Staining lichen

[MikeBradley]

I plan on examining some foliose forest lichen sections and I'm wondering what stains are being used for this? I have seen online references to lactophenol cotton, iodine and Meltzers reagent being used and I will probably try all of these. Are there other recommendations that have proven to be particularly useful in practice? Thanks
Michael?

[mrsonchus]

Hi Mike - what a coincidence! Only this afternoon I've been examining what I think is 'jelly lichen' - Collema.fasciculare found amongst some moss samples while peering through my stereo 'scope. I took a few images the other day, see below, and today had a go at crudely halving the 'round things' and staining them.

Stills from a few days back,


Most was this blackish colour - perhaps at a stage where photosynthesis not needed or simply deceased? Some was still the expected greenish colour as first image.


Unsure though of ID as Lichens are really not my area as yet, mosses are my main subject in non-flowering plants.

Today I took some video of my clumsy explorations and hacking-up of said Lichen, including an attempt to stain the 'live' tissue, first with toluidine-blue (limited success) then with safranin (very little staining) and finally with the go-to lactophenol 'cotton' blue as used with fungi in general - with reasonable results. This was a really rubbish attempt though as I was really just practicing with my pretty-recently acquired 'scope and (HDMI industrial C-mount) camera... I water-mounted the pieces with coverslip and spacers to hold the coverslip off a little - but the rather rushed results were pretty poor....

[MicroBob]

Hi Mike,
on our website you can find Schömmers "Kryptogamenpraktikum": http://www.mikrohamburg.de/Schoemmer/Schoemmer.pdf
The name of the book is difficult to decipher even for me as a german, but the book itself is an insider tip.
When you look for "Flechten" = lichen, you will find the right chapter and can translate it with DeepL or Google translator.
Special about this book is that Schömmer apparently tested all the recipes and didn't just copy them from other sources as it happens frequently.
On 16 pages you will find lots of recipes for every step of the preparation for different kinds of lichen.

Bob

[MichaelG.]

Hi Mike,
on our website you can find Schömmers "Kryptogramenpraktikum": http://www.mikrohamburg.de/Schoemmer/Schoemmer.pdf
The name of the book is difficult to decipher ...

MichaelG.

[MikeBradley]

Thanks Bob and John for your responses, I took a look at the Schoemmer pdf, it’s certainly quite a tome! Eventually I decided to use Lactophenol CB (LCB) and Fuschin-Chrysoidine-Alcian Blue (FCA) for staining the foliose lichen. I’m very much a novice at all aspects of sectioning and staining but the results I obtained have been quite encouraging. I was especially happy with the unstained image of the Nostoc colony. (Images were taken on an Olympus BHS 10x SPlan20x na0.46 and SPlan10x na0.30 with NFK 1.67/Canon 90D), some images have been cropped).

Interestingly the sections that seem to be the “best” to my eyes are the LCB one, these had been sectioned before fixing, with the slices then placed in 70% EtOH overnight, then transitioned via 50 and 30% to distilled water. Staining was 1 minute on the slide and then washing – water and cover slip for mounting. Differentiation of the elements of the thallus was quite clear. For the FCA treatment however I had decided to fix the fresh material overnight in FAA first and then proceeded to sectioning and conditioning following the same procedure as before. I had expected “crisper” sections and “cleaner” differentiation, but was still happy with these early results.

I’d be very interested in hearing any observations you folks on the forum may have.
Thanks
Michael

[MicroBob]

Hi Michael,
thank you for posting these interesting results!
The FAA fixation shouldn't be a problem with Etzold FCA stain, but it has to be washed out thoroughly in 70% ethanol and the specimen has to be bought into water through steps of ethanol water mix, like 50%, 30%, H2O 2x. The Chryosidine takes hold a bit better when warming the staining solution for a moment. I have no experience with lichen, but with plant sections I heat the well slide with a blue flame lighter to short before bubbling.
Here: http://www.lichenes.de/technische-informationen/ embedding into PEG 1500 is recommended. with the crumbly structure of lichen this might help to get even undamages sections. What also might work is embedding in glycerine soap (just an idea, not tried myself):
-Soak lichen in water
-alcohol steps to 70%
-fix with FAA
-remove FAA
-alcohol steps down to water
-glycerine soap water mix, evaporating water slowly
-embed in block of warm glycerine soap
-section on hand microtome

I hope to find the time to give this a try.

Bob

[MicroBob]

Hi together,
I found that I have some Histowachs 2 which probably is PEG. It either has an extreme long term stability or is rubbish by now as it dates from 1983! It is water soluble and tomorrow I will try to use it to infiltrate a piece of lichen, taken from a tree which is swimming in FAA now.
I will report what happens!

Bob

[MikeBradley]

Bob, you've inspired me to have another run at these lichens and I think I'll try you soap idea as part of the project! We don't have any glycerine soap at hand at present so i'll try to find some tomorrow, I'll report back on results of course. I'm interested to hear whether the Histowax did the trick for you.
Michael

[MicroBob]

Hi Michael,
I have bathed a sample now:
- water + drop of dish washing liquid
- water
- alcohol steps into FAA, over night
- wash in 70% Ethanol,
- alcohol ladder down into water

then two methods:
1) 1:1 Histowachs - Water - solves nicely on the heater/stirrer at about 40°C, water is evaporating now
2) 1:X Glycerin soap - Water: A hardening foam resulted from gentle stirring, difficult to dissolve, a little Ethanol might have helped, I have to see what I will further do with this.*

Ok, putting it into a heat block at 55°C lead to dissolving the soap. Now I let it cool down a bit and evaporate the water.

My goal is to form a solid block that can be sectioned with some kind of microtome.

Bob

[MikeBradley]

Bob thanks for the glycerine soap idea> , I decided to try it out and found a random variety (BODYSENSE Jasmine) that melted in a dish on a coffee cup warmer. I diluted the soap by adding 30% liquid glycerin and poured the mix over the lichen. The lichen had been previously fixed in FAA and rested in 70% EtOH overnight, then laddered down to water. After 12 hours the soap block was sufficiently solid for me to be able to section the lichen. Setting the hand microtome to 30um resulted in clean slices but at 20um I couldn't get a good section. The sections looked rather like shavings from a wood plane (see images). I warmed the sections in distilled water to remove the soap and then washed and viewed them, They were very good sections, if a little thick of course, and my lab/office smelt of Jasmine. I was really pleased with the section that remained unstained, the various layers in the thallus are very nicely resolved (cortex, photobiont, hyphae), this image is a stack of 15.Once again the images i get after FCA staining are far less resolved, I need to get to the bottom of this!

My conclusion is that the glycerine soap method may have promise for delicate materials, especially with a "harder" soap block, I plan to try this soon!

Michael

[MicroBob]

Hi Michael,
nice to see that this has worked so well!
Here a few comments:

soap - I bought soap that is sold for DIY soap makers, apparently a kind of craft hobby with a lot of funny youtube videos. Compared to usual household soap this soap feels a bit soft and sticky, a bit harder would probably be an advantage.

cooling - the soap hardens when it is cooled down so one can place the hand microtome with block into the fridge and cool it down before sectioning

hand microtome and blade holder - This combination is mainly used for fresh plant sections of about 50µ, so you are probably on the limits of it's application here. It might improve a little with a cooled block. Also wetting the knive could improve sectioning.

stained section image quality - with twig or lead sections I often see that they deform in the fixing and staining process and have to be pressed to give a flat slide. I usually make permanent slides and use Euparal for this application. I dry the slides on a hot plate at 45°C with an M6x20 screw on to op the cover slip. A couple of hours is enough for a short peek with the microscope but I keep them on the hot plate for a week with screw on top. I think your section doesn't lie flat so this might be the main problem here.


I continued with my own experiment too. I used the described soap and this looks good but I have remelt and to orient the specimen better. I also used a PEG called Histowachs II with a melting point of 52-54 degrees. This gave a very brittle block that gives a snow like powder and not sections when cut. Im remelting and adding glycerine. 5% glycerine seemed to help a little bit, now I increase to 15%. The Histowachs is perhaps just too old.

Bob

[MicroBob]

Hi together,
today I added 10% glycerin to the PEG and it sectioned at least a little bit. Sectioned to 20µ with my blade holder on a cylinder microtome. The tight an brittle rolls unfolded well and the lichen material hed together well. Staining with Etzold FCA nad Wacker W3A Sim was not very successfull, but methylene blue and our new black stain gave a nice look.
I also made a glycerine soap block that looks much much better but will need cooling as it is a bit soft. The PEG melts and dissolves quicker, but the block looks like a heap of guano, So glycerine soap it probaly at least as promising as PEG.

Bob

[MicroBob]

And the sections, single shots from the freshly made permanent slides:

[MikeBradley]

Bob, I haven't tried PEG, it's not easy to get in modest quantities and I'm finding that the 100% glycerine soap works very well. Glad to learn that your soap experiments seem to have been successful too. I tried your suggestion and loaded the cover slips for 24 hrs after mounting in glycerine jelly. The result was definitely positive with a more uniformly focused "flatter" specimen under the microscope. I've attached an image from a 30um section stained on slide in 0.5% lactophenol cotton blue for 60 secs.
michael

[MicroBob]

Hi Michael,
that looks great now! When comparing our images it is now visible that our lichen are not the same. The glycerin jelly has a tendency to get mouldy, so it is better seal it with a paint ring.

Bob

[mrsonchus]

Hi all, this is an interesting post and I've been following it along with pleasure. Has anyone an image of the 'whole thing' with an idea of where the sections are made - I think I'd like to try the paraffin process and microtome method with them but know nothing about lichen - even what they actually look like or the best plane of section....

[MicroBob]

Hi John,
at the very to of my post from january 31th you can see the sample . I cut the lichen away from a cherry tree, including a layer of bark. It was placed in the block with the lichen layer vertically. This way I get cross sections through the lichen "leaves".
My blade was blunt after sectioning. With the same blade I have made a couple of hundred sections of plant stems or leaves with just a bit of stoppping on pure leather in between. Possible dust collects in the coars lichen structure. So you perhaps better don't use your best heirloom quality microtome knife.

Bob

[mrsonchus]

Aha! Thanks Bob.

So essentially a cut across the 'leafy parts' as with a liverwort. I'll have a go at this - just need to find some nice samples, which must be around somewhere in our gardens. The pieces of what I think are lichen that I found in an earlier post are blackish-brown in the leafy parts as well as the 'basin-like' structures that I assume are reproductive - images 1 and 2 in my post at the top of this thread. I'd like to use a thicker sample like yours - my above ones seem a lot thinner as a variety... I'll have a hunt today and see what I can find - it may be interesting to section and stain a few permanent slides of these intriguing plants.

I'm enjoying this thread a lot. Thanks for the help Bob!

[mrsonchus]

Right-ho, I've been snuffling around the garden and have found what I think are some lichen.



A few more images of my harvest... Includes a chunk of nice liverwort 'Lunaria.cruciata', which I made a few slides of years ago, but might take into processing also. If I go on to do this I'll put it into a new post.

I'll have a pick over these later hopefully, and try to select a few pieces that may be suitable to process for slide-making -

[MicroBob]

Hi John,
wow, that was a good harvest! I can imagine that England is a comparatively good place to look for lichen. From what I read there are very different species, so a topic with lots of opportunities and not much covered by amateurs. Do you already have an idea how you will stain your sections?

Bob

[mrsonchus]

Hi Bob - yes I gathered that bowl-full in the time it took our Jack-Russel to 'do her business' as it were...
Where I live, in Cumbria, is actually a bit of a paradise for wild flowers, mosses, and the lower plants too.

Staining, hmm, as usual it's a matter first of deciding just what I'd like to learn/show from these fascinating partners. My usual start is simply general morphology, for which I'd begin with say safranin, fast-green and maybe a touch of cotton-blue as a treatment for the fungal element. Although the yellow stains can perform very well also in combination, perhaps with alcian blue.

At the first dissection and probably some hand-sectioning (grossing?) I'll likely simply use safranin alone. This will give me a good idea of just how to orient the tissue at the embedding stage - if this is poor every section from the tissue will be a waste of time!

I'll move over to a new post with some first-look hand-sections and some basic staining, probably tomorrow. It should be quite a challenge to make slides from these tiny specimens, although my harvest did include some relatively large bracket-fungus. The bark-shaving substrate specimens are taken from an apple tree.

[MikeBradley]

John, welcome to the thread, I'm looking forward to hearing about your ongoing explorations!

Here's an image of the foliose lichen that I was looking at. I believe it is a Lobaria sp., a cyanolichen, common in the coastal cedar/hemlock forests in British Columbia. My sections were 1 cm pieces taken at various angles across the thalus to try to capture the key features (cephalodium etc). Nothing more rigorous than that I'm afraid.

Michael

[mrsonchus]

That looks a beauty, the 1cm sounds a good size to me too.

I'll start a new thread with further details - I can't jam this one with my own adventure.