Lichen Slide Making From Scratch

[mrsonchus]

Hi all, further to discussion over on the excellent thread "Staining Lichen" by Mike, I've started a new slide-making adventure to try to make some slides of Lichen, gathered-up from my garden the other day.

Here's a nice selection gathered...


Basically I roamed around our gardens here in Cumbria, U.K. with our dog and snapped a few Lichen-bearing small twigs from our apple trees and elewhere, even some Liverworts from between decorative slabs foun their way into the bowl. Slices of bark bearing Lichen also collected using a fruit-knife.
I know nothing about Lichen, but have found several species which I have tried to identify as best I can at this time - if anyone has any information or corrections please post them here and I can put any inaccuracies right!


I think this is Physcia.adscendens, see this Woodland Trust page...



I think this may be Xanthoria parietina but then... it could be the same as above?



Here's a nice piece of bracket-fungus gathered from the cut stems of a garden shrub, close to ground level,



Another similar-looking sample,



A rather fetching chunk of Liverwort which I'm pretty sure is Lunularia.cruciata may give some interesting sections also,




This frilly (foliose?) sample is I think Collema.fasciculare and has some features that may make interesting sections also,


Next stage is to prepare tissue pieces for further processing.... see next post.

[mrsonchus]

So, with some samples collected we move on....

I've had a pick over my samples this afternoon and have a couple of jars of FAA with pieces in for of course fixation.
I used 2 approaches today, one was to pre-clean and separate (from substrata) under stereo 'scope as best I could in the time. The other was to leave some of the samples upon the wood but merely to break the wood - very small twigs basically, into manageable pieces for immersion into FAA.

The 'sandwich' of the alga between top and bottom layers of fungal filament, is quite easily noticed as the samples lie in their jars, from above under the 'scope that is - a quick look only at this stage as an open jar of FAA is not a nice thing to be directly above during observation!

From a couple of days in the FAA I plan to carry out further dissection and cleaning as I move the pieces into their 1st dehydration, which will be at 50% IPA to match the alcohol concentration of the FAA in which they are fixing. Working with the 50% IPA (isopropanol alcohol) is no problem. From there I'll transfer what by then will almost certainly be far smaller pieces, into tissue-cassettes for further dehydration, clearing, infiltration then embedding. This avoids the tribulations and damage caused by forcep-handling the tissue through processing....

This should be an interesting slide-making adventure, certainly of a tissue that I haven't dealt with before, the closest to Lichen being some Liverwort thallus I made slides of several years ago, which I stained then mounted and now have in my slide collection.

I'll add a few images to this thread tomorrow of the dissected pieces to start the documentation of this interesting process off as it were.

[tgss]

Will be following with great interest mrsonchus!
Tom W.

[HowardHopkinson]

I too am going to follow this thread with interest.

[MicroBob]

Hi John,
I'm following your project with great interest!
For the cleaning of the lichen I had an idea: A proven method to remove fresh spots from carpets is to soak them with carbonated water (right word?). The bubbling apparently loosens dirt and makes it easy to remove it. This might be just the right method to get much of that abrasive dust out of the lichens.

Bob

[TonyT]

I use a small ultrasonic cleaner to remove 'junk' from most insects (not lepidoptera). Rarely does any damage even to delicate antennae.
I suspect it would work equally well for lichens.
https://www.amazon.ca/17-ounce-Stainles ... 541&sr=8-5

[mrsonchus]

Hi all, thanks for joining this adventure!
Bob I think the bubbles are a really original idea - may give this one a try.
Tony, I tried an ultrasonic cleaner for coverslip, slide and tool cleaning quite a while ago now, but found it to be pretty ineffectual for those purposes. However for the tiny grits etc of my samples it may well help.

My usual method for cleaning (samples), mostly mosses at the smaller scale, is to use a series of tiny paintbrushes, which works very well indeed. However the mosses are quite an easy case as they're really more robust than one may imagine under water for cleaning. Another technique I use is simply to put the samples into a dish, place a few objects in with them and leave a gentle water flow into one end of the tilted dish and out at the other. The items, eg a small jar help to give a disturbed flow through the water as it travels through the samples on the way the the 'overflow end' of the dish. This works very well indeed also, especially if combined with some careful brush fiddling.

Now, having said that, I just changed the FAA in the 5 sample jars that I have with the aforementioned Lichens, bracket-fungus and Liverwort in to fix. This change isn't usually needed but as these samples are atypically dirt-laden (relative to the botanicals I usually process) I though I may as well, the FAA costs a trivial amount to make so why not...

Here are a couple of images, the first of the jars in the first FAA (placed into this yesterday) where a lot of chlorophyll can be seen in the liquid, as well as much other debris and other organisms also occupying the wood that the samples are on. The second image is after a 'quick swirl and rinse' then fresh FAA, a lot better at least to look at!

These will need about 48hrs or more to ensure optimal fixation in my experience, only another day 'til we can clean and move them into the first dehydration stage of 50% IPA.




Fresh FAA,


More soon!

[MicroBob]

Hi John,
nice series! It is obvious why you needed a new stereo microscope - the old one had a smaller foot!

Bob

[mrsonchus]

The next one's going to reach right across the desk!

[mrsonchus]

Hi all, well I haven't had a lot of time today unfortunately, but have still moved the samples into 60% IPA to be removed (dissected) from the twigs and substrata to which most are attached, before cutting into their final pieces for the rest of processing and ultimately to sectioning.

5 jars on the go, here are a few rather grainy images from 'Pot1' - I think this may be the common foliose lichen Physcia.adscendens
It's a grey (all images here on in will seem mostly grey actually as the action of processing decolours the tissue) lobed lichen with characteristically 'hooded' lobe-ends, as seen in these images. This however may be way off base as an ID - I really know nothing about lichen yet. Please correct if you know what it is most/more likely to be.

Possibly Physcia.adscendens...

Pot_1_09_02_21 (1).jpeg (76.61 KiB) Viewed 7970 times

The 'little legs' it appears to be standing up on are bundles of fungal hyphae with an anchoring (to substrate) funtion and are called 'rhizines', having no nutrient absorption or transport function, being only anchors which may or may not penetrate the substrate, sometimes quite deeply. These images are taken with a stereo 'scope and with the lichens a couple of inches below the surface of at this stage 60% IPA, but still acceptably clear enough to be useful.
It's these rhizines that I'll need to cut as close to the substrate as reasonable, my microtome would probably explode if I tried to section the wood that these are on! This should give me some pretty good samples to take into processing. A couple more images from 'Pot1'....

Here the lobe-end and rhizines are easily seen,

Rhizines growing down from thallus

lichen rhizines from thallus lobe.jpeg (44.72 KiB) Viewed 7970 times

About a month ago I was studying some old moss (Polytrichum.commune I think) when I came across what looked like a white spidery thing.... Which I now think was definitely a lichen, maybe even the one above, i.e. Physcia.adscendens. Point is, I further dissected and took some images, some stacked and some stitched, of what have I think turned out to be rhizines, mounted in water with coverslip and imaged through an Olympus BX50. The fungal filaments comprising the rhizines seem pretty clear in hindsight.

This is the 'strange thing' I saw back in January,

Strange creature on old moss

lichen on old moss.jpeg (34.33 KiB) Viewed 7970 times

A stitch of a few images,

Rhizine detail

lichen rhizines 1.jpeg (35.19 KiB) Viewed 7970 times

A close-in stacked image shows the fungal 'filaments' quite well... Also shown (as these were in water and not decolourised by any alcohol) is that they are as expected not green - many have black tips or are nearly all black, but not one has any green anywhere.

Rhizine detail

lichen rhizine stacked.jpeg (36.01 KiB) Viewed 7970 times

Anyway, those were from 'Pot1' - 4 other pots in the pipeline, the liverwort sample is however of quite poor quality - they were I think, looking back over some images, at their best several months back. The liverwort may have to go from this slide-making adventure, we'll see...

Tomorrow I'm pretty sure I'll have the samples parted from their twigs etc and ready to process further, and I'll post some images of the freed pieces as I think there may be at least a couple of different lichen species in there.
That's about all I can add today, but progress tomorrow as I'll have a lot more time then!

[deBult]

Enjoy your lichen adventure, keep them coming please.

[mrsonchus]

Now then, all Lichen has been cleaned and dissected to the right size and shape to continue processing and go on to make blocks for sectioning with my rotary microtome AKA 'The Mighty Shandon' - acquired about 5 years ago when I first started with a microscope, for a great price ands unused (at the time)!

Here's a shot of my desk this morning - looks chaotic but everything's under control!


Dissection, cleaning and trimming into pieces that may be placed and oriented nicely when embedded in wax are taken from the 5 pots of tissue currently in 60% IPA. The 5 pots had about 10X as much tissue as I needed so plenty to choose from...

First stage was to remove the lichen tissue from the (apple tree twigs) wood that it grows on, without breaking it into tiny shreds. Although the pieces are tiny - some down to about 1mm - they are, with a small scalpel and forceps, quite easy to remove from the wood substrate...


There is some very nice lichen in these samples, of more than 1 species I think. I haven't given attention to identification in this activity as my priority is to try to learn how to make slides of the lichens - which is a pretty complex and lengthy task as it is.


The bracket-fungus was particularly easy to prepare, a lot like slicing tiny loaves of bread! These pieces should orient pretty well for sectioning once processed and embedded in wax for the attention of my microtome.


Here are pieces of dissected thallus, again these look quite nice,


Some pieces are really, really small!




The tissue has now been placed into tissue-cassettes to enable them to be further processed without the need to pick up individual pieces with forceps for each transfer across the processing reagents. In all I have now 4 cassettes of tiny tissue pieces, which should - fingers crossed - be plenty for a couple of dozen tissue-blocks. So, from her they will move to 70% IPA, then 80% --> 95% to complete dehydration. After this they'll go into 'Histoclear', the limonene-based clearing agent that I always use. This is the ante-medium for wax-block casting, being a solvent of wax.



Back soon as these will likely be ready to go into the wax-oven tonight for overnight infiltration, wax-block making tomorrow all going to plan, the sectioning grows closer!

[MicroBob]

Hi John,
so far I have moved my plant parts individually with forceps, and often they were a bit too big for the cassettes. But these fine lichen bits really make the cassettes indispensable!
I'm looking forward to see the next steps of your process. There are fairly few amateurs who do paraffin embedding, but it really is doable, once everything is set up in a useful manner.

Bob

[mrsonchus]

Hi Bob, yes I also will move larger pieces such as whole (well, sliced maybe to 2/3ish to present a good infiltration pathway) plant buds or flower-heads of composites such as Sonchus.spp. However, if needed there are two further sizes-up of cassette that I occasionally use, but they're quite hard to find. Fortunately they're reusable of course!

There are the 'standard', as used in this adventure, the 'large' and the 'mega', also the std is available with slotted-holes rather than square, which are good for larger pieces and probably have a better circulation when submerged. I haven't any of those left at the moment, must get some actually, it's Botany-time again! However for these pieces the tiniest holes are as seen above, preferred.....


It's quite exciting when the process nears the blocking-out stage - this tissue should be OK but I've never processed of sectioned lichens before, so, fingers crossed. This tissue with it's composite structure rather than an actual true tissue, held together by a matrix, may well be prone to fragmentation 'under the knife'.... Still, I'll make sure I keep the blocks cooled, although in my experience the opposite may also be equally likey, and the blocks better softer during sectioning.... We'll hopefully be able to take a few rough sections sometime tomorrow...... perhaps, maybe...hopefully.... Great fun though and always a superb learning process!

Off now to move the cassettes on into the final (1 of 2 actually as it's repeated) 95% IPA to complete dehydration, then a couple of clearing stages in Histoclear and into wax overnight I think should be good....

[MicroBob]

Hi John,
these bigger cassettes look to be very usefull. I have just the small ones and use them as the base of the block. For bigger specimen I have two tea eggs. Especially nice is the one without paper clips as it fits in a fairly small round glass.

Bob

[mrsonchus]

Ah yes, the infusion thingies - a very handy improvisation for just about any immersion! The chain-hanging one as you say looks really very handy for smaller diameter containers.

I tried these a long whila ago now but just kept discovering new methods and equipment, purpose-made and of course 'repurposed' as is said these-days. Certainly true that I still find the kitchen-ware isle hard to pass-by when shopping.

My wax-oven's a mini table-top oven, my slide and glassware air-drying fan-assisted thermostatic tray unit is a fruit-drying item, coffee-warmer plates, stainless-steel measures and jugs for liquid wax..... so many items out ther for the home lab that cost peanuts.

Nicely-done old chap.

Update, the cassettes are now in their second and final clearing stage (Histoclear, wax ante-medium) and will go overnight into their first wax infiltration stage in about an hour. Another couple of wax changes tomorrow then onto blocking-out sometime tomorrow afternoon with luck!

[mrsonchus]

Back again!

Well, all went well yesterday and the day ended with the tissue in wax, infiltrating nicely overnight.
So, this morning I started to embed the pieces into wax-moulds to make the blocks that will then go to sectioning.
Two blocks in (bracket-fungus in fact) my trusty dry-block heater that I use for keeping everything liquid and warm, well, there was a loud bang, a jump of the whole machine and finally palls of black smoke - not exactly billowing but definitely enough to make me turn all off!
It showed no signs of stopping either...

Anyway, I took the dead heater outside and left it there - there's no way I'm going to attempt repairs to this old boy - it's served me well, cost me a pittance and is now an ex-heater, sadly.

This left me with something of a problem as you can imagine, tissues in liquid wax, moulds cooling, cassettes cooling and wax solidifying...
To cut a long story short I quickly fired-up my large hotplate and finished the blocks the old way, as I did years ago. I'm a little unsure how they'll be as the imposed workflow is very inferior to the one I use, but at least I managed to cast 23 blocks for sectioning, and every one of them came cleanly from the moulds when cooled.
Fingers crossed - I'm going to make some rough sections later and will report back!

No sign of burns or anything on the dead heater, no fuses in the house blew when it failed, I opened it up and found a shard of what looks like an old fashioned fuse casing made of glass, that's clearly blown! I've no intention of making repairs, this isn't my area and I'm a great believer in respect for electricty!

A couple of images of dead heater,









23 blocks ready to section.....





Well, so far so good! I may have got away with the blow-out of the heater, we'll soon see......
Back later hopefully, with some early sections......

[MicroBob]

Hi John,
good that you had an alternative in this situation! Most likely it was just an aged condensator that blew: https://www.digikey.com/catalog/en/part ... eries/4042
and all else could be ok. So if you had somebody with real electrics knowledge it could be a worthwhile repair even if you have to pay for it.
Just recently my small lab power supply let out the magic smoke and I scrapped it, not clear what was damaged, probably the transforer itself.
As a makeshift heat cabinet I use a cast aluminium cooking pot, isolated on the outside, with a flat underside made from heat resistant silicone. This works very well and melts nicely with a fan that circulates the air inside.

Bob

[hans]

No sign of burns or anything on the dead heater, no fuses in the house blew when it failed, I opened it up and found a shard of what looks like an old fashioned fuse casing made of glass, that's clearly blown! I've no intention of making repairs, this isn't my area and I'm a great believer in respect for electricty!

Those RIFA line filter capacitors were widely used in the 80s/90s and inevitably fail like that. A search for "rifa capacitor" will bring up lots of examples. They are not really essential to the function, mainly there so switching transients from the circuit controlling power to the heating element are not conducted/radiated via the power cord and the device can pass EMC testing, but if replaced should be replaced with equivalent safety-rated types.

[mrsonchus]

A Single Early Slide....

Hi all, well, after moaning and groaning about today's debacle with a popping block-heater and a rush-job with hotplate stand-in, I finally got around to some very early, rough sections. In fact I had time to make an early slide, stained and mounted.

Here are some images of a slide made (the first in fact) from the foliose lichen that has featured in this thread from the beginning. Sectioning rough (quick sections to assess the wax-block) did yield some promising sections considering the trouble I had just as I began to embed these tissues.

The following are images of the single slide I've been able to stain and mount this evening of a 10µ section through the thallus of the above lichen. Although this early section has not actually reached the part (depth) of the wax-block to give a full section, these images are useful for an assessment of this particular wax-block in terms of content, and of the set in terms of processing.

I've never even dissected a lichen before let alone made slides from them, so this adventure to make my first is a really enjoyable task for me! I hope those following are enjoying it as much as I am. Sectioned at 10µ - a good starting thickness although judging by the surprisingly rich structure this lichen shows, I'll surely be sectioning thinner also, in order to produce some high resolution slides for higher-power objectives to really 'see what's in there'. Probably I'll section at about 5µ for this, maybe thinner for finer detail if possible.

A few images, straight from the mounting process - mountant is still wet and I dread to think what it's RI is at this stage, but the section has imaged quite well. Stains used are again my first go-to combination of safranin (the red) with fast-green as a counter-stain. Great to start but I plan to try several different schemes with these slides to see which looks best, and which gives me the most structural detail/differentiation at the thinner end of the sectioning scale...

Here's the overall section, I was optimistic when I saw this one, all seems to be developing quite well.




It was immediately clear to me that these lichen sections may have a lot more interesting structure to study than I
Closer-in the details begin to get interesting, although I think I will need to back the staining off a little for subsequent slides, these are a little garish to my eye.




An area with a lot of algae,




This is the 'rhizine' that attaches a foliose lichen such as this one to it's substrate.


So, a pleasing beginning, looking forward to further sectioning (another 22 blocks lie waiting for slide-making - lovely!) and to increasing the quality and resolution of details as I go. Back tomorrow with some more early slides hopefully, maybe even with a couple of variations of staining.

[perrywespa]

John,
Your projects always fascinate me and your narratives are so interesting and instructive.

[mrsonchus]

Thank you for the kind comment - I'm pleased you like them.

I've just finished staining another 7 slides from the first-look batch of yesterday, with a few different staining protocols applied. More new images later when I can look over these new slides - still need to mount the coverslips this afternoon yet!