To embed or not to embed ?

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TonyT
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To embed or not to embed ?

#1 Post by TonyT » Sat Feb 13, 2021 3:09 pm

Undoubtedly, embedding in paraffin wax and sectioning using a rotary or rocking microtome gives the best sections. A hand microtome is possibly 2nd best.
However, paraffin embedding is a somewhat specialized and time-consuming technique.
It seems reasonable, before committing to embedding, to take a Q & D (quick and dirty) look at the sample to see if it is worth the effort.
The "double razor blade technique" detailed by Walter Dioni:
http://www.microscopy-uk.org.uk/mag/ind ... icerb.html
is a good Q & D method.
I took a flower stem from a Honeysuckle in our garden in November; this was almost certainly non-living and would fall of the plant during the winter. Stored it in 70% alcohol and sectionined it inFebruary 2021 using the above technique.
Stained in Safranin-O and Fast Green (stains from Brunel), mounted in Canada Balsam. Width of the section: 2.40 mm. Closeup view with 20x Nikon objective.

Proved difficult to get a thin section of equal thickness across the entire stem but at least at one corner the section was thin enough to see detail.
Overall, the double-blade technique proved useful.
honeysuckle 12ii21-1.jpg
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honeysuckle-2 12ii21-1.jpg
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mrsonchus
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Re: To embed or not to embed ?

#2 Post by mrsonchus » Sat Feb 13, 2021 4:15 pm

Hi Tony, very nice indeed with superb colours!

I often (very in fact) hand-section larger tissues such as stems and nodes to see which orientation would give me the view I'd like, or just to be able to anticipate what the tissue is like. Hand sectioning isn't 'as good' as embedded microtomed sectioning in terms of permanency, detail and study, but is of enormous value and 'better than wax-embedded tissue' when carried out for quick inspection or indeed to enable fast appreciation of a plant's fantastic structure.

The sheer accessibility to Botany afforded by hand-sectioning is of great value, and a source of great enjoyment. "Horses for courses" I'd say. Good hand-sections stained well with the right selection of stain/s (for permanence or at least longevity) are also easily mounted in a variety of mountants. I suspect a lot of folk veer more toward the alcohol-based mountants as the resin-based mountants with their requirement of total dehydration seem daunting. However it's not too difficult to for example move a hand section from alcohol over to a limonene-base clearing agent which will allow resin-mountant to be used for the mount....

A really nice example, keep them coming.

p.s. I learned all my basic techniques from the late Walter D by working through his excellent series of examples, great to know others have discovered him also - his work is still superbly valuable and enjoyable to peruse or study.
John B

TonyT
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Re: To embed or not to embed ?

#3 Post by TonyT » Mon Feb 15, 2021 2:48 pm

Hi John,
Thanks for your comments. I looked at a couple of other species and the stems had essentially the same structure with a concentric series of vascular bundles.
Such hand-cut sections are far too thick for serious study. I have a hand microtome and equipment for wax embedding just waiting for a blade-holder and blade; also some warmer weather!

I'm following your lichen studies with interest. One of my texts specifies 25 genera of green algae and 12 genera of cyanobacteria as photobionts in lichens.
The text also describes 8 types of fungal hyphae growth.
Obviously lots of potential and variety in lichens - more than a lifetime's work.
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Re: To embed or not to embed ?

#4 Post by MicroBob » Mon Feb 15, 2021 3:32 pm

Hi Tony,
plant twigs and leaf stems and needles and leaves can be sectioned evenly and nice to 30-100µ thickness with hand microtome and blade holder. With paraffin embedding you can go much thinner. Whether this is needed depends on plant and personal taste. With fresh material you are much more productive and the sections take up more colour. With thin paraffin sections you get more precision in the details and less artifacts from structures below the surface. Paraffin blocks last more or less forever so you can come back and make a few more sections, I just made sections from a 37 year old block :D. I probably is like with craftmanship: It is best to know all methods not just one! :lol:

Bob

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Re: To embed or not to embed ?

#5 Post by mazo4033 » Mon Feb 15, 2021 6:52 pm

Tony, it is an interesting method you bring up. With a lot of perseverance and experimentation, hand sectioning can yield pretty decent sections. Of course nothing beats the quality of a nice rotary microtome's cuts. But personally I do not own any microtome (I wish I did) and do all plant sections by hand, not even a fancy method like the one you linked, but I just take a razor blade, place the stem onto a surface, and cut it. Is it quite a janky set up? Definitely, but after lot of trial and error some good sections can appear, such as this one:

Image

Image

(a loss of quality in sharpness is from the camera, the section is nice and sharp looking through the eyepiece. But it could also benefit from staining that would make the features more visible :) )

Of course the kind of method I describe has its limitations, for example it becomes very difficult to section harder stems, and sections can fall apart or be stretched.

I write this all to say that I think a lot of amateur microscopists shy away from sectioning plants because it seems like you need to go through an intense process of wax embedding and procuring an expensive microtome, and of course that is what yields the best and most professional sections, but with some passion and time, a relatively good section can be made using the most basic tools. It's often the technique that counts, not always the tools! ;)

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Re: To embed or not to embed ?

#6 Post by mrsonchus » Mon Feb 15, 2021 7:35 pm

Exactly what I say - hand-sectioning is so accessible and enjoyable, especially as now it's so easy and cheap to take images for later study and comparisons etc!

A really good aqueous stain for hand sectioning is toluidine-blue, used at about 0.05%. It's easy to get, cheap and totally reliable. As a bonus it exhibits fine metachromasioa, especially-so with 'live' sections. Next in line, safranin again aqueous and at 1% for exactly the same reasons as for t-blue. The two don't mix well, but either is an excellent one-shot stain. Stain for say 30 sec, water rinse and enjoy!
John B

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Re: To embed or not to embed ?

#7 Post by mrsonchus » Mon Feb 15, 2021 7:37 pm

I like the last two images, seems to be an emergent petiole just beginning to expand it's lateral meristem output to build a lamina, excellent!

Here's an example of a safranin-stained hand section I made a long while ago and is in this post of mine from 2019...

The different shades are all from a single application of safranin, which like toluidine blue is metachromatic.
Image
This is sectioned without a microtome, just a sharp blade and a firm grip as I remember..

Have a go at some staining, it's far easier than you may think!
John B

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Re: To embed or not to embed ?

#8 Post by mazo4033 » Tue Feb 16, 2021 3:04 pm

John, thank you so much! It means a lot to get a compliment from you regarding a plant section photo, your threads are the ones that I looked at for guidance on how to hand section very well!! :D It is like getting an autograph from a favorite celebrity.

Yes, the pictures are of an emergent petiole, it is the petiole of a young leaf from Ligustrum (privet bush). I will try out the stain method, thank you for recommending it! There may not be any more of these leaves though, just got 12 in. of snow and I might have to wait until spring to section this leaf again :(

I'm impressed that you were able to section bamboo so well, it seems like a thick and hard stem to cut.

How do you identify the structures of the plants? Is there a book like in protistology?

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Re: To embed or not to embed ?

#9 Post by quantum » Tue Feb 16, 2021 3:22 pm

mazo4033 wrote:
Tue Feb 16, 2021 3:04 pm
Yes, the pictures are of an emergent petiole, it is the petiole of a young leaf from Ligustrum (privet bush).
I cannot help but note that in the Latin version of Harry Potter and the Philosopher's Stone (Harrius Potter et Philosophi Lapis), the opening sentence starts:

Dominus et Domina Dursley, qui vivebant in aedibus Gestationis Ligustrorum numero quattuor signatis. . .

which means

Mr. and Mrs. Dursley, living at four Privet Drive. . .

:)

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Re: To embed or not to embed ?

#10 Post by mrsonchus » Tue Feb 16, 2021 8:19 pm

mazo4033 wrote:
Tue Feb 16, 2021 3:04 pm
John, thank you so much! It means a lot to get a compliment from you regarding a plant section photo, your threads are the ones that I looked at for guidance on how to hand section very well!! :D It is like getting an autograph from a favorite celebrity.

Yes, the pictures are of an emergent petiole, it is the petiole of a young leaf from Ligustrum (privet bush). I will try out the stain method, thank you for recommending it! There may not be any more of these leaves though, just got 12 in. of snow and I might have to wait until spring to section this leaf again :(

I'm impressed that you were able to section bamboo so well, it seems like a thick and hard stem to cut.

How do you identify the structures of the plants? Is there a book like in protistology?

Hi, well, over the years since I started I've built-up quite a selection of books but there's one in particular that I always recommend when asked.

This book is a superb book if you would like to be able to identify structures found in plant sections (hand sections or microtome sections - relevant to both). This isn't a great thick technical tome, nor is it a dumbed-down guide - it's simply very well written and illustrated, loaded with photomicrographs and ties explanations of what the different tissues do, in with the forms that are seen. So much more than simply a book to leaf through until you find something 'that looks like what I have' - under 200 pages, very flexible and not about to fall to bits with constant use - a very well made book indeed.

Did I mention that it's inexpensive and easy to find?

Image



Image
Image

If you're interested in plant anatomy this is a great started!

I thought so - I have some privet tissue (flower-buds, leaves etc) in FAA that I collected last year... They should make interesting sections if I ever get around to them!

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Re: To embed or not to embed ?

#11 Post by MicroBob » Tue Feb 16, 2021 9:47 pm

Hi John,
thank you for the book recommendation! It is also avilable for free here: https://archive.org/details/AColourAtla ... tStructure
Maybe a tablet computer wouldn't be so bad, just for these books I would like to have at hand and not much desk space.

Bob

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Re: To embed or not to embed ?

#12 Post by mrsonchus » Wed Feb 17, 2021 12:05 am

Hi Bob, most welcome. The PDF I downloaded of the book is very handy as you say on a tablet. I must also say that the scans on your linked-to site are of a very high quality. Many thanks for this very handy link Bob.
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Re: To embed or not to embed ?

#13 Post by Placozoa » Thu Feb 18, 2021 4:54 am

Say, thats a really nice book. Thanks! :)

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Re: To embed or not to embed ?

#14 Post by mazo4033 » Thu Feb 18, 2021 6:12 am

John, thank you so much, this is incredibly helpful! And Bob, great link, also very helpful!
Dominus et Domina Dursley, qui vivebant in aedibus Gestationis Ligustrorum numero quattuor signatis. . .

which means

Mr. and Mrs. Dursley, living at four Privet Drive. . .
This is great, makes total sense though because scientific names come from Latin! Now I wonder what "privet" means... (if it has a meaning other than the bush)
Last edited by mazo4033 on Fri Mar 19, 2021 3:09 pm, edited 1 time in total.

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Re: To embed or not to embed ?

#15 Post by TonyT » Tue Mar 09, 2021 2:59 pm

I am still attempting to get decent sections with the double-blade technique (I have a hand microtome but have to find a way to anchor it to the bench).
With the double blade it is easy to get complete thick sections, top is of a lupine stem collected in the Fall and stored in 70% alcohol. About 1 in 10 are quite thin but these are never complete.
However, a thick section is useful to show overall pattern of the vascular bundles and the thin section allows to see more clearer details, bottom image.
Safranin-O and Fast Green.
I must emphasize that the cost of equipment, = 2 razor blades, is negligible compared to other methods.
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lupine stem x-section.jpg
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Re: To embed or not to embed ?

#16 Post by mrsonchus » Tue Mar 09, 2021 3:15 pm

Lovely sections and beautiful staining!

Really nice and as you say, great for morphological example.

If you take these to 'pure' alcohol, then into a limonene-clearant you'll be able to mount permanently to a resin-based mountant as used for the paraffin-method slides I make. I've done this and it works well, the stain will nto be lost either with a non-alcohol mountant.

Keep up the great work, very nice!
John B

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Re: To embed or not to embed ?

#17 Post by MicroBob » Tue Mar 09, 2021 4:03 pm

Hi Tony,
nice section and stain! The marrow sometimes is to fragile to section without embedding in any method. I attatch a photo of my hand microtome holder, th microtome is "permanently" fixed with doubles sided tape.

Bob
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Sarorius Zylindermikrotom mit Halter und Bessey Schraubzwinge.jpg
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Re: To embed or not to embed ?

#18 Post by TonyT » Fri Mar 19, 2021 12:17 am

Found a White Pine twig on the snow last week a result of very strong winds, Placed in 70% alcohol. Sectioned today with the two-blade technique.
Very difficult to cut, could not get a complete section but this one-half shows sufficient detail. The large and small holes in the xylem are resin ducts.
Fast Green gave the blue colour. The red is Safranin-O, a very aggressive stain, used very dilute and stains heavily within a few minutes.
Width across center = 1.40 mm
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Re: To embed or not to embed ?

#19 Post by mazo4033 » Fri Mar 19, 2021 3:11 pm

Tony, that's a great section especially with the two-blade technique.. it's really hard to section wood well by hand. But you've got a lot of good detail, especially with the xylem! Great stain as well. I like your other sections you posted above, too, very nice stains and detail on them.

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Re: To embed or not to embed ?

#20 Post by MicroBob » Fri Mar 19, 2021 6:07 pm

Hi Tony,
nice section!
Lignified plant parts can be really difficult to cut so this is probably the best you can acheive with the double blade technique. With your cylinder microtome, a ridgid carrot embedding and a slicing cut with the blade holde can produce nice 50µ sections over the whole width.
Safranine stains everything like bolognese sauce an needs differentiating. I use 70% Ethanol with a little muric acid for this.
The needles are nice obejects to cut with the double blade technique.

Bob

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Re: To embed or not to embed ?

#21 Post by TonyT » Fri Mar 19, 2021 6:23 pm

Thanks mazo & Bob
Bob: I still have to make a bench-top holder for my hand microtome. I think I have worked out how to do it but all my wood is in my outside shed whose door is blocked by 1/2 meter of snow/ice. I could buy some wood but i do have a shed full. I can wait!
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Re: To embed or not to embed ?

#22 Post by TonyT » Thu Mar 25, 2021 1:58 pm

I have been trying several techniques to get cross-sections of plant stems including 'hand' cutting with the double razor blade, using a hand microtome fitted to a bench with the plant held by a foam plug and with the plant embedded in paraffin wax and using a cutting aid.
All techniques gave inconsistent results but the simplest method, by far, was the double razor blade. The simplicity allowed one to make dozens of sections in a very short time; usually one or two of the sections were suitable for further processing.

We bought some fresh carrots, all the way from California, which still had the greenery attached. I cut up a green stem and placed in 99% alcohol for 3 days. Cut with the double blade. Got quite a few decent complete sections. One partial section was about 1 cell thick!
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24iii21 carrot xylem.jpg
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Re: To embed or not to embed ?

#23 Post by MicroBob » Thu Mar 25, 2021 5:14 pm

Hi Tony,
your grocery store seems to be an endless if unconventional source of especially nice sectioning objects - I have to put carrot on my to do list!
What I'm puzzled about is that you didn't get better and more consistent results with table microtome an blade holder. Is it possible that your carot green was not overly fresh? One important lesson I learned when cutting with he blade holder: A section is the result of two successful cuts. The first cut has to produce a proper section and the second cut the section one wants to use. If the first cut just surfs over the object the second cut will give a double sized and damaged section.

I'm looking forward to your next grocery store find!

Bob

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