An improved double-embedding technique, finally!

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mrsonchus
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An improved double-embedding technique, finally!

#1 Post by mrsonchus » Sun Apr 10, 2016 1:12 am

Hi all, as you probably know I've been working on my 'double-embedding' technique for the encapsulation of plant tissue in agar before final embedding into the usual (paraffin) wax prior to sectioning etc.
The first two attempts made were very successful in terms of the aim to improve the orientation of tiny or awkwardly-shaped tissue pieces during the embedding procedure with molten wax. However the agar itself proved to be a problem or at least a sub-optimal material as the final sections showed many ugly 'chunky-looking' 'bits' throughout the agar, which when sectioned and mounted spoiled (aesthetically speaking) the desired clean-look of my slides.

Anyway, I must be brief here as I've got to get some sleep before a busy day ahead so here are a few pictures that are from a new set of doubly-embedded tissue - the leaf of a Geranium pot-plant in TS. The surrounding agar is now completely homogeneous and virtually invisible! Finally. :D

A few quick pictures focused (literally) on the many tiny glandular-hairs that completely cover a Geranium plant. When a plant is handled these tiny 'flasks of essential-oils on the ends of hairs' are broken open and we smell the strong and distinctive odour of a Geranium plant...

Notice the almost invisible nature of the doubly-embedding agar surrounding the sections! I've stained with a basic Toluidine blue as it's very reliable and also doesn't stain the agar, as Fast green surely and readily does when used in the classic Safranin + Fast green combination stain used so often by me for my sections. The emphasis at this stage of the 'first sections off of the block' was to examine the 'smoothness' of the agar - which I'm very pleased to see has been achieved in this my third trial of this excellent and powerful technique! :D

Some quick pictures, slides not dried yet so these are optically not at their best, but good enough to illustrate the improvement in the agar, and some very interesting glandular trichomes found on the upper surface of the geranium's leaf blade.

Interesting to see two sizes - heights really, they are all of roughly the same diameter, a result it seems of structure (rather than simply more growth and therefore bigger..) - the larger 110µ-ish ones have an additional 'platform' of cells upon which they appear to stand, unlike the shorter 50µ-ish ones that have no such structure at their bases... Interesting and worthy of future investigation.

Enough of my usual blather, hope you find these interesting - I know I do! :D

The background has been evened-out in tone but the agar/wax/section details are untouched and are represented truly here. The agar is so clear and above all homogeneous that it's border with the wax that seeps into and around the tissue during infiltration is barely noticeable as a 'ghost outline' around the section. A far better result than my earlier attempts, things are starting to look very good for the use of this technique! :D
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Here are some quite nicely sectioned 'flask' cells atop their trichome 'stalks' - two distinct heights seem to be present.
First are the shorter 50µ versions,
ws_09041601-x40-glandular-t.jpg
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and the 106µ version with it's extra base of cells giving it it's superior height,
ws_09041602-x40-glandular-t.jpg
ws_09041602-x40-glandular-t.jpg (188.03 KiB) Viewed 3646 times
Now then, a bonus discovery occurred as I ran a quick polarizer-peek over these slides - the polarizer (my cheapo home-made version) revealed that the Geranium tissue contains our old acquaintance Calcium Oxalate, usually found in the form of the needle-shaped 'raphides' as in Daffodil tissue for example (the ovary walls are lined with them!). However, in the Geranium leaf's parenchyma are found crystals in the form of 'flower-like' clusters called Druses (sing druse) rather than needles as with raphides!

Here's a section with druses barely visible with no polarizer at x60,
ws_x60-crystal-no-polarizer.jpg
ws_x60-crystal-no-polarizer.jpg (196.94 KiB) Viewed 3671 times
and as the polarizer reaches extinction magic happens.... :D
ws_x60-geranium-leaf-druses.jpg
ws_x60-geranium-leaf-druses.jpg (47.51 KiB) Viewed 3671 times
My 2mp Toupcam doesn't really do them justice, but it's good enough for the forum-sized images I think. When seen through the 'scope every 'leaf' of the druse's 'crystal flower' can be seen, with amazing effects as the polarizer is rotated somewhat! Beautiful little gems these! What a nice surprise I had! :D

Anyway, that's my lot tonight, sorry to be brief with the technique, I'll concentrate on the production of these results in another post rather than just pictures. Hope you like them!

Back soon! :D
Last edited by mrsonchus on Sun Apr 10, 2016 9:56 pm, edited 2 times in total.
John B

DaveH
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Re: An improved double-embedding technique, finally!

#2 Post by DaveH » Sun Apr 10, 2016 6:34 pm

Superb as usual, great images.

Dave

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billben74
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Re: An improved double-embedding technique, finally!

#3 Post by billben74 » Sun Apr 10, 2016 8:04 pm

The toluidine blue is really lovely and well done on your normal industry in improving the double embedding.
I'm sure I'm not alone on the forum in keeping an eye on your progress with the intention of using your hard earned skill myself - standing on the shoulders of giants. :)
Like the crysal too.

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mrsonchus
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Re: An improved double-embedding technique, finally!

#4 Post by mrsonchus » Sun Apr 10, 2016 9:43 pm

Thanks Bill & Dave, great fun I'm having. :D I also 'stood upon the shoulders' of the late great Walter Dioni (contributor of an excellent series of articles to the magazine).

His superb material launched my own adventures. I still look at and derive pleasure from my first W.D.-instructed onion epidermis slides mounted in many different media; sugar, glycerin, glycerin-gel, nail polish, Euparal, etc as detailed by the great fellow. :)

Nice to know folk are with my adventures, every time I begin an investigation another 10 spring to mind as I 'work'! What a fine thing a microscope is! :D
John B

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billben74
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Re: An improved double-embedding technique, finally!

#5 Post by billben74 » Mon Apr 11, 2016 9:43 pm

I'll join the wax lyrical about the joy of the microscope...

I think I have at least ten years of projects "in the pipeline".
Trying to get some "fancy" staining attempts being my present most burning short to medium term one...

In addition to doing some double embedding...

...making better photographs...

...getting LED light system...

...doing some simple diy fluoresent microscopy...

do you see what I mean? ;)
Last edited by billben74 on Tue Apr 12, 2016 7:52 am, edited 1 time in total.

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mrsonchus
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Re: An improved double-embedding technique, finally!

#6 Post by mrsonchus » Mon Apr 11, 2016 10:41 pm

Haha! It's never-ending once you get the taste for it Bill! :D So many possibilities.... :D
John B

Interference
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Re: An improved double-embedding technique, finally!

#7 Post by Interference » Tue Apr 12, 2016 12:39 am

Great stuff John
I must get round to GCSE biology one day. I didn't do any at school :(
Interference

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