I'm about to organize the ingredients and will give it a try then.

Bob

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Re: Embedding in glycerin soap - recipes and experiences?

#16 Post by MichaelG. » Tue Nov 20, 2018 8:19 pm

MicroBob wrote:... E. Mead Wilcox (In Michaels linked document) reports that he has better success with glycerin and alcohol as a solvent. This seems to be the most promising recipe so far, even if it is from 1898. Right now I'm about to organize the ingredients and will give it a try then.
Just one small caveat, Bob

As a previously enthusiastic user of Pears transparent soap, I must mention that it is now not what it was. ... and that may well be common to other products. The original was truly transparent, and the 'last remaining sliver' of one bar would nicely fill the the recess in the new bar; bonding perfectly.
E.M.W. wrote:I have employed Pears' Transparent Soap owing to the great uniformity of this product and the resulting superior transparency of the imbedding mass.
I do hope that you can find a soap with the same excellent character.

MichaelG.
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Re: Embedding in glycerin soap - recipes and experiences?

#17 Post by MicroBob » Tue Nov 20, 2018 8:37 pm

Hi Michael,
there are enthusiasts of all kind, even soap enthusiasts! :D
The glycerin soap I got is quite transparent: Through 2cm of soap and the plastic foil around I can barely read fat text in normal print size.
To me this looks ok. The soap is made for people who make soap pieces with whatever in them as a hobby, so this is perhaps a source for a better quality soap.

Bob

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Re: Embedding in glycerin soap - recipes and experiences?

#18 Post by MichaelG. » Tue Nov 20, 2018 9:31 pm

MicroBob wrote:Hi Michael,
there are enthusiasts of all kind, even soap enthusiasts! :D
:lol:
.
Your product sounds good ... Where do you get it ?

MichaelG
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Re: Embedding in glycerin soap - recipes and experiences?

#19 Post by MicroBob » Wed Nov 21, 2018 8:18 am

Hi Michael,
I bought it here:
https://www.ebay.de/itm/Glycerinseife-R ... :rk:1:pf:0

It is made in Turkey.

What did you use the Pears soap for that you had so high demands?

Bob

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Re: Embedding in glycerin soap - recipes and experiences?

#20 Post by MichaelG. » Wed Nov 21, 2018 8:44 am

MicroBob wrote:What did you use the Pears soap for that you had so high demands?
:? Washing myself [personal hygiene]
My grandparents, on both sides of the family, preferred it; and it had all the requisite properties.
https://en.m.wikipedia.org/wiki/Pears_(soap)

MichaelG.
.
P.S. in case it was not immediately obvious; the quote that I included was from E. Mead Wilcox
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Re: Embedding in glycerin soap - recipes and experiences?

#21 Post by MichaelG. » Wed Nov 21, 2018 8:47 am

MicroBob wrote:Hi Michael,
I bought it here:
https://www.ebay.de/itm/Glycerinseife-R ... :rk:1:pf:0

It is made in Turkey.
Thanks for the link, Bob

MichaelG.
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Re: Embedding in glycerin soap - recipes and experiences?

#22 Post by MicroBob » Wed Nov 21, 2018 9:01 am

I haven't tested this soap yet. So I can't confirm that it is usable for this purpose. It is transparent and has a suitable hardness but the real test will probably follow when melted and mixed.

MichaelG.
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Re: Embedding in glycerin soap - recipes and experiences?

#23 Post by MichaelG. » Wed Nov 21, 2018 10:46 am

MicroBob wrote:I haven't tested this soap yet. So I can't confirm that it is usable for this purpose. It is transparent and has a suitable hardness but the real test will probably follow when melted and mixed.
It certainly looks promising
... I will be very interested in your findings.

MichaelG.
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