Liverworts, a new adventure unfolds...

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mrsonchus
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Liverworts, a new adventure unfolds...

#1 Post by mrsonchus » Sat Jul 30, 2016 11:27 pm

Hi all, well, about those Liverworts I posted about a while ago, the ones from the 'mystery bag' which turned out to have the amazing elater-spirals that flung spores about as they danced their strange dance as shown in my short video in this post -> viewtopic.php?f=6&t=3402....

Well, I've since taken a small piece of newly-grown (Marchantia I think) Liverwort thallus (the green 'plate' that is the body of the tiny plant) that is immature and bears no spore cases, only the 'gemmae cups' that contain tiny little independent 'buds' of tissue ('propagules') that are scattered by water, e.g. rain, and are thus spread to pastures-new, although perhaps not far away from their original gemmae cups!

These are genetically identical to the 'parent' plant and are therefore effectively 'clones' not the result of sexual-reproduction and it's resultant genetic mixing of two different individual parent plants. So, no genetically new individual is formed, more a 'cutting' from the original - not ideal but a means of perhaps increasing this individual plant's size, range and chances of survival?

This you may remember is the original 'big chunk of green-stuff' that I brought home and started to investigate, it turned out to be a Marchantia liverwort with an amazingly complicated structure for such a tiny and primitive (it's an 'early' non-flowering land-dwelling plant - evolved before flowering plants I think) plant....

The mature Marchantia 'clump' looks like this, complete with all stages of reproductive growth, including gemmae cups..
This is what I brought home in a plastic bag!
This is what I brought home in a plastic bag!
ws_gametophyte_entire.jpg (250.39 KiB) Viewed 6452 times
This is the tiny piece of thallus I've been working on with a view to trying to process, section, stain and permanently mount as slides.
Two gemmae-cups - these will make interesting slides I think!
Two gemmae-cups - these will make interesting slides I think!
ws_gemmae-cups-0004.jpg (198.4 KiB) Viewed 6452 times
This piece bears two gemmae cups; numerous pores are also visible on the surface - they enable air (and maybe water?) to enter the tissue as would be the case with the more 'advanced' stomata of a flowering-plant. They appear not to have the 'open & close' capability of a stomate with it's shape-changing 'guard cells' however, and appear essentially to be simply 'holes' in the epidermis. With luck these will be visible in the sections too...

Remember I'm no expert and may be horribly wrong - two weeks ago I knew nothing about liverworts at all - and the little I think I know now is only what I've read in that time in the precious tomes!

I thought these would be a good place to start the adventure, so I set about putting them (as two pieces after cutting the main piece in half) through the full histological process, using a new 15-hr 'extreme' protocol that I thought may be suitable for these tiny pieces of tissue - I got lucky, very lucky...

After full processing through the new 15-hour 'extreme' protocol the two wax-blocks, each with one tiny gemmae-cup and surrounding tissue embedded within, are cast.....

It's tricky as the tissue has to be held upright (actually 'on edge') with forceps, with the mould standing upon an ice-pack to solidify the wax from the bottom-up.
As the solidifying wax 'grips' the bottom of the piece of tissue being held upright (to achieve optimal orientation for TS sectioning) the tissue will begin (hopefully! :D ) to stay in place.
The forceps may then be removed quickly and carefully and a tissue-cassette placed upon the mould's top before more molten-wax is added to fill the mould and ensure the wax-block is attached to the tissue-cassette body that will be clamped into the jaws of the 'Mighty Shandon' (my Shandon 0325 rotary microtome). Phew - sounds harder than it becomes with practice! :oops:
tissue (1 gemmae-cup) into wax mould for embedding
tissue (1 gemmae-cup) into wax mould for embedding
ws_marchantia_embedding.jpg (89.68 KiB) Viewed 6452 times
After they have cooled and completely set the blocks may be 'knocked-out-of-the-mould' to give, all being well, nicely firm wax-blocks ready to feed to the ravenous 'Mighty Shandon'! Hussar! :D :D
Finished blocks - each contains a tissue-piece with gemmae-cup!
Finished blocks - each contains a tissue-piece with gemmae-cup!
ws_marchantia_wax_blocks_re.jpg (85.65 KiB) Viewed 6452 times
Now things get both exciting and a tad nerve-wracking - it's 'make or break time'. The tissue will simply be destroyed rather than give silky-sections if the 15-hr protocol (or my competence :oops: ) fails!

To the 'Mighty Shandon' - and after 'roughing' the blocks to reach the tissue 8µ beautifully-cut sections begin to form a ribbon from the blade's edge! Looks good! I start to breathe again! :D :D

Here's one of what looks like a very promising set of 7 sections cut initially from the virgin blocks! On the slide, still 'wet' and yet to undergo at least 24hrs drying. This 8µ sections looks cleanly-cut (from the very starting-edge of the tissue almost) and to have excellent integrity - as do the others thankfully!
A nice section through the edge of an epidermal-pore maybe - seems to show very good structural integrity...
A nice section through the edge of an epidermal-pore maybe - seems to show very good structural integrity...
ws_mystery_in_wax.jpg (72.91 KiB) Viewed 6452 times
Exciting, and I had to wait patiently for 24-hrs before I could de-wax, stain and mount the sections! That's my 5 pictures and the next post after this one will have the lovely resulting sections that I managed to get time to make 4 of this evening - fully stained and mounted!

Back with some pictures I think you'll enjoy in the very next post after this one... See you soon...

What an adventure - liverworts are fascinating little plants indeed! :D :D
John B

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Re: Liverworts, a new adventure unfolds...

#2 Post by mrsonchus » Sun Jul 31, 2016 12:31 am

Now then - the main feature of this liverwort adventure - the slides!
I processed 4 slides using two different staining protocols. I just couldn't resist trying both for comparison.... :oops:....
Two slides were stained whilst still 'in the wax section' upon the slide - that is to say the dried slides with the wax-sections upon them were placed into Toluidine-blue (0.05% aqueous) where they were stained for 10 minutes (in-wax staining takes about 10-times as long as de-waxed conventional staining, and doesn't always go well!) before being rinsed with tap-water to remove excess stain, then cabinet-dried for 45 minutes at approx 38 deg C.

After which they were de-waxed and mounted directly, as from the de-waxing 'Histoclear' they need no further treatment - Histoclear is compatible with the resinous mountant that I use and enables the coverslip and mountant to be applied directly. This method is good to reduce the 'trauma' that delicate or as in this case 'very freshly cut' tissue has to go through as it's stained often with an aqueous and an alcohol-based stain!

The other two slides were stained with what I think is the most beautiful, reliable and complementary bi-stain combination that I use - that of (red/pink) Safranin O (aqueous) for nuclei and lignin, followed by 'Fast-green' (alcohol-based) for cytoplasm as a contrasting and complementary 'background stain' - a superb combination as far as I'm concerned... :D :D

So, the result was 4 slides, two of each method, and here are a few pictures that I hope you enjoy..

First, this is a Toluidine-blue stained section showing masses of structural detail, including a good TS through a gemma-cup with gemmae at it's bottom, epidermal pores, what looks like an almost 'palisade' spongy mesophyll below the epidermis, the list is endless! For the first attempt and a roughing section I'm a very happy fellow this evening!
x4 - Toluidine-blue stained Marchantia TS
x4 - Toluidine-blue stained Marchantia TS
ws_marchantia_ts_2.jpg (99.72 KiB) Viewed 6446 times
Here's the same slide closer-in, with detail of one of the epidermal pores (that are seen as whitish dots on the surface of the living plant). It's clearly similar-to but not the same as a stomatal pore - intriguing! :D :D
The 'air-chamber' below and spongy-tissue is clearly discernible also - lovely definition.
Toluidine-blue in-the-wax stain has 'done me proud' I think!
Good contrast and definition of a pore in TS - Toluidine-blue triumphs!
Good contrast and definition of a pore in TS - Toluidine-blue triumphs!
ws_marchantia_pore_1.jpg (83.12 KiB) Viewed 6446 times
I also noticed that the rhizoids - not 'proper roots' as such but a means of the plant anchoring itself to the substrate upon which it is living, have an interesting structure - not easy to see as they are truly tiny. They are as expected essentially 'tube-like' but have an amazing series of what look almost like 'teeth' or 'hooks' if they were on the outside.
They appear to be outgrowths of the cell-walls (or should that be 'ingrowths'..) and presumably have the effect either strengthening the rhizoid somewhat or indeed increasing it's (inner) surface area (or maybe both and more...)? I have no idea, but intend to find out!
So many new mysteries - all in such a tiny inconspicuous 'green lump' walked over every day! Amazing! :D

Here's a close look at rhizoid fragments,
Interesting structure - I really need to go to the tomes re liverworts!
Interesting structure - I really need to go to the tomes re liverworts!
ws_rhizoid_projections.jpg (62.86 KiB) Viewed 6446 times
Now - I bet you're wondering what happened to the Safranin + Fast-green slides; well, here are a couple of pictures, the first is a x4 'overall' of the Marchantia stained with Safranin + Fast-green for comparison. Gemmae are clearly visible as are a multitude of other structural details, as is the case with the Toluidine-blue stained slides pictured above - I love the colours of these!
x4 Marchantia TS, Safranin + Fast-green, gemmae clearly visible at bottom of cup..
x4 Marchantia TS, Safranin + Fast-green, gemmae clearly visible at bottom of cup..
ws_marchantia_TS_1.jpg (103.47 KiB) Viewed 6446 times
Finally for picture 5, a close-in look at the above slide - so much to see and investigate! Truly wonderful... :D :D
Super detail of the actual gemmae in the cup-bottom....
Super detail of the actual gemmae in the cup-bottom....
ws_gemma_pan_1.jpg (226.57 KiB) Viewed 6446 times
I think the large-ish, round-ish heavily-stained details may be some kind of channel or duct, maybe even a primitive vessel-precursor? They are also visible in the Toluidine-blue stained slides but at this time I've no idea what they actually are!
I've an awful lot of reading about liverworts to do! These sections are so full of detail I could spend a month slide-surfing them with a few good tomes to hand!
I intend to make a 'full set' from these (well, new tissue that is) to cover all the many features as seen in the pictures of the living plant - so much to include! A great project for the coming Winter months I think - I'm going to be very busy!
That's before I look at them or the live tissue with phase contrast and dark-field with my new Swift 'scope (especially those intriguing rhizoid 'inner teeth') - when I get to this I'll post a phase-contrast vs darkfield vs brightfield comparison - should be very interesting as I'd expect each method to reveal or obscure different features of the tissue! What fun! :D :D

Anyway - I've rambled-on for too long. I hope you find these as fascinating as I do - back soon with more pictures of the many details to be discovered with this amazing little plant! :D :D :)
John B

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Re: Liverworts, a new adventure unfolds...

#3 Post by mrsonchus » Sun Jul 31, 2016 2:34 am

Hi all, I've found out what the deeply stained 'round-ish' areas are - they're oil bodies typical of Marchantia!
A small piece of the puzzle has been out into place!
:)
John B

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Re: Liverworts, a new adventure unfolds...

#4 Post by zzffnn » Sun Jul 31, 2016 3:46 am

Very nice work, John B!

Wouldn't oil bodies be stained with alcohol-based Fast-green, instead of red/pink Safranin? Aqueous solution (Safranin) usually won't stain lipid well?

I would be interested to see archegonium, antheridium and sporangium as well. http://www.vcbio.science.ru.nl/en/virtu ... patophyta/

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Re: Liverworts, a new adventure unfolds...

#5 Post by vasselle » Sun Jul 31, 2016 5:24 am

Bonjour.
Très beaux clichés et comme habitude du très bon travail.
Merci pour le partage
Cordialement seb
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Re: Liverworts, a new adventure unfolds...

#6 Post by billbillt » Sun Jul 31, 2016 10:04 pm

Hi John,

WOW!.. This just gets better and better!!... Wonderful work!..

BillT

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Re: Liverworts, a new adventure unfolds...

#7 Post by mrsonchus » Sun Jul 31, 2016 10:14 pm

billbillt wrote:Hi John,

WOW!.. This just gets better and better!!... Wonderful work!..

BillT
Hi Bill, thanks for joining the adventure old chap - there's more to come soon as I get a couple of hours; we've had a very busy weekend! :)
John B

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Re: Liverworts, a new adventure unfolds...

#8 Post by mrsonchus » Sun Jul 31, 2016 10:14 pm

vasselle wrote:Bonjour.
Très beaux clichés et comme habitude du très bon travail.
Merci pour le partage
Cordialement seb
Thanks Seb - you're very generous, pleased you like the adventure! :D :D
John B

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Re: Liverworts, a new adventure unfolds...

#9 Post by mrsonchus » Sun Jul 31, 2016 11:26 pm

zzffnn wrote:Very nice work, John B!

Wouldn't oil bodies be stained with alcohol-based Fast-green, instead of red/pink Safranin? Aqueous solution (Safranin) usually won't stain lipid well?

I would be interested to see archegonium, antheridium and sporangium as well. http://www.vcbio.science.ru.nl/en/virtu ... patophyta/
Hi there an my friend! Ah... if only stains acted so selectively! :D :D
Most stains (at least the few 'basic ones' that I've used up to now) are quite happy to stain everything in their 'default' use if you like.

It's only when they're differentiated that they become more specific as the less 'attractive' tissue sites lose stain before the more specific and attractive sites for which the stain may be best used. That's my understanding for 'regressive staining' - whereas the 'progressive stains' may perhaps be added ('progressed') to jus the right point to achieve highly selective staining - which is often only visible and so must be controlled (basically 'add then check, add then check.....) whilst viewing the process through a 'scope.

This has evolved as quite distinct workflows for me, the essentially 'morphological' sectioning and staining vs the more selective and accurately targeted (hopefully at least.... :D ) 'HQ' (sectioning and) staining such as the mitotic-spindle differentiation process which I just about managed to get the basics of under control! :D

What the stains won't really do is reliably selectively stain based on tissue chemistry alone - this makes simple staining unreliable for such chemical analysis of tissue with the 'ordinary' light-microscope stains I think.
But there are 'specialist' stains that may be used to stain fats & oils, starch etc to a greater specificity I believe, although I've not started to use those yet - I certainly plan to as my staining is able to become more 'targeted' for want of a better term, as has been the case with my tissue processing and sectioning, as I learn the dark-craft. :D :D
An example of this is that I am now often able to approach sectioning from the 'what plant tissue do I want to section?' rather than as in my earlier days 'what plant tissue gives me the greatest chance of success at this time?'. I'm approaching the staining processes in this same incremental (experimental and about 80% heuristic) manner as early failure is the quickest route to loss of confidence.

It's even possible to enable two supposedly differently-targeted stains to differentiate 'the other way around' if certain factors are altered (often pH at staining-site I think), such as the differences with 'acid stains' vs 'basic stains' which under 'normal' circumstances may be expected to target their favoured tissue as predicted but are quite easily persuaded to act quite 'out of character' depending on various factors - one factor I find very relevant is the type of stain that is added second in a two-stain protocol - the colour of Fast-green for example varies greatly from a bright light green to shades of blue - often following Safranin with Fast-green will have this effect, whereas following Safranin with the similarly 'acid stain' Light-green results in the Light-green stain retaining it's 'true' (originally applied) colour.

Obviously there are ways to detect fats/lipids/oils etc in tissue but will they still be present in the tissue after the often harsh processing that brings them to the stage of staining - I use 'std' fixative and processing protocols for plant tissue at the moment but know there are many techniques to detect in a reliably diagnostic manner tissue chemistry.

So what I may consider is that the staining as seen cannot be trusted to be indicative of the presence or absence of in this case oils/lipids as an indication that these structures are definitely 'oil bodies' or otherwise. As may be seen both Toluidine-blue and Safranin have deeply stained their locations.

I've been persuaded that these may be oil-bodies (but not necessarily with oil/s present) by their structural appearance, which upon closer examination does indeed appear 'tubular' to an indeterminate extent, and my further reading that has brought to light the fact that liverworts are definitely known to possess such structures.
At first peek under the stereo 'scope of those TBO sections stained in the wax the deeply stained areas looked to me like simple 'blobs of excess stain' that as I then attempted to 'rinse away the excess' stood their ground and made me reconsider, in that they were structures actually stained but unknown to me.

I then saw exactly the same event when examining the Safranin-stained sections during their regressive differentiation with alcohol - the tissue again simply held on to the stain indicating a genuine staining reaction had occurred rather than a simple 'excessive pooling' of stain.... Fascinating to see and ponder as their presence was revealed and then confirmed by the two different staining methods!

That is then to say that the staining can't really be used to indicate the presence of oil as an aid to identification of oil-bodies with this tissue and my protocol and stain choices.

So I'm left with the morphological observations such as they are meaningful to my very limited expertise and the 'supporting evidence' Botanical texts -
I'll see if I can find something, also I have somewhere a fascinating paper that actually examines correlation of the staining methods such as I have used here, with the very specific 'chemical tests' involving fluorescent markers - some stains did correlate very well but not exclusively when examining I think lignin with fluorescent staining - this is one of the papers I think, http://library.umaine.edu/MaineAES/Tech ... n/tb53.pdf I'll have a look for the others if you would like to have a peek at them, it's a fascinating area but not one that I'm ready to enter yet....

Oh, the structural evidence, here's a very informative paper-abstract (the article in full is way beyond my pocket! :D :D ) http://www.tandfonline.com/doi/abs/10.1 ... 013.765765

and there's some perhaps clearer info here - this picture really looks like the sections albeit live tissue...
http://blogs.ubc.ca/biology321/files/20 ... tled-6.png

Oh, I also took some more images of them tonight and these are a little better but not I think definitive.....

Here are those couple of pictures of them closer-in - looking at their relative size I'm really not convinced that they're oil bodies at all - although I suppose some may be larger per species - more to investigate...
Looking less definitively an 'oil body' I think...
Looking less definitively an 'oil body' I think...
ws_IMG_5556.jpg (118.61 KiB) Viewed 6390 times
and,
Hmm -- not at all sure....
Hmm -- not at all sure....
ws_IMG_5557.jpg (116.53 KiB) Viewed 6390 times
Hmmm - the more I look and ponder the less convinced I am. Still - these are the initial 'rough' sections from just one block - over the next few days I'll cut some far better sections than these, and I'm going to dissect the full set of antheridia, archegonia and sporangia (interesting to section the spores and elaters in their capsule.. :D :D

Maybe a quick examination for oils with some hand-cut sections would be helpful if I have the time also.

I've a lot of material to choose from as I also found two nice patches (gemmae cups and thallus only unfortunately) in my garden today - as well as a quick trip to the place I found the fully mature specimens I should be OK for the full tissue set I think....

This will be a very interesting exercise indeed - techniques learned here may well give me a good praparation for mosses also - so much to do! :D :D

Thanks for the input Fan, there's a lot of material and mystery in these tiny plants, not to mention beauty. If I can get anywhere near to some of the truly stunning sections I've seen online I'll be delighted! There's only one way to learn! Onward-ho! :D :D :)
John B

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Re: Liverworts, a new adventure unfolds...

#10 Post by zzffnn » Mon Aug 01, 2016 12:35 am

Thank you, John B, for your very detailed explanation. I was just curious. What you said makes sense.

Fixation may have changed all tissue chemistry. Even if you don't fix tissues, I suppose you could still break/empty an oil body if you cut it thin enough in the right orientation.

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Re: Liverworts, a new adventure unfolds...

#11 Post by mrsonchus » Mon Aug 01, 2016 12:45 am

zzffnn wrote:Thank you, John B, for your very detailed explanation. I was just curious. What you said makes sense.

Fixation may have changed all tissue chemistry. Even if you don't fix tissues, I suppose you could still break/empty an oil body if you cut it thin enough in the right orientation.
I think you probably could - it's an interesting exercise to interpret these structures - I was also reading in the article about their coatings of protein - these may respond to stains such as those known to stain nuclei selectively? I think I'll try my Haematoxylins on a couple of sections with the next session - these can be 'backed off' very accurately I find - perhaps Harris' or Ehrlict's as these are the two I use - interesting to try them out next batch.

Thanks for the interest Fan, an intriguing time ahead with perhaps some better sections - these are a little poor for orientation I think - mind you the other block is still to be tried also! :D :D :)

Lots to do - lots to learn! :D :D
John B

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Re: Liverworts, a new adventure unfolds...

#12 Post by vasselle » Mon Aug 01, 2016 9:00 pm

Bonjour
Très belle images
Cordialement seb
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Re: Liverworts, a new adventure unfolds...

#13 Post by billbillt » Mon Aug 01, 2016 9:44 pm

I SURE LOVE FOLLOWING THIS ADVENTURE!!..

BILLT

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Re: Liverworts, a new adventure unfolds...

#14 Post by Culicoides » Mon Aug 01, 2016 9:52 pm

This is a masrteclass in the botany of a little-studied group of plants. I just wonder how many can appreciate the skill and expertise involved in this study. Awesome!

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Re: Liverworts, a new adventure unfolds...

#15 Post by billbillt » Mon Aug 01, 2016 9:59 pm

Culicoides wrote:This is a masrteclass in the botany of a little-studied group of plants. I just wonder how many can appreciate the skill and expertise involved in this study. Awesome!
It sure is!.. John could write a textbook on this!...


BillT

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Re: Liverworts, a new adventure unfolds...

#16 Post by gekko » Tue Aug 02, 2016 12:04 am

Wonderful work indeed! And images to match.

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Re: Liverworts, a new adventure unfolds...

#17 Post by c-krebs » Wed Aug 03, 2016 6:26 am

A lot of work, but the results are excellent. And if you enjoy the entire process, all the better.

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