Apple Twig Hand Section, Stain & Mount

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mrsonchus
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Apple Twig Hand Section, Stain & Mount

#1 Post by mrsonchus » Fri Apr 03, 2020 1:26 am

Hi all, I've been having a clean and tidy in my 'lab' and whilst doing so had a quick go at some crude hand sectioning of some of the small twigs on one of our dwarf apple trees, which are just starting to elongate their buds.
Only really did this as I was distracted by said tree while taking the dog for a quick visit to the garden this morning, but got a little carried away and spent about 4 hours elbow-deep in stains, mountant and of course apple tree parts! Great fun!

Anyway, here are a few images, I'll fill-in some more details and images tomorrow also, but for now here are a few images from today's adventure!

Once I'd started, there was no turning back, and pretty soon my main desk was awash!
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A couple of blades used - the coarse-cut with the stiff-backed razor, the finer slices with a used microtome blade, a tin of which I keep for hand sections.
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Crudely-cut sections - no hand microtome, just a blade down the back of a fingernail - good enough to explore the internal structure for an indication of whether I may make some 'proper' microtome-cut sections of the tissue. This basically depends on what aspect of plant anatomy I'm studying at the time, and how interesting or just plain beautiful the anatomy is...
These are wonky and thick, but they did the trick today. It can be seen in the asymmetry of these sections that lateral branch-traces or leaf-traces (vascular) are traversing the cortex towards their ultimate emergence from the stem's surface, having grown away from the vascular cylinder whence they originated
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Rapidly (and likely poorly - this was only a quick playtime really..) fixed in alcohol then transferred to TEP - triethyl phosphate. I used TEP to attempt to mitigate some of the usual nasty shrinkage almost always seen with rapid alcohol dehydration - a technique I use for my microtome slides when alcohol dehydration either distorts the tissue too much or compromises, even removes, prior staining.
Also, TEP is fine as an ante-medium for Histoclear, which itself is an ante-medium for the resin-based mountants that I use for permanent slides, namely Histomount or Ominimount - almost exclusively Histomount these days as I'm not keen on Omnimount's properties...

I thought I'd start with some Safranin (red) for the vasculature and fibers, then mix a little acriflavin (yellow) with the always reliable alcian blue to give the alcian blue a nice greenish-tinge, and also to act as a fine differentiator for any excess safranin. As it turned out a little of the acriflavin persisted in it's own colour around the lenticels seen in some of the following images. A rather pleasing effect.
I grabbed a selection of stains from my cabinet and considered my approach briefly, here's a handful before I selected the aforementioned trio of stains.
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A fair amount of staining, rinsing, staining, followed, finishing with stained sections in Histoclear, ready to attempt a resin-mountant slide with these ultra-thick sections.
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Because of the thickness of the sections I couldn't of course simply mount and coverslip in the manner used for a 5µ section - so 'packing' had to be used to create enough depth for the setions to be encased in mountant and coverslipped. I 'mounted' a pair of coverslips onto the slide first, with the Histomount resin. This left a space between them for the section to be mounted and a coverslip (and more mountant) placed over, sitting on the two coverslips and sealing the section's position - well that's the idea anyway - this method is not one I've really tried before. I used whole coverslips for one mount, and pieces of broken coverslip for another, just to see what happened really - both seemed to go reasonably well as far as the mounting process was concerned.....

This is the whole-coverslip version with a section in place - already slightly alarmed at the mess I was making.
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Mounted final coverslip and ready for the dryer (60 deg air-dryer) for about 4hrs should be stable enough to enable viewing - some excess will need to be carefully removed also!
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This is the broken coverslip pieces version, same treatment essentially.
Strangely satisying to pulversise a coverslip - oh dear,
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Mounted, hugely messy, but my first go so not too bad.
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Final coverslip over gives this mucky devil,
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A sneak preview through the stereo 'scope - looks OK-ish.
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Here are a few images of the mounts from the dryer, after about 2 hrs or so. The dark dots in the central pith are I think air, this mount will never be permanent, considering also the very basic preparation used and a total lack of clearing of chlorophyll for example, but I'm sure this could be extended to give perfectly good resin-mounts for a reasonable degree of permanency - I strongly fear that the stains would fade also in this method - but this too is probably modifiable and able to be corrected if hand sections that are permanently mounted are desired.

In brightfield,
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Epidermis or probably bark in fact, hence the presence of lenticels - bark cannot contain the flexible stomata found in softer epidermal tissue, so the 'great big hole' approach is employed with the formation of lenticels. There's also what looks like a differentiated close-packed hypodermis beneath - again not sure of the facts without checking.
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A 'great big hole' that is a lenticel!
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and
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Some beautifully vivid fiber-caps, safranin has stained exclusively in the bundle centers, I think acriflavin has 'mingled' in the bundle outer regions to give a rather nice orange-red effect surround.
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Some secondary xylem with rays (which transport radially AKA laterally rather than axially as in xylem) between - these rays will pass through both xylem and phloem to complete the transverse pathways across the stem.
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A nice view of an emergent vascular bundle, probably a leaf-trace making it's way to the surface, where it will enter the base of the leaf's petiole.
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Another fine vascular bundle,
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In polarised to extinction - many, many crystals in cortex, mostly of druse ('flower shaped') form.
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Druse crystals both sides of the (red stained) fiber-bundles that cap the phloem,
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Some crystals are cuboid - but these may or may not be artifacts - not sure,
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Quite pretty with the addition of a lambda retardation plate, very colourful!
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Well, that's about all I can manage tonight - getting tired. If anyone would like to see a few more images mainly of the prep, here's a link to my shared G-Photos folder containg these images - feel free to have a peek.

I'm pretty certain these mounts will never be 'permenent', but they were fun and I'll be interested to see how long they take to begin to go bad.

Hope you like them, I had a lot of fun messing about with them today!
John B

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Seb28
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Re: Apple Twig Hand Section, Stain & Mount

#2 Post by Seb28 » Fri Apr 03, 2020 1:55 am

Great work.Very informative,thanks.
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MicroBob
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Re: Apple Twig Hand Section, Stain & Mount

#3 Post by MicroBob » Fri Apr 03, 2020 4:46 am

Hi John,
thank you for this interesting report! It really is no bad idea to do free hand sections here and there as it is quite a productive method and easier to fit in a busy schedule. A while ago I have made my first paraffin sections and liked it a lot. But I also had a lot of fun with my free hand sections for the fluorescence experiments. Our next decentral/virtual group meeting will be about stomata and how to make slides to observe them. I will present this topic myself and have to get going with the preparations and advertising.

Bob

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Re: Apple Twig Hand Section, Stain & Mount

#4 Post by Hobbyst46 » Fri Apr 03, 2020 8:59 am

John B., mrsonchus, what a combination of skill, knowledge and art in plant anatomy!
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Re: Apple Twig Hand Section, Stain & Mount

#5 Post by jimur » Fri Apr 03, 2020 9:35 am

I always look forward to and enjoy your posts mrsonchus. Thanks for taking the time to share your sessions.
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tgss
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Re: Apple Twig Hand Section, Stain & Mount

#6 Post by tgss » Fri Apr 03, 2020 11:52 am

Excellent, informative, visually compelling, keep 'em coming!
Tom W.

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Re: Apple Twig Hand Section, Stain & Mount

#7 Post by daruosha » Fri Apr 03, 2020 12:42 pm

I really appreciate when somebody goes to the trouble to explain the preparation process with visually appealing photos.

Thanks for sharing.
Daruosh.

Chris Dee
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Re: Apple Twig Hand Section, Stain & Mount

#8 Post by Chris Dee » Fri Apr 03, 2020 1:13 pm

Thanks for explaining the process, much appreciated.

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Re: Apple Twig Hand Section, Stain & Mount

#9 Post by MichaelG. » Fri Apr 03, 2020 2:18 pm

All the preceding accolades ... endorsed !!

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Re: Apple Twig Hand Section, Stain & Mount

#10 Post by MicroBob » Fri Apr 03, 2020 6:30 pm

What I always find funny: We aim at making a tiny object observable, a couple of mm at max. And we devastate at least a whole desk surface, maybe a whole room, each time! :lol:

I now have the added problem of working in home office or better office/lab. This brings a lot of additional stuff that has to be placed and stored. For general stuff my laptop and pressing a couple of keys is enough, but for difficult tasks I need a bigger monitor, telephone and materials to carry out tests. This interferes with the less than stable order in my offic/lab room...

Bob

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