Richard said:
I have managed to get hold of a microscope and have done a couple of spore prints and looked at the spores. One was a Common Bonnet and the other I have yet to id (I may have to give in and ask for help!). Do you / does anyone know of a source of info for spore microscopy? Technique tips would do for starters. I can't find anything on the net and wonder if I need to purchase a book.
Richard R
Not easy to answer this.
First, the book that first taught me some mycological microscopy is the book I first learnt fungi with, and I still think it has the best simple advice on examination of fungi, simple chemical tests, etc.
The book is the original
Collins Guide to Mushrooms & Toadstools, by M. Lange and F.B. Hora, long out of print but quite often sitting on the shelves of second-hand bookshops at no great price.
There is a slim American book (though with one of the authors British), entitled '
How to Identify Mushrooms to Genus III: Microscopic Features', by Largent, Johnson, Watling, published a number of years ago (no date on my copy) by the Mad River Press, but still in print I think - though maybe at not such a slim price. It is excellent, assumes no prior knowledge and is full of practical advice for examining spores and the various types of tissue, has an excellent glossary etc.
It also occurs to me that a parts of a microscopy laboratory manual I wrote for our own undergraduate students could be adapted. I shall have to think about that.
Anyhow, some quick tips.
1) Be sure the microscope is correctly set up, notably the sub-stage condenser lens is in the right position and the sub-stage iris is also correctly adjusted. This really makes a lot of difference to the definition.
2) The smaller the amount of material on the slide the better, otherwise all kinds of optically bad things happen.
3) Read tip 1 again, it is very important. Also tip 2.
4) For examination of spores, best is to lay a clean glass microscope slide under the cap so that at least part of the spore print is dropped onto the slide. Do not use this directly for microscopy as the spores will be massed together and distorted, but having the spores on a glass slide is convenient for subsequent handling or comparing with colour charts (as is sometimes useful).
Take another clean glass slide, add a drop of mountant (presumably water) to the slide. A small drop. As the glass is clean you can touch the slide with the dropper without contaminating anything and it is easier to control the size of the drop.
Then scrape up a tiny amount of the spore print on the tip of a needle and transfer
into the drop of mountant. (N.B. transfering any mycological material into the mountant reduces the amount of air that is trapped.)
Add cover slip and examine spores.
5) If spores are colourless ("white") and thin-walled, then ideally use Nomarski Differential Interference Contrast to make them more visible, but if, for some reason, you are disinclined to add some £3000 to the cost of the microscope, try a stain instead.
What stains will work will depend on the spores, and on whether the walls take up stain or whether the stain simply provides a better, contrasting background. There are various mycological stains but once, in emergency, I raided my parents' larder and tried a green food stain. It worked superbly well. Ensure any such colouring is truly dissolved and not granular.
Stains and other chemicals may slightly affect the degree of swelling of spores, so accurate measurements should be done in water.
6) Spore ornamentation requires careful microscopy and best possible definition, so read tip 1 again.
A x100 (oil immersion) objective ideally is needed, but it needs to be of high optical quality. A duff x100 objective may show less than a good x40 objective. (Note, on standard microscopes, total magnification is obtained by multiplying the power of the objective by the power of the eyepiece, so a x40 objective with a standard x10 eyepiece gives a magnification of x400. This may not be true with more advanced microscopes with extra insertions in the light pathway, or with old microscopes with adjustable tube lengths.)
Note that dilute ammonia, often used as a mountant, can dissolve some types of spore ornamentation.
7) Hydrophobic spores (e.g. from earthballs, puffballs and the like, in which spore examination is sometimes vital for reliable ID) trap air-masses when mounted in plain water. Smear a thin film of photographic wetting agent on the slide before adding the water droplet, or just mount in very dilute 'Fairy Liquid'. (Also works well with some 'moulds', such as Penicillin Mould.)
8) When examining spores, always try to use spores from a spore print rather than directly examining spores on a fragment of gill. That way you (mostly) get mature spores. Spores still on the gills may include some that are immature, deformed, over-sized, etc. This is particularly important with waxcaps (
Hygrocybe), in which the very thin-walled spores show a lot of variation - though the variation in mature spores may still be considerable and important for identification.
Examination on the gill surface is important, however, for determining whether the fungus has two- or four-spored basidia (especially essential information in, e.g.
Conocybe and
Coprinus).
9) Read tips 1 & 2 again!
I hope this helps!
Alan
