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Nobitte
Student


Registered: 02/20/08
Posts: 493
Loc: Biosphere
Last seen: 6 years, 7 months
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Lichen Culture!
#18449640 - 06/20/13 10:03 PM (10 years, 10 months ago) |
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Hi Chaps/Chapettes,
So recently I decided to make a few plates up using small samples of a lichen I found on my roof.
It might be Austroparmelina pseudorelicina think or some other related genus maybe a Xanthoparmelina? Which is mainly on the basis of commonality in their gross morphology and occurrence.
I lack the resources for chemo-typing, and at the moment the time to check some of the reproductive cups out with the scope, although im not sure they will yield anything of use anyway.
Ill describe it for the moment, if anyone expresses interest, ill take some photos. I took small fragments of two sections of the larger colony, that I describe as being either 'Foliose' or 'Spikey'. I put them on some 2% MDEA and forgot about them.
Two weeks later, the plates I made up with samples from the spikey all appear to have some other inhabitants in them. The ones I made of the foliose sections all appear to have produced the same sort of mycelium without any visible intruders (they colonized the plates and excluded them or have grown over the top of them). The mycelium appears to spread radially from the point of inoculation, if it is indeed the fungal component of the lichen colonizing, then it has not brought the photobiont with it... for the moment.
Forgot about them for another week or two and came back to them today. The spikey plates look real gross, but I haven't thrown them out yet, I want to see what happens. The foliose plates on the other hand are now fully colonized and have begun forming small primordia-like structures, evenly about the surface, now if it was the usual green mould contaminant that get around here, it would have already vigorously sporulated and made its presence known by now, which leads me to believe it might be something different.
Anyway, not sure if its a trick of the light (I did check it in several artificial lights and natural sunlight) but I believe there is a green tinge developing at the interface of two of the sections, that are now overgrowing their partitions, it doesn't look like a spore mass. Im hoping that what im seeing on the foliose plates is the first stage of thallus development, if something really really interesting happens ill take some photos as well, at the moment I have exams to study for =3.
Any who, has anyone tried anything similar, what should I expect, will lichen thalli form on an agar plate like that? So many questions... as I said before, if you want photos, ill take them, but for the moment I am preoccupied.
XXX
Nobitte
-------------------- First we must learn... Then... WE CAN TEACH
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Nobitte
Student


Registered: 02/20/08
Posts: 493
Loc: Biosphere
Last seen: 6 years, 7 months
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Re: Lichen Culture! [Re: Nobitte]
#18458530 - 06/22/13 09:48 PM (10 years, 10 months ago) |
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A little update on how this is going, the green growth seems to have overtaken most of the foliose plates, the plates made from 'spikey' samples have stayed white.
Had a look at a sample of some of the green growth under the scope today, although my lack of experience makes it hard to differentiate between green spore and a cyanobacteria/algae/photobiont.
A sample of the surface of the colony showed numerous small spherical objects that were about 1/100th of the FOV when using 400x (that's 40x objective with 10x eyepiece), that im going to assume are mould spores (until I can get the oil immersion working).
Now, I was thinking that if they were a photobiont, id be able to see some sort of visible chloroplast or thylakoid complex in their cytoplasm. Which I cant at the moment, cant even see a nucleus in them, just a field of tiny little clear spheres on a green background.
More to the point, im having trouble working out where the green is coming from it might be the spores/bacteria, by themselves they appear clear, but are green when aggregated.
Anyway, if anybody feels like helping my out on my journey of discovery, im sort of one of those people that learns from screwing around. Im starting to believe I cultured something that was commensal with the thallus now, as opposed to the lichen itself, or something from my lab, as the growth pattern on agar doesn't seem like anything I've had around here before.
When my new stock of dishes comes on Monday im going to make a few sub samples, see if I can isolate different species from it, or attempt to culture the spores on a media to see if they display growth more typical of the green coloured (polyphyletic I know, I just don't have the skills to determine which is which at this point) mould contams I have around here.
Last point, and I really hope someone is listening/reading. Does anyone have a guide or tips to using 10x100x or 20x100x oil immersion, im having issues, they never really showed me at uni, and I keep getting the cover-slips stuck to the objective trying to figure it out. Further than that, will I need to use a stain to see the hyphae or reproductive structures, or should they be visible under white/led light unstained.
Anyway, moving forward from here, I'm going to make a bunch of slides to compare the typical radial green mould that I find(that I assume is T. harzianum or something similar) with what I've got growing on the plate, to see if they share any commonality.
Any help with this is greatly appreciated, even though I know it is a little esoteric and/or useless in the context of run of the mill mushroom cultivation/mycoculture.
Much love,
Nobitte
-------------------- First we must learn... Then... WE CAN TEACH
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matsc
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Registered: 09/17/12
Posts: 681
Loc: Arizona
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Re: Lichen Culture! [Re: Nobitte]
#18459018 - 06/23/13 12:17 AM (10 years, 10 months ago) |
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Even with a 40x objective its really quite difficult to discern cell components, unless theyre stained in some way.
Its possible that your lichen has a cyanobacteria as the photobiont. Or, the other (admittedly less likely) possibility is you have a tripartite lichen, and its only the cyanobacteria that is growing out. Ive personally never cultured lichens so im afraid my practical knowledge here is limited.
EDIT:
Oil immersion is a pain in the ass, in my opinion. I have to use it when taking publication photos, and it aggravates me more and more every time I touch it. Make up the slide as normal, keeping the sample as thin as possible, get it in position using a lower res objective, then rotate the objective half way to the 100x. Lower the stage a little to give you some room and then put a tiny drop of oil dead center on the slip. Rotate the 100x into place and make sure the lens was low enough to move into place, and picks up the oil. Then move the stage back up and use the fine adjust to get things in focus. Once youre here you can move things X and Y a little but hunting the whole slide a painful option at this point. The objective itself should more or less be in contact with the cover slip, so only use the fine focus here or you run the risk of cracking slides or damaging the lens.
When youre done lower the stage, rotate the objective, and use lens paper (not a kimwipe, they can scratch) to wipe the oil off the lens. And then spend the next 20 minutes trying to wash that damned oil off your hands.
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Edited by matsc (06/23/13 12:25 AM)
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Nobitte
Student


Registered: 02/20/08
Posts: 493
Loc: Biosphere
Last seen: 6 years, 7 months
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Re: Lichen Culture! [Re: matsc]
#18459798 - 06/23/13 07:39 AM (10 years, 10 months ago) |
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Thanks for the tips, I'm feeling game to give it another go when I get the time to do so. I'm going to let the colony grow out a bit more before I torture it again, see if it gets up to anything interesting. After doing a little research it seems possible that the fluffy aerial growth it is displaying could be a load of soredia-like structures.
Its funny, when look at it under the scope, I can see little partitions in the groups of spores that look like they should have hyphae, but are mostly clear.
I guess some searching with regards to the sorts of stains I need to acquire is in order at some point too.
I might as well take some photos of it at some point, but I'm not sure I can do it justice.
-------------------- First we must learn... Then... WE CAN TEACH
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matsc
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Registered: 09/17/12
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Re: Lichen Culture! [Re: Nobitte]
#18461200 - 06/23/13 02:37 PM (10 years, 10 months ago) |
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Yeah you can see hyphae without stain but the contrast is terrible. You can try dimming the light a bit to see if it helps but you wont see all that much.
If you want to see any sort of detail you need some help, either a phase contrast filter on the scope, or a stain. The most common stain we use is lactophenol cotton blue. It works well for coloring, though personally I find its high glycerol content annoying. Its very syrupy, and somewhat dangerous given its high phenol content.
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Nobitte
Student


Registered: 02/20/08
Posts: 493
Loc: Biosphere
Last seen: 6 years, 7 months
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Re: Lichen Culture! [Re: matsc]
#18463403 - 06/23/13 11:59 PM (10 years, 10 months ago) |
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Im more than sure its some kind of mould now, I think my sample/inoculum preparation needs some work, I might also consider experimenting with media. I say this, as I just spent a few more hours getting accustomed with my scope. I took your advice, messed with the contrast and tried a filter on it.
Surprise surprise I was able to see a lot more, including my samples from the other day. Would appear that the mass on the surface is a whole bunch of spores (or some microscope particle of similar size and dimensions) which were about 1/20th of the FOV at 800x (so about 2-4um?).
When I examined the thallus samples again, I found that the visible photobionts in the tissue were approximately twice to three times the size and a different colour relative to the samples from the plates (which I will henceforth refer to as 'the spore plates'). In addition to this, it seemed that when I was able to separate one or two of them from their homes and have a look, that they had well defined cell walls. Leading me to believe that what I have cultured was not the lichen, but some other hungry chap that was looking for an easy feed.
Interestingly, even after an hour or two of searching through hyphae and spores, I was unable to see a structure that looked responsible for the spores(conidia, asci, basidia etc), which sort of puzzled me, if they were in such high numbers and I could see both spores and hyphae, I thought I might catch a glimpse of one.
Two hours no luck im afraid. Might not have been searching the right place, but I made a number of smears from different sections and nothing, they might just be hard to differentiate. Any suggestions?
Although I did find a number of hyphae with green spheres inside of indeterminate number (3-16ish).
I initially thought they might be asci, but I figured that the spores in an asci would be of a more uniform size (some of the blobs in the hyphae were 2-3 bigger than their neighbors) in addition to this, I figured they were also too numerous in some instances.
Galun et al 1971 managed to culture a number of soil lichen species from Israel using inorganic ion/citric acid supplemented silica media and had thallus development. Their method puts emphasis on washing the samples thoroughly beforehand, I might try this next time, ill post some pictures of the main colony later just for kicks, since it really is -so- pretty.
I was going to include some scope pictures, but they were a little amateurish, im sure y'all seen 2-4um spherical spores before.
xxx
Nobitte
-------------------- First we must learn... Then... WE CAN TEACH
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matsc
Stranger



Registered: 09/17/12
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Re: Lichen Culture! [Re: Nobitte]
#18463503 - 06/24/13 12:33 AM (10 years, 10 months ago) |
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Its not uncommon, especially with the ascomycetes, to have trouble seeing spore bearing structures. Asexual spores are often on very fragile structures that release their spores easily and get lost in the background. Its also possible your particular fungal friend has "arthroconidia". This is a special strategy where hyphae themselves differentiate into conidia. The valley fever fungi, Coccidioides spp. use this method.
Its also possible you have a bacteria on the plate along with the fungi, making a mess of things and confusing the field. Ah the joys of microbiology.
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Nobitte
Student


Registered: 02/20/08
Posts: 493
Loc: Biosphere
Last seen: 6 years, 7 months
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Re: Lichen Culture! [Re: matsc]
#18463601 - 06/24/13 01:12 AM (10 years, 10 months ago) |
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I'll keep the plate around, although ive decided to start opening/examining it in a smaller environment (tent inside lab) with the air filter running, I accidentally copped a little whiff of it before without a mask on... something like a gross anise smell.
I had thought that it could be a bacteria proliferating over the top of it, although I have a feeling that they are indeed spherical spores. Only because I didn't see any in various states of fission as you would expect (unless they are mass producing endospores, in which case they might very well be dormant) if they were bacteria. Just a bunch of spheres of uniform shape, size and state, with a few septate hyphae in amongst them.
They could very well be arthroconidia now that you mention it, although the spheres inside them didn't appear to be defined by septa, but were rather green 'chloroplast like' objects floating around inside the hypha, that were about 1/2 the width of the hyphae most of the time.
You've made me want to go and take another look now =3, also invest in a microscope camera, my neck is killing me.
It is actually a lot of fun, it really makes me regret not making the move to a uni where I could major in Microbiology.
-------------------- First we must learn... Then... WE CAN TEACH
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