First steps

First steps

Postby Dave H-Tranter » Sat Jan 31, 2009 6:08 pm

Being a newcomer to microscopy I would love a few refreshment tips on what to do as I am sure others will do as well. Right then - the microscope is all set up - I have taken a spore print....

What next?

After the question has been fully answered then I will add more detailed questions so as to see how people deal with those tricky microscopic moments.

Fungalpunk Dave
Dave H-Tranter
 
Posts: 210
Joined: Thu Jan 29, 2009 10:42 am

Re: First steps

Postby Neil » Sun Feb 01, 2009 12:06 am

Hi Dave,

Well I guess first you scrape the spores together to have a go at the colour, then you dispose of the majority and decide whether to use a stain or plain water before applying the cover slip (the thickness of which should be suitable for your microscope)
In certain cases it is better to apply the stain by drawing it under the coverslip.
Having removed any excess solution I would go straight to a 40X objective for a general scan, finally using the 100X to take a measurement.

Over to you Dave !

Neil.
Neil
 

Re: First steps

Postby Neil » Sun Feb 01, 2009 9:12 am

I just realised the above is not much help to those who take a spore print on a piece of half black, half white card - I never bother, I place the specimen directly on a glass slide.
So if using card, all you do is lick your finger, dab the card, then dab it on a glass slide and then carry on as above.

I think I'm right in saying the outer shell of spores are made of chitin - a very hard substance found in beetle wing cases, but I still find depending on what mounting solution you use, that spores can break up when under a cover slip after a while.

Just in case I read the original question completely wrong, like I often do, you cannot proceed much further without good reference books, and basic tools such as very fine tweezers (I sharpen mine to a very fine point on an oil stone) a scapell and a small needle pushed into the end of a solid twig.(handy for pulling apart your sample)

The secret is not to take too big a sample and knowing where to take the sample from. It is necessary to squash the sample under the cover glass while you try to squeeze out any air bubbles. This is done in order to separate the cells and also the flatter the sample the more will be in focus.

One particular problem you will come across is woody brackets and the breaking of many cover slips, in this case it is better to scrape off a sample rather than cut off.
Another problem is rubbery Jelly Fungi like Exidia spp. - these tend to ooze from under the cover slip without actually breaking up and I'd be interested to know how other members cope with this frustrating problem.

Neil.
Neil
 

Re: First steps

Postby Dave H-Tranter » Mon Feb 02, 2009 11:59 am

Great explanation and hopefully gets everyone off on the right footing. More questions to follow ha, ha. Appreciations.

Fungalpunk Dave
Dave H-Tranter
 
Posts: 210
Joined: Thu Jan 29, 2009 10:42 am

Re: First steps

Postby JohnG » Mon Feb 02, 2009 2:22 pm

Did you have any joy with the cell slide for jelly fungi, Neil?

What about making permanent mounts of spores? I've not tried it but would be interested in the procedure.

All the best,
John
JohnG
 
Posts: 15
Joined: Thu Jan 29, 2009 8:35 am

Re: First steps

Postby Dave H-Tranter » Mon Feb 02, 2009 7:49 pm

Totaly agree - to build up a collection of spore slides would be good for reference. But...do spores stand the test of time? Do they need a preserving agent?

Fungalpunk Dave

www.fungalpunknature.co.uk
Dave H-Tranter
 
Posts: 210
Joined: Thu Jan 29, 2009 10:42 am

Re: First steps

Postby JohnG » Mon Feb 02, 2009 10:17 pm

A quick look through the "Mrs Beaton" of Microscopy - Bolles Lee's "Microtomist's Vade Mecum" - shows dozens of "recipes". They could be grouped into 3 main methods:
1) aqueous stains and gelatine mounts
2) aqueous or alcohol-based stains, dehydration and mounting in balsam or synthetic resin
3) mounting in lactophenol with cotton blue stain

I guess that the small structures in many fungi - and I'm not talking of making mounts of whole caps! - make them rather good subjects as there is no need for sectioning and trying to lay out the bits in a pleasing pattern! Must give it a try soon.

John
JohnG
 
Posts: 15
Joined: Thu Jan 29, 2009 8:35 am

Re: First steps

Postby Dave Jewsbury » Thu Feb 05, 2009 9:01 pm

Hi Dave,
I am at the same point as you, or more of a beginner, so I'm very interested in the thread. I've taken a look at spores and they seem easy enough to see. I tried a squash of a gill edge of Tubaria furfuracea, and although I think I could make out the essential point of the shape of a cystidium, it wasn't very visible, until I'd closed the diaphram a long way.

I haven't got phase contrast, so I supose staining is the way to make it more visible. Has anybody got any advice on stains for this job, or any good references. The only thing I've found is some online fungal resources for schools on the BMS website.

http://www.fungi4schools.org/Microscopy_page.htm

I would really like a simple reasonably priced book, concentrating on fungi, to guide me through the first steps.

Thanks, Dave J
Dave Jewsbury
 
Posts: 11
Joined: Tue Feb 03, 2009 10:40 pm
Location: Basingstoke, Hampshire

Re: First steps

Postby admin » Fri Feb 06, 2009 10:02 am

Dave
The use of stains and how to learn about the techniques is not particularly well documented and when it is the books are not always in English. However, the Forayer over recent years has run a series of good practical articles on the use of microscopic stains (and incidentally on the setting up of a microscope). Somebody might be willing to photocopy these for you. I think more or less all the stains you are likely to want to use have been explained, plus some that you may not get around to using. It is likely that these articles will continue in future issues but you need to be an ABFG member to receive the Forayer. Members can also obtain back issues and/or parts of back issues at cost.
MJ
Michael Jordan
Forum moderator
admin
Site Admin
 
Posts: 532
Joined: Wed Jan 07, 2009 11:43 pm

Re: First steps

Postby Leif Goodwin » Fri Feb 06, 2009 10:25 pm

I found information hard to find, and often slanted towards other areas of study rather than mycology. So I wrote a few notes to summarise some information I have gleened from elsewhere. You can find it here:

http://www.leifgoodwin.co.uk/General/Microscopy.html

I don't claim that it is authoritative or complete, or even free from error.

There is somewhere a useful article explaining how to set up a microscope for Kohler illumination which ensures optimum performance.

I do recommend that you understand the role of the condenser as it is as important as the objective and the eyepiece. It acts rather like the aperture in a lens and an incorrectly aligned condenser can reduce the resolution of the microscope. In fact with a x100 objective you really need to oil the condenser to the slide in order to get the correct cone of light into the objective.

I toyed with writing some notes about microscopes, but I wasn't convinced I had anything to say which was not already out there in internet land. In fact a quick Google found these:

http://www.bio.umass.edu/microscopy/doc ... nation.pdf
http://www.micrographia.com/tutoria/mic ... cb0400.htm
Fungi and Nature Photography: http://www.leifgoodwin.co.uk/Fungi/Fungi.html
Leif Goodwin
 
Posts: 1080
Joined: Mon Feb 02, 2009 5:41 pm

Next

Return to Microscopy

Who is online

Users browsing this forum: No registered users and 1 guest

cron