Wall-Ivy Tissue Prep For Slide Making

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mrsonchus
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Wall-Ivy Tissue Prep For Slide Making

#1 Post by mrsonchus » Sun Jul 28, 2019 11:29 am

Hi all, just thought some may find the very first step of the long & involved process of permanent-slide making interesting.

It all begins with gathering of tissue and the cutting of this tissue into suitable pieces that will then be placed into a 'fixative' that will kill and preserve the tissue pieces basically for as long as you like. This time-out I'm 'grossing' - the term used for this stage, some rather lively Ivy growing up one of my house walls. The fresh and quite soft growing shoots make ideal candidates for the slides I have in mind.

I'm hoping to make some nice longitudinal sections/slides of the growing-tips.

This is a very interesting area that will not only give a good low magnification view to the interested, of the way a shoot develops and branches, supplying the side-shoots and leaves with a vascular system as they develop (by vascular system I refer to the xylem and phloem vessels that carry water from root to plant, and carry sugars and hormones from photosythetic sites such as leaves to the roots, for storage maybe and the rest of the plant as an energy supply, respectively).
A lot of morphology is able to be seen and enjoyed at the lower levels of magnification within the capability of almost any microscope and user-expertise.
At the higher, perhaps 40x objective +, levels the actual cellular arrangement of the very tip, an area called the 'apical meristem' or 'shoot apical meristem' AKA 'SAM' may be seen and studied. The SAM is a very complex area that produces the new cells that will defferentiate into all the other tissues and organs of the plant. Every shoot, side-branch, leaf, flower etc has a meristem.

Anyway, here's a quick look at this type of section made by me back in 2017, of a Coleus shoot-tip, to give an idea of what I'm hoping for from this time, wall-Ivy.

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So, back to the tissue-chopping!

The Ivy didn't see me coming....
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The Ivy is easily persuaded to let-go of the wall at it's new shoots, giving a big untidy pile,
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The tiny roots that spring from along the stem to hold the Ivy against the wall are interesting and an 'added bonus' as I think their apical meristems may be sectionable too, enabling comparison of the shoot-tip and root-tip structure....
Here are a few roots along a stem piece,
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I also took stem/leaf node and leaf tissue for sectioning,
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Closer-in,
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A selection of pieces, including pieces for stem-sections, leaf-sections and stem-leaf-node sections as well as the roots and shoot-tip SAMs.
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I cut enough pieces to use 3 of the plastic processing-cassettes that I put small tissues into for processing (beginning of course with fixative) - larger pieces are simply put loose into the fixative,

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Closed and ready for fixative,
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Larger pieces are loose in fixative,
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The jar containing 3 cassettes and loose pieces, the tissue will be usable and preserved almost indefinitely in this fixative.
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No need to hurry their processing from here, but the Ivy will be one of my very next slide-making projects, along with some other tissues I've been grossing recently....
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Sooo, that's how a slide begins to be made, from the actual cutting and grossing of live tissue, into fixative.....
John B

MichaelG.
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Re: Wall-Ivy Tissue Prep For Slide Making

#2 Post by MichaelG. » Sun Jul 28, 2019 12:38 pm

Another brilliant masterclass commences ....

Thank you, John

MichaelG.
Too many 'projects'

Sauerkraut
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Re: Wall-Ivy Tissue Prep For Slide Making

#3 Post by Sauerkraut » Sun Jul 28, 2019 3:04 pm

Thank you for posting this. Very nice slide image. Anyone who's ever tried to make a slide knows how difficult it can be to get good results.

What specific fixative do you recommend for plant tissue?

Heather

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mrsonchus
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Re: Wall-Ivy Tissue Prep For Slide Making

#4 Post by mrsonchus » Sun Jul 28, 2019 3:35 pm

Sauerkraut wrote:Thank you for posting this. Very nice slide image. Anyone who's ever tried to make a slide knows how difficult it can be to get good results.

What specific fixative do you recommend for plant tissue?

Heather
Thanks Heather - here's a link to my Coleus sections folder that you may like to have a look through.

I always use 'FormolAceticAlcohol' AKA 'FAA' with the alcohol (isopropanol) content at 50%.

Here's a bottle of my home-made FAA-fixative, the fomula is on the bottle,
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The formula, all v/v:
'95% isopropanol' - 50%
'glacial' acetic acid - 5%
'formalin' (which is about 37% formaldehyde and readily available) 10%
water (I use de-ionised battery topup water) 35%

A mix it a litre at a time, it takes only minutes and is very inexpensive to make.
Last edited by mrsonchus on Tue Jul 30, 2019 11:02 am, edited 2 times in total.
John B

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Crater Eddie
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Re: Wall-Ivy Tissue Prep For Slide Making

#5 Post by Crater Eddie » Sun Jul 28, 2019 3:40 pm

I love these procedural adventures!
CE
Olympus BH-2 / BHTU with Olympus E-P1 MFT camera mounted
LOMO BIOLAM L-2-2
LOMO POLAM L-213 / BIOLAM L-211 hybrid
LOMO Multiscope (Biolam)

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mrsonchus
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Re: Wall-Ivy Tissue Prep For Slide Making

#6 Post by mrsonchus » Sun Jul 28, 2019 4:01 pm

Hello Ed' - me too!
I've as you may have seen above, several batches of tissue in fixative (not to mention a couple of dozen in my cabinet) - including some rather nice 'broad-bean' 'beans' grossed about an hour ago...

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Cotyledons are seen here in LS,
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Here the cotyledons are seen in LS and the root-to-be is seen in TS, the shoot between the cotyledons isn't visible here but should appera in LS in other sections at different orientation through the bean,
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The 'stalk' that feeds the developing bean (the 'funiculus') will also be of interest, and I have put several of them into the fixative also,
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I've removed the seed-coat from the beans - leaving just the embryo - as the embryo is my target for slides and the seed-coat in this case - i.e. a fully developed embryo - is not of interest and would make sectioning a little more problematic possibly...
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Another nice jar!
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Perhaps I should post the Ivy sections here when they are ready about a week from now for further processing and eventual sectioning as that was the one I started the thread with! :D :D
John B

Sauerkraut
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Re: Wall-Ivy Tissue Prep For Slide Making

#7 Post by Sauerkraut » Mon Jul 29, 2019 6:06 pm

mrsonchus wrote: Perhaps I should post the Ivy sections here when they are ready about a week from now for further processing and eventual sectioning as that was the one I started the thread with! :D :D
Many thanks for the FFA recipe. And yes please, keep us posted on the sectioning progress.

Heather

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Re: Wall-Ivy Tissue Prep For Slide Making

#8 Post by tgss » Tue Jul 30, 2019 1:23 am

This looks to be an awesome thread - really looking forward to following this one! Please don't feel you have to skip any of the details ;) (No pressure of course!)
Tom

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