Slide-surfing surprise - fungus! - maybe.....

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mrsonchus
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Slide-surfing surprise - fungus! - maybe.....

#1 Post by mrsonchus » Sat Apr 22, 2017 10:08 pm

Hi all, I've been slide-surfing three slides I made a coupe of days ago from an old tissue-block (paraffin-wax) with the purpose of practicing the balancing of staining-strength to section thickness of some relatively thick 'morphological' sections sectioned at 12µ and 15µ and stained with Safranin - a good all-rounder stain for such a trial.

Anyway, I sectioned a Sonchus.oleraceus ('smooth milkweed' - a common garden weed here in the UK) stem and leaf block for the tests - the stem in LS and the leaf in TS - and found quite a surprise lurking within the tissue of both the stem and the leaf. At least I think it's a surprise - I think it's a fungal infection that seems to occupy the region (created by the presence of the fungus - ordinarily there is continuity between the tissues in question) between the epidermis and the usually adjacent mesophyll of both leaf and stem.

I thought I'd put up a few pictures to see if anyone can shed a little light upon the mystery invader - is it actually a fungus? May it in that case be a type of 'Albugo' perhaps - it looks like a picture of one in one of my books.... :oops: It looks like the multi-cellular spore-bearing sporophytic stage - but I know nothing at all about fungi, and wonder if anyone here has any idea?

Anyway, here are some pictures you may like to peruse - any ideas welcome - I think I'll pursue the plant/fungal condition a little this year, it looks like an interesting adventure to be had!

Pictures from tonight's slide-surfing....

Here's the area seen on the leaf TS that first caught my eye,
Image

and,
Image

closer in...
Image

even closer,
Image

This is a normal leaf TS section for comparison - same leaf, different place along section,
Image

Now, I saw the same thing when looking at the stem LS - also on the same slide, from a different viewpoint of course. Also the fungus, if that's what it is, is in the same location, between the epidermis and the mesophyll,
Image

and,
Image

closer-in,
Image

Not much fine detail I'm afraid - the purpose of the thicker 'morphological' sections at 12-15µ is to show overall anatomy whilst preserving the cellular contents of cells such as the mesophyll - which has indeed been preserved. To see fine detail of the supposed fungal spores/sporophytes I'll need to section at between about 5 and 2µ I think - a good project over the next week or so I think!

I'll present the thinner sections when I have their slides made - should throw some light upon the finer structure of these features - providing of course that some of them are still left in the wax-block used here! I'll let you know soon.

Meanwhile, any idea if these are fungal?

Slide-surfing always throws-up something interesting! Such fine fun, mystrey and another opportunity to investigate and learn from another Botanical mystery! :D :D

John B :)
John B

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mrsonchus
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Re: Slide-surfing surprise - fungus! - maybe.....

#2 Post by mrsonchus » Sat Apr 22, 2017 10:34 pm

Hmm, after looking at some online images it looks a candidate for Albugo.candida at least - apparently a mould rather than a fungus too! Interesting stuff! :D :D :)
John B

JimT
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Re: Slide-surfing surprise - fungus! - maybe.....

#3 Post by JimT » Sat Apr 22, 2017 10:59 pm

Hmm, after looking at some online images it looks a candidate for Albugo.candida at least
Well you are the resident expert but regardless, god surfing.

billbillt
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Re: Slide-surfing surprise - fungus! - maybe.....

#4 Post by billbillt » Sat Apr 22, 2017 11:10 pm

mrsonchus wrote:Hmm, after looking at some online images it looks a candidate for Albugo.candida at least - apparently a mould rather than a fungus too! Interesting stuff! :D :D :)
Hi John B.,

These are very interesting slides for study.. Very good work!.. Thanks for sharing!....

BillT

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Re: Slide-surfing surprise - fungus! - maybe.....

#5 Post by zzffnn » Sun Apr 23, 2017 2:13 am

Very interesting indeed. Thank you JB for sharing.

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mrsonchus
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Re: Slide-surfing surprise - fungus! - maybe.....

#6 Post by mrsonchus » Sun Apr 23, 2017 4:58 pm

Thanks for looking chaps. It's a pity I hadn't sectioned these a lot thinner, maybe about 3-4µ - very little beyond a basic outline is visible here - thick and heavily-stained they show little structural detail.... :cry: .
This is a choice made for morphological detail and as such it works well at 12µ - nice plastid details and intact cell-contents - but I'll try thinner sections tonight if there's 'any mould left' within the tissue wax-blocks from which these were cut.... I'll let you know.
Thanks for looking, John B. :)

p.s. The planapo 1.32 n.a. objective is so-far a pretty awful performer - I'm better using the x54 oil or the x50 water I think. Maybe I'll look around for a replacement for the x100. I've tried it now with a 1.25 and a 1.4 condenser, neither option really more than acceptable in their resolution..... I may nee to take a very close look at the objective - it may well have problems... delamination maybe?

Still, an interesting exercise to encounter this mould none-the-less.... :D
John B

kinase
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Re: Slide-surfing surprise - fungus! - maybe.....

#7 Post by kinase » Mon Apr 24, 2017 3:08 am

If you look at root parts you should be able to find fungus pretty easily, pretty much all plants have symbiotic fungi. Leaves, probably not so much.

Have you ever considered microwaving your samples while staining or the stain itself? I know people do that for EM and sometimes with giemsa for regular light stuff. It at least accelerates a glutaraldehyde fix for EM.

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mrsonchus
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Re: Slide-surfing surprise - fungus! - maybe.....

#8 Post by mrsonchus » Mon Apr 24, 2017 3:47 am

Hi kinase, no I've never tried MW - can you tell me a little more - I may just have a try at this.
Thanks for the input old chap. :)
John B

kinase
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Re: Slide-surfing surprise - fungus! - maybe.....

#9 Post by kinase » Mon Apr 24, 2017 7:38 pm

I'm not too familiar with it. But at the very least, fixation and staining are chemistry based and the rates at which those things occur are subject to temperature. Typically shortened stain and fixation times reduce cellular deformations and increase penetration into paraffin sections. Usually I see it in the context of human tissue samples. Although it seems people are able to do it with fungi.

I do know that kitchen microwaves aren't the same as scientific microwaves in terms of the way the microwaves are generated. Kitchen ones work in cycles of the magnetron being on/off. Plus I'm not so sure you'd want stain fumes in your kitchen microwave. That said, I bet if you garbage picked a microwave for the purpose of science it'd work well enough.

This appears to be a pretty good overview https://www.hindawi.com/journals/ijcb/2016/7076910/ Here as well http://microscopy.berkeley.edu/Resource ... al2001.pdf . I guess there's lots of air spots in leaves and plants that could impede the movement of stains/fixatives.

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Re: Slide-surfing surprise - fungus! - maybe.....

#10 Post by mrsonchus » Mon Apr 24, 2017 7:56 pm

Thanks for the link kinase - a good read for tonight. I shouldn't have much trouble with air-pockets as I usually begin fixation with a vacuum applied to the fixative + tissue jar. Several careful apply-then-release cycles of a vacuum I find really does degass the tissues quite well, not to mention the fixative (I use FAA 50) itself.

I'll have a look into it - as you say, a cheap microwave is easy to find - worth a try I think - it may aid consistency and predictability also, as well as shortening protocols and perhaps reducing tissue artifacts.

An interesting little adventure that I'll look into sooner rather than later I think.

Thanks again.

John B. :D :D :)
John B

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vasselle
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Re: Slide-surfing surprise - fungus! - maybe.....

#11 Post by vasselle » Thu Apr 27, 2017 3:42 pm

Bonjour
très beaux sujet avec de belles photos
Microscope Leitz Laborlux k
Boitier EOS 1200D + EOS 1100D

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mrsonchus
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Re: Slide-surfing surprise - fungus! - maybe.....

#12 Post by mrsonchus » Thu Apr 27, 2017 9:32 pm

Thanks vasselle, pleased you like them. :D :)
John B

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