Microscopy

  1. Introduction
  2. Stains
  3. Examining Spores
  4. Examining Hyphae
  5. Photomicrography
  6. UK Equipment Suppliers
  7. Further Information

Introduction

There is a limit to the fungi that can be identified by examination of macroscopic features alone, partly because most fungi are extremely variable, but also because some species are almost identical at the macroscopic level. A microscope allows the identification of a species by examination of microscopic features, namely the spores, and the hyphal tissue.

Stains

A specimen is normally stained before it is examined under the microscope in order to enhance otherwise hard to see features. A stain is simply a chemical dye which preferentially attaches itself to certain structures, such as those which contain starch.

For general mycological use I recommend Congo Red dissolved in 10% ammonia solution. The latter is sold in many chemists, sometimes under the name Household Ammonia. It is a rather nasty substance, and should not be allowed to come into contact with the eyes, or skin. Do not attempt to smell the undiluted solution as the vapour is extremely unpleasant. Note also that Ammonia solution is corrosive, and should not be allowed to come into contact with the microscope, especially the objectives.

Melzer's reagent is another excellent stain but it is rather difficult to obtain as one of the ingredients - Chloral hydrate - is a controlled substance.

Alternatively Lactophenol cotton blue may be used. It is not as good a stain as the others, but it is easy to obtain and non corrosive.

Examining Spores

Spores may be collected by placing the cap or fruiting body on a microscope slide for a few hours. It is best not to collect too thick a layer, otherwise they tend to swim around on the slide. The slide can be examined under the microscope with a low magnification to locate some spores, then a few drops of stain placed on the slide, followed by a cover slip. Excess stain can be mopped up with a tissue. A magnification of x400 should suffice to show the spore shape and ornamentation although a magnification of x1000 is preferable when measuring the spores.

The spores of fungi vary considerably, both in size and shape. They may be spherical, sub-spherical, elliptical, angular (with flat sides), mitriform (shaped like a bishop's hat), sausage shaped, knobbly, spiny, warty, reticulated, or decorated with wing like appendages. Some are filled with oil drops, whilst others are septate (having compartments), and the number of compartments may be critical to the identification.

Examining Hyphae

It is usual to cut a thin section through the specimen. There are many techniques for sectioning fungal tissue, including using a special tool called a microtome. The simplistic method is to use a sharpened kitchen knife and a chopping block. I find this method works quite well though it does require a fair degree of manual dexterity. Another method is described here. The tissue section is placed on the microscope slide, followed by a few drops of stain and a cover slip. Gentle pressure on the cover slip using the tweezers helps to flatten the section. It may take several minutes for the stain to permeate the specimen, and attach itself to the features of interest.

The fungus tissue consists of intertwined strands, or hyphae. In gilled and poroid fungi - basidiomycetes - specialised hyphae - known as basidia - carry the developing spores and the number of spores on each basidium and the form of attachment are important features. Most basidiomycetes have hyphal strands known as cystidia which extend out from the surface. Cystidia have different names according to where they are found: cheilocystidia are found on the gill edge, pleurocystidia are found on the gill face, dermatocystidia or pileocystidia are found on the cap surface and caulocystidia are found on the stem surface. The shape of these varies between fungi and may be important. In another group of fungi - ascomycetes - the spores develop in a long tube - the ascus - from which they are shot when mature. (In fact if you tap the surface of many ascomycetes, especially cup fungi, you might be rewarded with a cloud of smoke-like spores as the acsi simultaneously discharge spores. It is also worth placing a cup fungus next to the ear, as the discharge of spores can sometimes be heard as a fizzing sound!)

Photomicrography

Photomicrography is the taking of photographs through the microscope.

Perhaps the simplest method is to place a camera (with lens) next to the eyepiece. The image produced by the microscope is imaged by the camera lens onto the camera sensor (or film). Many small digital cameras are ideal for this purpose. Adaptors for various cameras are available from specialist dealers.

The alternative is to replace the normal eyepiece with a projection eyepiece, which forms an image directly onto the camera sensor, without the need for an intermediate camera lens. The advantage is that the image quality is potentially higher. Trinocular microscopes are ideal for this purpose. These instruments have two eyepiece tubes for visual use and a third eyepiece tube for photographic use. The phototube usually has a T2 mount at the top end, allowing the attachment of a camera via a camera specific adaptor. Vibrations are the biggest problem when taking photographs through a microscope. Vibrations can be reduced by using the camera's mirror lock up (MLU) feature (if present) in combination with a cable release. It also helps to turn the microscope's light source to the maximum setting so as to reduce the exposure time, and if neccessary increase the ISO setting on the camera. I find that an exposure of 1/1000" used with MLU is sufficient to avoid vibrations.

UK Equipment Suppliers

In the UK stains, slides, slides, cover slips, and other supplies may be purchased from Brunel Microscopes. Stains can also be purchased from the Association of British Fungus Groups though you will have to join first, and most stains can be purchased more cheaply elsewhere.

Further Information

Futher information can be found at the following sites:

  1. Microscopy-UK
  2. Fungi 4 Schools
  3. Yahoo Microscope Forum
  4. Brunel Microscopes