PEG embedding

Here you can discuss sample and specimen preparation issues.
Post Reply
Message
Author
mete
Posts: 82
Joined: Thu Aug 26, 2021 8:31 am
Location: Switzerland

PEG embedding

#1 Post by mete » Wed Nov 24, 2021 7:35 am

I want to try PEG embedding with plant samples, I dont have any experience with paraffin and I was avoiding it, PEG seems to be an easier/simpler and safer process. Following up the reply https://www.microbehunter.com/microsco ... 80#p114180, I have two questions:

- I see from 1000 up to 4000 molecular weights are used. As far as I understand higher ones have higher melting temperature and becomes more hard when solidified so they can be sectioned thinner ? but they are difficult to handle because of a need of high temperature. Is there anything else I need to be aware of ? Should I try 1000 or 1500 or something else first ?

- Can I use a rocking microtome or do I need a rotary or sliding one ?

ldflan
Posts: 21
Joined: Wed May 22, 2019 11:36 pm

Re: PEG embedding

#2 Post by ldflan » Wed Nov 24, 2021 6:02 pm

Hi again.

Haven't used the procedure with PEG in water as mentioned in the post you reference.

I do have substantial experience with Steedman's wax, though (that is, PEG 400 distearate and cetyl alcohol flakes mixed at 9:1). I can tell you that the biggest problems with Steedman's wax (and perhaps also with the water-based PEG method described in the post?) are: (1) it is very, very difficult to get sections to adhere to slides for processing; (2) flattening sections on the slide is tricky; and (3) quality of sectioning and the ability to form ribbons depends very much on ambient temperature of the room and equipment.

Also I found it hard to source the distearate PEG 400. If after looking into Steedman's wax you are interested in getting some to work with, send me PM.

The adhesion problem with polyester wax is the biggest issue. Charged/silanated slides don't help much at all. None of the traditional adhesives work. The only really effective method is a solution of amylopectin in water, which Steedman himself came up with. The trouble with that as a section adhesive is that it leaves microscopic dots of starch that are quite visible at moderate to high magnification, and which interfere with any dye that indicates starch, such as PAS or Lugol's.

. . . .

As to microtomes, my suggestion would be that whatever you get, equip it with a disposable blade holder that fits properly! Sharpening microtome knives is tedious and dangerous, and it's easy to ruin them by doing it wrong. There are problems with disposable blades, too, but in my opinion the convenience far outweighs the problems they introduce. And no microtome will give really good results unless the knife blade is perfect.

The go-to microtome for botanists has always been the sledge type because you can deliver a lot of force, and botanical samples can be woody or harden very substantially in processing. Sledge microtomes are hard to come by, though, and are inconvenient for ribboning. I have an old Reichert sledge microtome, but don't use it nearly as much as my ancient rotary AO820 from the late 1950s.

A used rotary type microtome works pretty well for all but the hardest samples (seeds, wood, etc.) if it and the knife are in good shape. I would get a used one of whatever was most popular in your area from the 1960s on. There are a lot of old AO820s in the US especially, and it's a fine machine, very durable. Whatever you get, make sure that your disposable blade holder will work on it. The inexpensive holders from India do not fit the AO820 knife mount, for example (learned that from hard experience...) Sometimes you can find a used rotary microtome that is already equipped with a proper disposable blade holder for a good price.

For starting out, a whole lot can be accomplished with a high quality hand microtome and a straight razor with disposable blades. Cheap, too. Something to think about.

Leonard

MicroBob
Posts: 2966
Joined: Sun Dec 25, 2016 9:11 am
Location: Northern Germany

Re: PEG embedding

#3 Post by MicroBob » Wed Nov 24, 2021 8:57 pm

Hi Mete,
I would like to suggest an uncommon type of microtome: A base sledge microtome like the Leitz 1300. They are not popular among amateurs but i can't really undestand why. The weight is about 40 kg, a little more than a Jung HN 40 sledge microtome, less than some heavier rotary microtomes. They are fairly easy to take apart, clean and lubricate. They are fairly safe to use as the knife stays in place and the moving hand doesn't come close. The knife can be set at different angles, unlike on most rotarys. If you find one on offer there will be little competition, they sometimes go for under 100€ in good condition.

For plant sections e.g. of stems a cylinder microtome with a blade holder works well.

Bob

mete
Posts: 82
Joined: Thu Aug 26, 2021 8:31 am
Location: Switzerland

Re: PEG embedding

#4 Post by mete » Thu Nov 25, 2021 7:17 am

ldflan wrote:
Wed Nov 24, 2021 6:02 pm
I do have substantial experience with Steedman's wax, though (that is, PEG 400 distearate and cetyl alcohol flakes mixed at 9:1). I can tell you that the biggest problems with Steedman's wax (and perhaps also with the water-based PEG method described in the post?) are: (1) it is very, very difficult to get sections to adhere to slides for processing; (2) flattening sections on the slide is tricky; and (3) quality of sectioning and the ability to form ribbons depends very much on ambient temperature of the room and equipment.
I have read about (1) and (2), naturally it is not very clear to me how difficult it is without actually trying. About (3), that is why I was wondering which PEG to use, I was thinking because of melting point it might be difficult to handle PEG 1000. I saw PEG 400 DS has melting point around 36C, it is similar to PEG 1000. PEG 1500 is higher around 45C. Maybe it is easier to process than PEG 400 DS because of this.
ldflan wrote:
Wed Nov 24, 2021 6:02 pm
Also I found it hard to source the distearate PEG 400. If after looking into Steedman's wax you are interested in getting some to work with, send me PM.
Thanks, at the moment I solved my sourcing issues, so I can get pretty much anything as long as it is not very dangerous.
ldflan wrote:
Wed Nov 24, 2021 6:02 pm
The adhesion problem with polyester wax is the biggest issue. Charged/silanated slides don't help much at all. None of the traditional adhesives work. The only really effective method is a solution of amylopectin in water, which Steedman himself came up with. The trouble with that as a section adhesive is that it leaves microscopic dots of starch that are quite visible at moderate to high magnification, and which interfere with any dye that indicates starch, such as PAS or Lugol's.
Interesting. If I am not wrong, traditional adhesion methods work with PEG, at least that is what I read in a few places.
ldflan wrote:
Wed Nov 24, 2021 6:02 pm
As to microtomes, my suggestion would be that whatever you get, equip it with a disposable blade holder that fits properly! Sharpening microtome knives is tedious and dangerous, and it's easy to ruin them by doing it wrong. There are problems with disposable blades, too, but in my opinion the convenience far outweighs the problems they introduce. And no microtome will give really good results unless the knife blade is perfect.
I definitely have no interest in trying to sharpen the knives :) I will use disposable ones.
ldflan wrote:
Wed Nov 24, 2021 6:02 pm
The go-to microtome for botanists has always been the sledge type because you can deliver a lot of force, and botanical samples can be woody or harden very substantially in processing. Sledge microtomes are hard to come by, though, and are inconvenient for ribboning. I have an old Reichert sledge microtome, but don't use it nearly as much as my ancient rotary AO820 from the late 1950s.

A used rotary type microtome works pretty well for all but the hardest samples (seeds, wood, etc.) if it and the knife are in good shape. I would get a used one of whatever was most popular in your area from the 1960s on. There are a lot of old AO820s in the US especially, and it's a fine machine, very durable. Whatever you get, make sure that your disposable blade holder will work on it. The inexpensive holders from India do not fit the AO820 knife mount, for example (learned that from hard experience...) Sometimes you can find a used rotary microtome that is already equipped with a proper disposable blade holder for a good price.

For starting out, a whole lot can be accomplished with a high quality hand microtome and a straight razor with disposable blades. Cheap, too. Something to think about.
At the moment, I avoid the woody samples until I am comfortable with normal samples (I mean not only for sectioning but for the whole process). I will check ebay for models. Is it possible to work with a hand microtome with a Steedman/PEG/paraffin block ? I mean not as a primary method, I wonder if the block stays because I dont know maybe the contact time is too long etc.

mete
Posts: 82
Joined: Thu Aug 26, 2021 8:31 am
Location: Switzerland

Re: PEG embedding

#5 Post by mete » Thu Nov 25, 2021 7:36 am

MicroBob wrote:
Wed Nov 24, 2021 8:57 pm
I would like to suggest an uncommon type of microtome: A base sledge microtome like the Leitz 1300. They are not popular among amateurs but i can't really undestand why. The weight is about 40 kg, a little more than a Jung HN 40 sledge microtome, less than some heavier rotary microtomes. They are fairly easy to take apart, clean and lubricate. They are fairly safe to use as the knife stays in place and the moving hand doesn't come close. The knife can be set at different angles, unlike on most rotarys. If you find one on offer there will be little competition, they sometimes go for under 100€ in good condition.

For plant sections e.g. of stems a cylinder microtome with a blade holder works well.
Thanks Bob. I think I should find and browse through some old catalogs to make myself familiar with all the models, I only know what the different types are conceptually but I have almost zero idea of the actual models.

By cylinder microtome, do you mean hand microtome ? I have one but I havent used it much yet so not very comfortable with it. I usually use single or double double edge razor blades directly. Paraffin embedding process seemed too complicated and/or a bit unsafe. I recently heard about using PEG for embedding, so if it is reasonably simple and it gives consistent results compared to using a hand microtome with the sample sandwiched in carrot etc., I thought it might be a good idea to try.

MicroBob
Posts: 2966
Joined: Sun Dec 25, 2016 9:11 am
Location: Northern Germany

Re: PEG embedding

#6 Post by MicroBob » Thu Nov 25, 2021 1:42 pm

Botanic sectioning with a hand/cylinder microtome and a blade holder is probably the most productive and least involved method. As long as 50µ sections are fine enough and th specimen holds together well I can recommend to make sections from fresh or fixed material with carrot embedding. You quickly have a watch glass full of nice sections and can start with staining and mounting. But for more fragile specimen you will need embedding in paraffin or else. For paraffin embedding it is possible to develop a compact setup that can be used at home. In my eyes the best start would be the hand microtome, blade holder, some stains, Euparal.
Attachments
Bogenhanf 633 1280.jpg
Bogenhanf 633 1280.jpg (421.26 KiB) Viewed 176 times
BA 01 Ginster W3A Sim DSC_1085.jpg
BA 01 Ginster W3A Sim DSC_1085.jpg (275.92 KiB) Viewed 176 times
Schnitte Übersicht.jpg
Schnitte Übersicht.jpg (213.5 KiB) Viewed 176 times

Microscopy_is_fun
Posts: 11
Joined: Sun Nov 21, 2021 6:11 pm
Location: Austria

Re: PEG embedding

#7 Post by Microscopy_is_fun » Thu Nov 25, 2021 9:23 pm

mete wrote:
Wed Nov 24, 2021 7:35 am
- I see from 1000 up to 4000 molecular weights are used. As far as I understand higher ones have higher melting temperature and becomes more hard when solidified so they can be sectioned thinner ? but they are difficult to handle because of a need of high temperature. Is there anything else I need to be aware of ? Should I try 1000 or 1500 or something else first ?

- Can I use a rocking microtome or do I need a rotary or sliding one ?
As Bob showed in his nice sections above, you can process many plants without embedding. PEG is in my view very helpful for rather soft and "squishy" samples which are hard to slice without embedding, or if you need rather thin samples (<25µm). Usually, it is recommended that embedding medium and sample should have similar hardness. That's why in my view PEG 1500 is a good starting point. By blending in PEG 4000 you can tune the hardness, if required. Smaller molecular weights are probably not useful, since PEG will be liquid around MW of 800.

I agree with Leonard that PEG slices are hard to transfer directly to a slide, therefore I do not see PEG as a real alternative to paraffin, where a direct transfer is possible.

In any case,PEG is worth a try and it's not too much additional effort.

Here is an example of a sliced plant stem from a cypress surge (Euphorbia cyparissias), 35µm thick and embedded in PEG 1500 prior to slicing. These loose tissue structures in the middle of the stem would not work without embedding.
P1001982-Pano_scale.jpg
P1001982-Pano_scale.jpg (274.36 KiB) Viewed 145 times
P1001989_scale.jpg
P1001989_scale.jpg (308.14 KiB) Viewed 145 times

mete
Posts: 82
Joined: Thu Aug 26, 2021 8:31 am
Location: Switzerland

Re: PEG embedding

#8 Post by mete » Fri Nov 26, 2021 10:47 am

Many thanks for all of your replies and nice images. I think I will try to improve my technique first for sectioning by hand, and get better at using stains. Meanwhile I keep PEG 1500 in my mind and probably try it in the future.

Post Reply