Preparing slides for hard material

Preparing slides for hard material

Postby Richard Scott » Mon Jul 26, 2010 5:00 pm

I am trying to get a microscopic view of Piptoporous betulinus and I am having problems preparing a thin enough specimen to see anything.
I have tried crushing between two slides but the material does not want to crush even with a lot of force.
Is there some trick to preparing hard material for microscopy?
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Re: Preparing slides for hard material

Postby Neil » Mon Jul 26, 2010 5:23 pm

I'm assuming you do not have a dissecting microscope to compliment your compound microscope.
Try buying a cheap pair of powerful reading glasses, say 3x, and with a fine pointed scalpel, remove just 2 or 3 tubes and mount on a slide. Working with KOH, available by post from the ABFG, will soften the material after a few minutes.
Hope that's of help.

Neil Mahler.
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Re: Preparing slides for hard material

Postby Mal Greaves » Mon Jul 26, 2010 5:51 pm

All I can suggest is take the smallest piece you can manage soaked in KOH and then teazed apart with needles.
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Re: Preparing slides for hard material

Postby Neil » Tue Jul 27, 2010 12:51 am

Just out of interest, in a BMS journal, Dr Martyn Ainsworth describes the technique he uses for obtaining a sample of Phellinus wahlbergerii in which he scrapes material away from the inside wall of a single tube (Field Mycology 9 (4) page 133)
First the hymenial layer (the tubes) were sectioned vertically so that the faces of the tubes had the appearance of microscopic gutters when viewed under the dissecting microscope. Then, with what must have been a very fine pointed scalpel, he scrapped away material which hopefully included setae to aid identification.

How the hell he was able to transfer this sample on the tip of a scalpel to a glass slide, and place a cover slip over it without loosing site of it, is totally beyond me !!
I guess he used the same technique when P.wahlbergii was found in an ancient wood in Suffolk last November during the annual BMS foray.

My chance to try this technique came when The Ancient Tree Forum held their 2010 Field Trip in Suffolk last June. Just before we were to be led by the great Professor Oliver Rackham for a guided walk in the nearby Oak wood, I was asked if I could go ahead and try to find the Oak Polypore - Piptoporus quercinus, to show the audience. I knew it was a bit early for Oak poly' to be showing, but it gave me the chance to search for P.torulosus or P. wahlbergii of which both were found last November in this very special wood.

My luck was in, for when I came upon only the 6th ancient Oak I examined, I found a fungal bracket inside the hollow close to the ground. It had all the appearances of a Phellinus spp. so I tried carefully to remove a sample, but unfortunately the whole fruitbody came away as it was only loosely attached. Knowing that this potentially could be only the third collection for mainland Europe, I told very few people back at the venue.

As soon as I got back home that night, with the 'instructions' from Field Mycology in front of me, I tried to obtain a sample. Things went well to start off with - I obtained a scrape (I think)! from the inside of a single tube, tapped the blade onto a glass slide, added a drop of KOH, placed on a cover slip, and promptly lost sight of my tiny sample. :cry: :cry: :cry:
I repeated the exercise, but kept on loosing the material when adding KOH.

In the end I just prised away 3 pores, added KOH, allowed it to soak, squashed the material with another slide with a bit of grinding for good measure, popped on a cover slip, placed under the scope and after scanning for a full half hour, found my first seta. It was more curved than hooked, so not really sufficient to convince me this was P.wahlbergii, so I pressed on.
Finally, after scanning the slide for over 1 hour, I had a total of 4 hooked setae (Unique to P.wahlbergii)

The bracket was then sent to Martyn Ainsworth, now working at Kew, who confirmed my findings.

So, in retrospect, I would guess the answer is to keep your sample within sight under the dissecting scope at all times - even when adding the KOH solution, and even adding the cover slip. Probably easier said than done. :P

Neil Mahler.

NB. Seta: a stiff hair, generally thick walled and dark coloured. The size is probably a little bigger than a basidium.
Neil
 

Re: Preparing slides for hard material

Postby Mal Greaves » Tue Jul 27, 2010 11:49 am

A couple of tips I was shown by Alan Hills were one how to transfer these very small pieces across to the slide using a very fine paint brush. I have tried this and found it just as difficult but the second tip I use all the time. It is to put a drop of mountant on the slide whether it is congo or KOH or whatever not on the material but to one side. Then using a needle (pushed through a cork to allow you to work without stabbing yourself) move the material into the liquid. In this way you don't risk flushing the sample to somewhere else and not being able to find it again.

Mal
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Re: Preparing slides for hard material

Postby Richard Scott » Thu Jul 29, 2010 11:42 am

I feel I may be sometime developing my method but thanks for the help chaps, intersting story Neil. Richard
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