Skeletonized Maple Leaf

discomorphella · Post by **discomorphella** » Sat May 14, 2011 3:07 pm

This is a piece of skeletonized maple leaf from under a nearby maple tree. Most of the tissue was already removed by natural processes, sparing me the usual work of microtoming, mounting sections, staining etc. I mounted the leaf in PVLG (polyvinyl alcohol, lactic acid and glycerol, a standard botanical mountant) after soaking in ethyl alcohol / glycerol 1:1 to help remove bubbles. The first 2 shots are stacks, the final one shows one slice from the tardigrade stack in the middle photo. You can see what I believe is an encysted rotifer and perhaps an egg, the middle has a tardigrade and the bottom is a lower mag shot showing the tardigrade encysted (or dead, not sure which, the leaf was very dry when I found it) with detail from its "jaw". I'll post other shots as I find more fungi, metazoa, algae, birefringent plant structures...this leaf has it all...
Optics: Orthoplan DIC with 25X and 40X NPL Fluotar ICT objectives, Vario-orthomat shutter, Variozoom photoeyepiece, 0.32X relay and Nikon D300.

David

Mitch640 · Post by **Mitch640** » Sun May 15, 2011 4:19 am

Very nice set of shots. And you've given me another lead on where to hunt bears.

discomorphella · Post by **discomorphella** » Sun May 15, 2011 6:59 am

Thanks Mitch,

Yes, its (water) bear season. This one was likely still "hibernating" in its cryptobiotic state since this leaf was pretty dessicated when I found it, plus I've had it sitting in a petri dish indoors for a while before I processed it (we haven't had any dry weather in the NW for many months now). But you're right, it must have been teeming with rotifers and tardigrades to judge from how many encysted ones I've found looking at just a cm^2 or so.

David

Mitch640 · Post by **Mitch640** » Sun May 15, 2011 7:18 am

I use to live near Portland/Vancouver. I know all about the rain. I'm now in Wisconsin and we get more cloudy days in a year here, but less rain, except in the spring. So right now, we have a lot of leaf litter and ferns in the shade moldering away and growing up right now. I think I will be checking some of that for bears soon.

discomorphella · Post by **discomorphella** » Sun May 15, 2011 7:32 am

Hi Mitch,
Check out leaf litter, and also moss. I've had better luck hunting bears in moss than almost any other source. I was surprised at how many types of rotifers, fungi and algae were present on what was apparently a very chewed up dried leaf; you might find a lot more than just tardigrades in your leaf debris. I didn't pick up this leaf thinking it would be a good source of rotifers etc, it was the vein pattern that initially attracted my attention as a birefringent sample. Good hunting.

David

Charles Krebs · Post by **Charles Krebs** » Mon May 16, 2011 5:26 pm

the leaf was very dry when I found it

David,
Are you sure you live in the same NW as I do? I don't think I've seen a dry leaf in 8 months! You did indeed find a good one. I've always liked the patterns in "skeletonized" leaves, but never found the interesting critters. Nice job on the tardigrade stack. Hard to get a shot where you can see all the the nifty "toes".

discomorphella · Post by **discomorphella** » Mon May 16, 2011 9:53 pm

Thanks Charlie--

I do indeed live in the same sodden NW that you do, in fact maybe even a tad more sodden since I'm in the coast range. You are crediting me with far more efficient sample processing than I actually have. When I found this leaf it WAS 8 months or so ago, august...I put in in a petri dish in my sample cabinet where it joined hundreds of other samples in dishes, vials of formalin and 70% IPA and other preservatives waiting for me to get to...and there it sat until a few weeks ago. One thing about leaf veins, they appear to be thin, but to a high NA objective, they're actually a bit thick, so stacking is kind of unavoidable. Also, to avoid trapping obnoxious bubbles under the leaf, it helps to soak it in a small beaker of 100% alcohol (ethyl, methyl, isopropyl, take your pick) and glycerol (~1:1 or 2:1), and let the alcohol evaporate until the sample is saturated in glycerol. Then you can pick up your leafy bit, or mite or other specimen, in a small puddle of glycerol on a slide, and work all the bubbles out of / around it using a dissecting needle. After which you can apply a coverslip with some PVLG (great stuff) or other water or alcohol soluble mountant and have a bubble-free mount. I'll post more as I get new stacks of the other critters.

David

rjlittlefield · Post by **rjlittlefield** » Mon May 16, 2011 11:12 pm

Really nice images, David. I especially like the aesthetics of that first one -- reminds me of being under water looking up at the sky.

PVLG -- as in HERE and HERE?

--Rik

discomorphella · Post by **discomorphella** » Mon May 16, 2011 11:56 pm

Hi Rik--

Thanks, and yes, the first PVLG recipe is the one I used. Its basically about 8% polyvinyl alcohol in a mixture of lactic acid and glycerol. I don't think the degree of hydrolysis of the PVA matters all that much either. I am not sure of the refractive index once its finally dried, but I haven't noticed a huge amount of spherical aberration using oil lenses, so it's probably pretty close to glass. It's convenient for things like this dried leaf since its water/alcohol soluble. I haven't tried any really long-term mounts with it yet (I have some slides with Permount that I made 40 years ago by contrast).

David

lauriek · Post by **lauriek** » Tue May 17, 2011 11:28 am

Lovely shots. The slice from the Tardigrade stack shows much more context, I don't suppose you could post an un-cropped stack could you? (I like the context the leaf veins give!)

discomorphella · Post by **discomorphella** » Tue May 17, 2011 11:34 am

Thanks Laurie--

I'll redo the stack at lower mag, I want to try and get both the claws and the jaws visible and I don't think the 40X can do it. I'll redo the stack with the 25X obj and if it turns out ok would it be within guidelines to repost it on this thread?

David