Final Lichen Images - Apothecia!

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mrsonchus
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Final Lichen Images - Apothecia!

#1 Post by mrsonchus » Thu Feb 18, 2021 10:18 pm

Hi all, if you've seen my previous posts on the adventure of making Lichen slides you may remember that I was hoping to make some 2µ sections having already cut 10µ then 4µ sections. With a view to seeing if they'd section at 2µ and if any extra detail may be seen I tried to section at 2µ. Well, they don't section too well at 2µ (no real surprise there though worth a try). I have made a couple of slides at 2µ that I don't really rate though and have yet to take images from that I like, but after a few days they may well improve as the mountant dries or at least stabilises somewhat.

Anyway I did make quite a few new slides at 5µ, which for this lichen and the paraffin technique, limited also by my own ability, has proven to be the optimum both in terms os section-quality and staining results. They are quite good really and have some new detail! I was hoping to 'drill-down' into the wax blocks that I posted early images from at the start of sectioning, in earlier posts.

Well, I pleased to say that I have been able to section through the lichen's (sexual) reproductive 'apothecia' - the cup-shaped structures that house the spores - 'ascospores' as I think they're know,n several of which are enclosed by their container - an 'ascus' - I think....

Here are a few images, first the macro image of the lichen as seen through a stereo 'scope.
The cups (apothecia) are easily spotted on the thallus of the lichen.
Image


Image

Then we move over to the slides made only the day before yesterday and stained with safranin and fast green or safranin and alcian-blue, as I often try several stain combinations to see which suits the slides as made....


This section through an apothecium is stained with safranin and fast-green and shows the macro-structure of the 'cup' - like a tiny volcano!
Image


Moving in closer structures start to become visible, although not as clearly as I'd like by a long-shot....
Image

Closer-still, the ascospores (as far as I know that's what these are - please correct if wrong...) show structure also, and this image I find quite pleasing, the slide is quite nice, not perfect, but O.K. to keep.
Image


Quite nice to see a little detail too of some of the fungal-hyphae of the lichen, although again not enough clarity and detail for my liking...
Image


I aslo switched briefly to phase-contrast although I have only basic phase objectives at 10x and 40x (oh and a recently acquired 100x oil).
A different view but with limitations, although I haven't really had a good look over them yet for a proper assessment.

The vertical fungal strands knwn as paraphyses when in this role of enclosing (protecting?) the asci are though quite well deliniated if not particularly detailed...
Image


A different view,
Image

Soooo, no decent 2µ images yet, but at least I reached the apothecia, I've quite a few blocks still unsectioned that I may have a go at in the next few days with a view perhaps to improving quality at the 4µ-5µ defacto optimum if I can't get any thinner with quality intact.

Hope you find these interesting, I certainly have as I've never even looked at lichen before this exercise began!
John B

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Re: Final Lichen Images - Apothecia!

#2 Post by perrywespa » Thu Feb 18, 2021 10:29 pm

Wonderful and inspiring images, John.
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Re: Final Lichen Images - Apothecia!

#3 Post by mrsonchus » Thu Feb 18, 2021 11:24 pm

Many thanks, pleased you like them.

I've been having a look on t'internet for lichen spore form info and think I may have found some interesting info with images at this fine website....

I thought at the outset of this adventure that the lichen featured here may be the common foliose-lichen called Xanthoria.parietina (see my earlier post images). This Australian website seems to support this when looking at the spores. They are said to be of a 'polarilocular' form. They're 'septate' in that they have a wall. This wall is very thick/wide and separates two cells. A connecting channel runs through the wall to connect the two cells...

When comparing my images with both websites it seems that the form of the spores is a very good match, from a couple of aspects too.
Looking closely at the spores the two polar cells are visible as is the thick wall between them, together with the channel through the wall! I think......
I added labels to this enlarged portion of an earlier image - see arrows in white...

Image

Who would think that such a tiny and common thing such as a liverwort on a tree twig could hold such delights!
John B

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Re: Final Lichen Images - Apothecia!

#4 Post by mrsonchus » Fri Feb 19, 2021 12:06 am

Crikey, things just became even more fascinating!

I looked back over some hand-sectioned/dissected lichen I had under the 'scope back in January - I didn't make permanent slides back then, but I did take images. This lichen I think was a common 'jelly lichen' called 'Collema.fasciculare' and was growing on some limestone outcrops in our garden....

Interesting to see that the ascospores (as I now think I know they are called....) are visible and of a different structure to the 'polarilocular' spores of the above featured Xanthoria.parietina lichen.... The ascospores of the Collema appear to be multi-cellular and this is also suggested in this free-to-download article at one of my favourite sources, the ReasearchGate site. Images within this paper show the ascospores of the C.fasciculare to be very similar to those in the following images from my January dissections....

This is quite a black lichen, but not at all times....
Image


Here's a hand-section I took through an apothecium 'cup' at the time, and stained with toluidine-blue for a quick look...
Image


Closer-in the ascospores begin to emerge from the gloom....
Image

I added a little oblique to try to give a little texture to the images, all of water-mounts with coverslip, as seen through an Olympus BX50 compound 'scope...
I should have posted this dissection I suppose - but at the time I was looking at the mosses surrounding these lichen....
Image


Finally another oblique - different direction - created by simple cardboard 'Matthias-arrow' as discovered years back when I was studying the late W.Dioni's superb articles...
The different structure - i.e. multicellular and unlike the previous images of Xanthoria.sp ascospores is quite clear to see here,
Image

Lichens are really quite ineteresting! :D
Last edited by mrsonchus on Fri Feb 19, 2021 2:53 pm, edited 1 time in total.
John B

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Re: Final Lichen Images - Apothecia!

#5 Post by deBult » Fri Feb 19, 2021 12:16 am

Fascinating, thanks for sharing John.

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Re: Final Lichen Images - Apothecia!

#6 Post by PeteM » Fri Feb 19, 2021 12:18 am

Bravo, John.

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Re: Final Lichen Images - Apothecia!

#7 Post by apochronaut » Fri Feb 19, 2021 1:21 pm

John. I am constantly amazed at the paced precision you approach your samples with. Lichens are fascinating but I must admit that I have only briefly looked at them in a stereo microscope. I must get closer to some, after seeing this fine work. Mostly I watch them from afar as they "bloom" after a rain or on certain rocks that have seemingly been there forever. And, apparently, they can survive exposed to the vacuum and temperature upheaval of space! One more piece in the puzzle of Panspermia.

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Re: Final Lichen Images - Apothecia!

#8 Post by mrsonchus » Fri Feb 19, 2021 2:56 pm

Many thanks for your kind comments chaps, really pleased you like the adventure.

Apo' - it surprised me too to see just what lichens are and how they're 'built' as it were!
It's very nice to receivce such kind compliments, thanks all. :) :D
John B

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Re: Final Lichen Images - Apothecia!

#9 Post by Hobbyst46 » Fri Feb 19, 2021 3:30 pm

Very beautiful images (as usual, John B) of the stained lichens.

Might be interesting to compare the info yielded by the phase contrast vs the stained specimen. Especially since the sections are so thin and flat.
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Re: Final Lichen Images - Apothecia!

#10 Post by KurtM » Sat Feb 20, 2021 1:25 am

John, your slide making is simply glorious!
Cheers,
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Re: Final Lichen Images - Apothecia!

#11 Post by mrsonchus » Sun Feb 21, 2021 10:34 pm

Thanks hobby and kurt, pleased you like them!
John B

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Re: Final Lichen Images - Apothecia!

#12 Post by mrsonchus » Sun Feb 21, 2021 11:31 pm

Hi, I cut some new 2µ sections with the blocks chilled in the deep-freeze, which hardened the wax-blocks considerably and helped a lot with sectioning at the 2µ level. Still there was a measure of folding where the tissue was I think harder than the wax (I need to buy some more that has a 58 deg mp, I'm only using 52 deg at the moment, which is fine, but not hard enough I suspect in this case. Harder wax would offer a more equal resistance to sectioning and would likely at the very least mitigate the folding if not cure it....

Anyway, it turns out as I sectioned into more blocks that I had quite a few apothecia (the 'cup' that houses the fungal reproductive ascus, which in turn contains the ascospores) and was able to take some very nice 2µ sections. These showed great detail after staining with safranin and fast-green. This is my preferred choice of staining protocol as I found the use of alcian-blue (also with safranin) to give too much brilliance, and this obscured detail in photomicrographs, which the fast-green counter-stain does not in this case...

Here are a few quick images from slides made this afternoon - as you'll see the detail is very nice, especially considering these slides are not anywhere near set yet. I'm looking forward in say a weeks-time, to using an oil 100x objective to hopefully look into even greater detail.

Here's an apothecium sectioned through the cup-overhang only, showing in this 20x stitch image a lot of resolution - makes the earlier 10µ sections look poor, and even betters the previous 4µ slides I made in earlier posts. See what you think,

Whole-width section of an apothecium, 20x objective stitched...
2micron lichen section (10)_stitch.jpeg
2micron lichen section (10)_stitch.jpeg (85.79 KiB) Viewed 182 times

Closer-in, ascospore measures at about 10µ on it's longest axis...
measured ascospore 2micron lichen section (12).jpeg
measured ascospore 2micron lichen section (12).jpeg (84.68 KiB) Viewed 182 times

Closer-in,
2micron lichen section (2)_stitch.jpeg
2micron lichen section (2)_stitch.jpeg (143.46 KiB) Viewed 182 times
Not much time tonight, hope you like these, I'm pretty pleased with them.
I now have over 2-dozen lichen slides!
John B

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Re: Final Lichen Images - Apothecia!

#13 Post by MicroBob » Mon Feb 22, 2021 7:26 am

Hi John,
very impressive sectioning and staining! It is interestig to see how much clearer the image becomes with thinner and thinner sections. Mike sectioned to 30µ, I to 20µ, and you to 10µ and have now have reached 2µ - and the clarity has developed accordingly. I have a Leitz base sledge microtome which should be able to section this thin, but I haven't done it so far. Yesterday I spent two hours walking around a moor but no interesting lichen to be found. :roll: probably too far away, I have to look closer to my door step. :lol:

At what concentrations do you use the safranin and fast green? I experimented with safranin for a while now and found it to stain very strongly. I have fast green but haven't used it so far. Compared to thicker fresh sections the very thin paraffin sections can probably take a little stonger staining?

Bob

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Re: Final Lichen Images - Apothecia!

#14 Post by mrsonchus » Mon Feb 22, 2021 4:49 pm

Hi Bob, thanks for the kind comments!

My go-to working solution of safranin is 1% aqueous. The aqueous stains tend to be pretty simple to use in general I'd say. Addition of a counter-stain, or even a third and occasionally even a fourth to the tissue is where the complications begin, and the learning starts in earnest!

Safranin as you have found is a very vigorous stain in general, and is, in almost every tome I've ever perused, said to be easily removed/reduced/differentiated by the use of alcohol, or acidified alcohol - not particularly true however... I remove safranin this way with between 1 and 5 % acetic-acid. HCl is also good but very, very fast acting, to a difficult degree in fact... It's quite possible to have safranin 'stuck in' the tissue where the aforementioned method simply doesn't work - the saviour then is to switch to HCl, which makes short-work of the safranin, but only as a last resort....

There are several ways to mitigate safranin's strength, one is of course to lower the concentration, say to 0.5% or 0.25%. Also the time may be adjusted but if the required time begins to get so short as to be impractical - usually because the human-error margin in the application and removal becomes a significant part of the total time. Try staining in safranin for 5 seconds then water-rinsing, not easy with more than say 1-2 slides!

The above 2 methods aside, the addition of a percentage of alcohol inhibits safranin and is quite a good method as often it will be followed by an alcohol-based counterstain such as fast-green in this case... 1% safranin in 50% alcohol is pretty useful.

I use fast-green almost exclusively at a 0.25% in 85% alcohol formula. If fast green is used in water it simply won't stain, it's just too mobile. In alcohol only it will also stain the slide itself and be hard to remove! At this concentration I stain by dropper-application (unlike the safranin that I apply in staining-troughs) for 5sec intervals - 5sec has become my basic unit for fast-green. It's easily removed quickly with a dropper and pure alcohol.
If fast-green is rinsed with water it will be totally removed!

Hope this helps - I've quite a few more sections on slides ready to stain and mount, of varying thicknesses - more images soon.

lichen slides ready to process.jpeg
lichen slides ready to process.jpeg (117.63 KiB) Viewed 95 times
John B

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Re: Final Lichen Images - Apothecia!

#15 Post by MikeBradley » Mon Feb 22, 2021 6:54 pm

John, these images just keep getting better, thanks for pushing the boundaries for us!

I've been looking for a supplier for Fast Green and see that Brunel carries it in Cellosolve, is this a suitable medium for the counterstaining you do?

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Re: Final Lichen Images - Apothecia!

#16 Post by MicroBob » Mon Feb 22, 2021 7:02 pm

Hi Michael,
Magnacol in England would be an alternative supplier, they sell it it powder form.

Bob

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Re: Final Lichen Images - Apothecia!

#17 Post by mrsonchus » Mon Feb 22, 2021 7:22 pm

Hi all, yes as Bob says, I've been buying my dyes from the same superb supplier - scuddlebut AKA Magnacol, for about the last 5 years. I began with Brunel Microscopes and as I learned more I naturally widened my scope for a growing selection of lab requirements.

This is the fellow I buy virtually all my stains now from now. The prices are excellent and the stains/dyes are absolutely fine in my long experience.

This link takes you there...
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Re: Final Lichen Images - Apothecia!

#18 Post by MikeBradley » Tue Feb 23, 2021 6:02 pm

John, unfortunately scuddlebut do not ship to Canada or the US due to some insurance issues however I have been able to source the Fast Green powder from a US supplier now. As far as these stains being sometimes supplied dissolved in Cellosolve, is that an impediment for botanic staining?
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Re: Final Lichen Images - Apothecia!

#19 Post by mrsonchus » Tue Feb 23, 2021 8:28 pm

MikeBradley wrote:
Tue Feb 23, 2021 6:02 pm
John, unfortunately scuddlebut do not ship to Canada or the US due to some insurance issues however I have been able to source the Fast Green powder from a US supplier now. As far as these stains being sometimes supplied dissolved in Cellosolve, is that an impediment for botanic staining?
Thanks
michael
Hi Michael, certainly not an inhibition. I suspect that the inclusion of cellosolve makes the formulation (together with other ingredients) closer to some of the oft-quoted classic formulations for fast-green. Ruzin's book is a good example for this I think.
Personally I started with the intention of reproducing the time-honoured recipes but almost immediately found that for example cellosolve is unable (by me at least....) to be found here in the U.K. for sale. I also started with Brunel's versions of safranin and fast green - both I think have cellosolve... They're nice to use but I found them to be prone to the growth of algae after a while in the bottle..... Also the number of slides I make - most of which are intermediate results towards what I'm trying to achieve, soon became impractical for the use of these little bottles.

I soon switched to the powders and made my own, basically never looking back from there...

In a nutshell, try both and see which (with or without cellosolve) suits you best. I love the ease of use, cheapness and results from the formulations I've settled into for my own use.

Here's a link to a disussion re this from 2018 which may also help.
John B

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