A few images from today - hand sectioned ivy

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mrsonchus
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A few images from today - hand sectioned ivy

#1 Post by mrsonchus » Sun Aug 04, 2019 7:03 pm

Hi all, it's been raining constantly here today so I've been confined to the lab! :D

I've been taking a few 'quick & thick' sections by hand of some very tough ivy (not the same species as the ivy prepared in my previous post) growing all over a drystone wall at home. This species of ivy, yet to be identified, is very tough and very hard to cut with a razorblade. I'm considering preparing (grossing) some of this one to make slides for comparison to the ivy grossed in my earlier post.

So, I took a few hand sections, necessarily quite thick as this is one tough stem! Looking at the sections, which I stained briefly with Safranin and Alcian-blue, it's easy to see why. This stem is full of hard fibers - sclerified-cells with thick lignified secondary walls, staining pink in the following images. The Xylem are a little disappointing in these images, but the sections are so thick that it was unavoidable I think.

So, a few images; quite dark and heavily-stained compared to the thin microtome-cut sections I make..
Image

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The Xylem (I think), with massive fiber-bundles either-end,
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Within the cortex are what I think are ducts - with a recognisable pattern of surrounding-cells in a 'ring' almost,
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This is I think collenchyma which is reinforcing below the epidermis, but is not totally rigid like lignified fibers and so is able to twist and bend during growth without compromise.
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An almost 'glowing' fiber-cap above the blue-stained phloem (I think this is phloem - these sections are pretty difficult to interpre without a very good hard look). To the right of this image is the blurred-outline of a duct within the cortex, to the left is the slightly darker blue cambium and further left the latest (pink) xylem, which looks like fiber rather than vessel-elements.
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Now, I referred at the top of this post to the fibers with the term 'massive' - and these two images, the same image only one in mono, help I think to portray this - massive in the sense of bulk rather than sheer dimension, first in somewhat lurid colour,
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and in a rather dark and heavy-looking mono,
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Those fibers make this a very tough stem indeed - I wonder what the Might-Shandon will make of them! :D

Finally I've been studying morphology of different types of vascular bundle as related to plant adaptation to habit and habitat and though you may like to see a 'normal' Sunflower-stem vascular bundle, again topped with a large fiber-cap (nowhere-near as tough as the ivy above though). This is a 'closed' vascular bundle in that it has no vascular cambium and is not undergoing any secondary growth - in essence it has 'done expanding'...
This is a fully stained and permanently mounted microtome section cut at about 5µ. The cellular contents have been removed prior to staining and mounting by the application of NaOH (I usually use 6% or thereabouts) to give a clearer representation of cellular arrangement without the need for details of cellular contents. Most often I don't treat sections this way as I usually like to study the cellular contents also - but not in this case. This also makes the staining cleaner, clearer and less lurid.
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Here's a stitch of the Sunflower leaf in TS. The obliquely-sectioned leaf veins are seen also - no nice clean parallel-veins to section across as with a typical monocot's leaf!
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This is the central 'mid' or 'main' vein of the same leaf. This slide was stained with the excellently-metachromatic Toluidine-blue stain.
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Just though a few colourful images would brighten-up this dismally rainy and dark day in the U.K.

Hope you find them interesting. I think I'll go ahead and take some tissue for sections of this big-tough ivy, it has an interesting structure. Also, these images are from this year's stem-growth - the older stem preceding this on the plant shows 'growth rings' from it's perennial growth, last year etc.
Last edited by mrsonchus on Sun Aug 04, 2019 9:49 pm, edited 1 time in total.
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Wes
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Re: A few images from today - hand sectioned ivy

#2 Post by Wes » Sun Aug 04, 2019 7:30 pm

Thats very interesting, thanks for posting! So Safranin stains the sclerified cells because its preferentially binds to lignin or some other component? They have such thick cell walls, very appealing visually. The sunflower sections are amazing.

Have you ever considered studying the cellular changes upon treatment with different plant hormones or noxious agents?

geo_man
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Re: A few images from today - hand sectioned ivy

#3 Post by geo_man » Sun Aug 04, 2019 7:34 pm

wow, you are a master! great pics and descriptions, thank you!

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mrsonchus
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Re: A few images from today - hand sectioned ivy

#4 Post by mrsonchus » Sun Aug 04, 2019 8:38 pm

Wes wrote:Thats very interesting, thanks for posting! So Safranin stains the sclerified cells because its preferentially binds to lignin or some other component? They have such thick cell walls, very appealing visually. The sunflower sections are amazing.

Have you ever considered studying the cellular changes upon treatment with different plant hormones or noxious agents?
Hi Wes, The Safranin will actually stain all tissues - a 'general stain' one may be tempted to say. Staining though is a very difficult area, especially-so with permanent slides sych as the Sunflower section. With living tissue or perhaps 'non processed' tissue, staining is in my experience far easier. A bright & colourful result to show general morphology (as detailed work will require the far thinner microtome-cut sections) is easily obtained almost by just adding stain, rinsing, adding a contrasting staine and so-on....

BUT, preferential-staining is another story altogether! In the case of Safranin in the above hand-sections it's more a case of staining with Safranin (which stains everything) then 'chasing away' or displacing some of this Safranin with either the addition of a solvent such as acidified alcohol (so-called differentiation) or a competing stain, in this case Alcian-blue. The affinity, not true selectivity I think, for stains such as these two enable them to stain different types of tissue, but not to the very strictest sense of 'stain A will only stain Tissue a'.

Staining for selectivity and differentiation is a very complicated and most, most definitely heuristic process that takes years to master. I've been making slides now for about 4 years and have only scratched the surface of staining.
This is a massive subject about which I could go-on for days my friend! :D A look over some of my earlier posts, starting in 2015 I think, will give you a good idea of what's involvedin the making of permanent slides.

Hormones - yes I'm actually reading right now about the role of hormones and signalling within the plant body. In particular I'm studying the 'phase changes' of a meristem, for example when a vegetative-phase shoot-apical-meristem happiliy producing leaves and axial-buds is triggered into a phase-change to the reproductive stage where a flower will be produced (or a whole branching-system of flowers - an 'inflorescence') and the shoot will terminate by the using-up as it were of the apical meristem.
There are hundreds of signals both inhibitory and stimulating, long and short range, ariving at the meristem in the form of hormones etc that first prepare the meristem for it's floral phase/stage (once it has achieved 'competence')....
In fact, here's a link to a shared folder of papers on this subject that you may find interesting.

A fascinating subject - an understanding, albeit in my case a very basic one, of the way meristems, both apical and lateral (lateral meristems produce secondary tissue that thickens rather than lengthens the shoot, such as secondary xylem, phloem), produce new cells (initials) that then produce cells that will differentiate into various specialist cell-types and the arrangements and sequences in which this occurs, helps me to understand the tissues and structures that ultimately end-up on my slides!

The paper (one of those in the linked-to folder) on epigenetic regulation of gene expression is a real revelation and of course complements the signalling from environment or other internal events of course - a subject that really does bolster understanding of a plant's form I find.

I'm also now able to see even finer detail with my BX40 and this is really helping me to differentiate the different cell-type of for example xylem, which not only has vessel-elements with several different secondary wall thickening (lignin) types, which are easily seen, but has parenchyma, fibers, tracheids etc - which in section and with a light-microscope can be very hard to confidently tell apart, without a good resolution of the cell-wall pit-types. This area in particular is benefitting from my change to Olympus from Leitz, at least with my brightfield use (and a little basic phase contrast - also very good for cell-walls).

Botany is a truly fascinating subject to me, and all available at my tatty old desk! Wonderful! :D
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Re: A few images from today - hand sectioned ivy

#5 Post by Hobbyst46 » Sun Aug 04, 2019 8:43 pm

Very attractive images, mrsonchus! personally I like the sunflower, yet the IVY are important. Our local (not native) ivy digs hard into the exterior of houses and brutally clings to the plaster - not to mention how it covers and dominates whole grown-up trees.
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mrsonchus
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Re: A few images from today - hand sectioned ivy

#6 Post by mrsonchus » Sun Aug 04, 2019 8:44 pm

geo_man wrote:wow, you are a master! great pics and descriptions, thank you!
geo', you're very generous, thank you. It's easier than it looks though, to take a few hand sections. I'd always recommend hand-sectioning - just with a single-sided razor and maybe a strong food dye or stain such as Safranin which is so cheap to buy and simply mixed with water at 1%.
I often investigate a considered tissue with hand sections before I commit to the full-on histological processing and production of permanent slides for my collection.

Most any type of Curcurbita (squashed etc) is very good as everything's so large - very large phloem sieve-plates easily seen in hand sections for example.

When yoou chop veg for tea - save some for the microscope!

Thanks for your interest geo_man.
John B

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Re: A few images from today - hand sectioned ivy

#7 Post by mrsonchus » Sun Aug 04, 2019 9:08 pm

Hobbyst46 wrote:Very attractive images, mrsonchus! personally I like the sunflower, yet the IVY are important. Our local (not native) ivy digs hard into the exterior of houses and brutally clings to the plaster - not to mention how it covers and dominates whole grown-up trees.
Hi Hobby' - yes the Sunflower slides are one of my favourites too, here's a series of images I took a few days ago to show the different objective powers from 4x to 60x, of a Sunflower stem sectioned at about 8µ nad stained with Toluidine-blue, permanently-mounted in resinous mountant 'Omnimount'...

The 4x view,
ws_4x sunflower stem.jpg
ws_4x sunflower stem.jpg (150.77 KiB) Viewed 1424 times
The 10x,
ws_10x sunflower stem.jpg
ws_10x sunflower stem.jpg (127.42 KiB) Viewed 1424 times
The 20x,
ws_20x sunflower stem.jpg
ws_20x sunflower stem.jpg (96.46 KiB) Viewed 1424 times
The 40x,
ws_40x sunflower stem.jpg
ws_40x sunflower stem.jpg (56.85 KiB) Viewed 1424 times
The 60x,
ws_60x sunflower stem.jpg
ws_60x sunflower stem.jpg (48.73 KiB) Viewed 1424 times
How about a longitudinal section through the very immature tiny florets crowded onto the Sunflower's head. Here are a row of florets, and the second image closer-in with the primordial individual floret (small flower) parts labelled,
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Close-up developing floret-parts,
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Each of these tiny florets will ultimately produce 1 seed each....
Here's an image or two of a LS through such a seed, with a developing embryo - on the way back to producing another monster Sunflower! The circle completes!
The whole seed sectioned legthwise,
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A very early-stage embryo inside the seed's ovule,
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Embryo at the later 'spherical' stage,
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The egg-sac is enclosed (early-on at least) by a 'nucellus' tissue - I think these are nucellar cells - the cytoskeletons holding the nuclei have been nicely fixed and are easily visible in their 'web-like' form...
Image

I have LOTS of images but I'd better stop there before I bore averyone to death! :D :D
John B

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Re: A few images from today - hand sectioned ivy

#8 Post by MichaelG. » Sun Aug 04, 2019 9:17 pm

mrsonchus wrote:I have LOTS of images but I'd better stop there before I bore averyone to death!
Keep 'em coming, John
... It's awe not bore

MichaelG.
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Wes
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Re: A few images from today - hand sectioned ivy

#9 Post by Wes » Mon Aug 05, 2019 7:34 am

mrsonchus wrote: A look over some of my earlier posts, starting in 2015 I think, will give you a good idea of what's involvedin the making of permanent slides.
Its amazing how you've progressed from simple hand cut sections to high-end microtome cut sections. Your enthusiasm is contagious and now I find myself thinking more and more about getting a microtome and the accessories gravitating around cutting fine sections.

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