Microscopy

  1. Introduction
  2. Examining Spores
  3. Examining Fungal Tissue
  4. Stains
  5. Microscopes
  6. Setting Up A Brightfield Microscope
  7. UK Equipment Suppliers
  8. Further Information

Introduction

Most fungi cannot with certainty be identified with the naked eye, and need to be examined under the microscope. Microscopy may seem rather daunting to the novice, but it is not too difficult given a little care and attention, and the correct equipment. You will need a microscope providing magnifications from 10 to 1000, a graticule (scale) in one eyepiece, some stains, pipettes, a razor blade or a sharp knife, some tweezers, microscope slides, cover slips and immersion oil (for the high powered microscope objectives).

Examining Spores

To collect spores simply place a cap or a fruiting body on a microscope slide for an hour or two. Once a visible layer of spores has been collected (it is best not to collect too thick a layer), place a drop or two of stain on the slide, followed by a cover slip (a square of thin glass), and press down gently. Excess stain may be mopped up with the corner of a paper tissue. Ideally the spores should be examined at a magnification of 1000x, and measurements taken using the eyepiece graticule.

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.

Figures 1 to 3 show some example spores

Figure 1: Spores from Sarcoscypha austriaca stained with congo red. Photograph copyright Leif Goodwin

Figure 2: Spores from Aleuria aurantia 'Orange Peel Fungus' stained with lactophenol cotton blue. Photograph copyright Leif Goodwin

Figure 3: Spores from Thelephora penicillata stained with Melzer's solution. Photograph copyright Leif Goodwin

Examining Fungal Tissue

To examine the fungal tissue, take a small tissue sample from the area of interest. Place the tissue sample on a microscope slide, and then apply a few drops of stain. Place a cover slip over the sample, and press down on the cover slip, using for example the flat of a tweezer handle. Excess stain may be removed with the corner of a paper tissue. It may take several minutes for the stain to permeate the specimen, and attach itself to the features of interest.

There are many ways to collect a tissue sample. I find the easiest is to use a pair of sharp nosed tweezers to rip off a tiny piece, the smaller the better. It is also possible to cut a thin section using a razor blade, or a freshly sharpened kitchen knife (high quality kitchen knives are very effective). In the case of very small fungi, it is often simplest to squash the entire fruiting body.

If you want to maximise the contrast between the sample and the background, wash it after staining. Once the stain has soaked in, which takes a minute or two, apply some drops of a clear fluid such as distilled water or KOH, then draw off excess fluid with a tissue. Apply a cover slip, and place under the microscope

Under the microscope you will see the various cells and strands of hyphae that make up the fungus.

In gilled and poroid fungi - basidiomycetes - specialised hyphae on the gills - known as basidia - carry the developing spores and the number of spores on each basidium and the form of attachment vary between species. Basidiomycetes are sometimes referred to as 'spore droppers' because they drop spores into the air, where they are then carried away by air currents. Most basidiomycetes also have hyphal strands known as cystidia extending out from the surface. Cystidia are named according to where they are found:

  1. Cheilocystidia: on the gill edge.
  2. Pleurocystidia: on the gill face.
  3. Dermatocystidia or pileocystidia: on the cap surface.
  4. Caulocystidia: on the stem surface.

The shape of the cystidia varies significantly between species. Terms commonly used to describe the shape of cystidia are as follows:

  1. Clavate: club shaped.
  2. Fusiform: spindle shaped.
  3. Urticoid: needle shaped, like a hair from a stinging nettle.
  4. Utriform: bladder shaped.
  5. Lageniform: flask shaped.

The function of the cystidia remains unknown though some authorities suspect that they serve to rid the fungus of excess fluid.

Figure 4 shows the edge of a gill of Bolbitius titubans. The structure at the right is a mature basidium with 4 spores attached. To its left is an immature basidium.

Figure 4: Basidia from Bolbitius titubans. Photograph copyright Leif Goodwin

In another group of fungi - ascomycetes - the spores develop in a long tube - the ascus - from which they are shot when mature. Ascomycetes are sometimes referred to as 'spore shooters' as the spores are forcibly propelled into the air, where they are then dispersed by air currents. 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. There may also be hyphal strands known as paraphyses between the asci.

Figure 5 shows a section through a piece of a cup fungus, Peziza repanda. The finger like structures are the asci, each containing eight spores (which are usually referred to as ascospores).

Figure 5: Asci from Peziza repanda. Photograph copyright Leif Goodwin

Figure 6 also shows a section through a specimen of Peziza repanda. The broad and thin finger like structures are the asci and paraphyses respectively. Notice that the paraphyses are septate, and not constricted at the septa, features which help distinguish the fungus from other Peziza species.

Figure 6: Asci and paraphysesfrom Peziza repanda. Photograph copyright Leif Goodwin

Other features that may be of interest include:

  1. The nature of the join between adjacent hyphal compartments. In some fungi there is a small overlap known as a clamp connection.
  2. The upper layer of cells on the cap known as the pileipellis.
  3. The nature of the hyphae in the body of the fungus. This is important for some polypores.

Specialist works describe in detail the features of interest for each species.

Stains

Fungal tissue is usually stained prior to examination with a conventional light microscope. A stain is a chemical dye which attaches itself to certain structures, such as those containing starch, thereby making them easier to see.

Congo red is a good general purpose stain. It is supplied as a powder, and although it may be dissolved in water, it soon precipitates out of solution, and hence cannot be kept for any length of time. Alternatively it can be dissolved in 10% ammonia solution in which form it will keep for many weeks. Household ammonia sold in many chemists and hardware stores is suitable. Ammonia solution is corrosive, and should not come into direct contact with the microscope.

Melzer's reagent is another excellent stain which has the property that the tissue or spores may change colour depending on the species. Unfortunately one of the components, chloral hydrate, is a controlled substance, consequently this stain is not easy to obtain.

The spores of some species undergo a diagnostic colour change when stained with Melzer's reagent as follows:

  1. Amyloid spores turn bluish black.
  2. Dextrinoid spores turn reddish brown.
  3. Inamyloid spores do not change colour.

Another useful stain is Lactophenol cotton blue which is easy to obtain, and as it stains chitin, it can show some structures, such as spore ornamentation, which are ignored by some other stains such as congo red (see the examples below).

Figure 7: Spores from Octospora rutilans stained with lactophenol cotton blue. Note the beautiful network pattern. Photograph copyright Leif Goodwin

Figure 8: Spores from Octospora rutilans stained with Congo red. Note the absence of the network pattern. Photograph copyright Leif Goodwin

Microscopes

The most widely used microscope for mycology is the brightfield microscope, typically providing a range of magnifications up to 1000x. There are other kinds of light microscope, including phase contrast and DIC, but they tend to be rather expensive, and for general use a brightfield microscope is more than adequate. In a brightfield microscope the sample is illuminated from below by a collimated beam of light from a bright lamp, and imaged from above by a serious of magnifying lenses.

The mycologist is faced with choosing a new instrument, or a second hand one. Unfortunately new research grade microscopes are very expensive. The big names are (in no particular order) Nikon, Zeiss, Leica and Olympus. According to some sources, the least expensive models from these manufacturers are often not as good as similarly priced instruments from competing companies, and many are rebadged Chinese instruments. An affordable alternative is provided by Chinese manufacturers, some sold under a Chinese brand, others rebadged by European companies such as Bresser, and Brunel Microscopes. Although some Chinese instruments are rather poor, the better ones are said to be well made, and good value. There are also some so-called second tier manufacturers such as Meiji, which make good quality instruments with reasonable price tags.

The alternative to a new instrument is a used one. Microscopes from the big names (including Leitz, which later became Leica) are extremely well made, and instruments from 20 years ago can still provide many more years of service. They also have the advantage that objectives and other accessories are widely available on the used market at significantly lower price than new items. These older instruments have one important advantage. Over the last decade or so the top tier manufacturers changed over to using so-called infinity optics. Current microscopes from Zeiss et al are not compatible with objectives and eyepieces from older instruments, and the user will find it very hard to find used accessories at affordable prices, whereas new items are often horribly expensive.

My own suggestion for the amateur mycologist is to buy a used instrument with non infinity optics, preferably one made by Zeiss, Leitz, Nikon or Olympus. It is best to buy from a reliable source, as older instruments do need checking to ensure that the optics and the mechanics are fully functional. Respected sellers include Brunel Microscopes. I also recommend that you search online for user reviews, and get opinions from a range of people.

Setting Up A Brightfield Microscope

Whichever instrument you buy, to get the most out it, the optics and the condenser need to be correctly set up for Kohler illumination as described here.

UK Equipment Suppliers

In the UK stains, slides, slides, cover slips, and other supplies may be purchased from Brunel Microscopes. The Association of British Fungus Groups can supply stains to its members, including the hard to obtain Melzer's reagent.

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