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Under the microscope

Although, with experience, you can identify many species or genera in the field, accurate identification requires at least some level of microscopic study. This page contains two sections; the first describes some of the main microscopic characters and the second gives tips and advice about how to view these structures under a microscope. As the Scottish Fungi hub grows, we hope that users will contribute photographs, diagrams and even videos to illustrate this page. Please use the comments form to let us know if you have something to contribute. You will also find useful information on Michael Kuo's site 'MushroomExpert.com'.

Microscopic characters in the basidiomycetes:

Flesh characters

  • Homoiomerous v. heteromerous  - most fungal flesh is composed entirely of cells that form filamentous hyphae and is called a homoiomerous trama. The flesh of Russula and Lactarius however, contains rounded elements called sphaerocysts as well as filamentous hyphae – this is called a heteromerous trama.

  • Gill tramas - filamentous hyphae are aligned into four basic patterns in the central part of the gills. These can be seen in thin sections of the gill ie the gill trama, and are a great help in identifying the genus when used in combination with the spore colour, gill attachment and the type of cap cuticle (see below for cap cuticle):

    • Regular eg Inocybe, Agaricus, Psathyrella, Entoloma, Lepiota

    • Irregular eg Omphalina

    • Divergent = bilateral eg Amanita. Boletales, Gomphidius, Paxillus

    • Inverse eg Pluteus, Volvariella

Inevitably there are intermediate forms particularly between regular and irregular gill trama. This can be clearly seen in some species of Hygrocybe e.g. H. quieta.

Gill tramas are often best observed at a lower magnification e.g. x 100.

  • Clamp connections – hyphae are composed of many individual cells. Often these are simple and the cross wall shows as a line across the hypha; these divisions are called septa. Sometimes however, clamp connections are present, appearing as a bulge near the septum, with the septum itself apparently ‘bent’. Clamps occur in many different situations are often difficult to find and you need to check several septa before concluding that they are absent. Check scalps and gill tramas of Inocybe species to practice locating some.


Cap cuticle

As mentioned above in ‘Structures in the flesh’, the cap cuticle can be useful in determining the genus (and occasionally species) of some fungi. There are two basic cuticle structures that you will be able to see by taking a scalp from the toadstool (see ‘Fungal microscopy techniques’ below):

  • Cellular – where the cap cuticle is composed of more or less spherical elements, arranged in a palisade across the cap e.g. Conocybe, Coprinus, Panaeolus, Psathyrella, Bolbitius

  • Filamentous – where the cap cuticle is composed of horizontally arranged filamentous hyphae e.g. Galerina, Cortinarius, Stropharia, Agaricus

As with the gill tramas there are some fungi that present intermediate forms particularly in the genus Pluteus. These structures are often best observed at a lower magnification e.g. x 100.

There are other characters within the cap cuticle that can be viewed by taking a section rather than a scalp. The scalp presents a ‘bird’s eye view’ of the structures, a section gives you a ‘side on view’.

Don’t forget that other features such as cap cystidia, veil remnants and scales might also require close examination.


Spores and basidia

Agaric and bolete spores are produced on special cells called basidia, which cover the gill (or tube) face and sometimes edge. In most genera each basidium carries 4 spores although occasionally some species will have 2 or 3 (eg some Laccaria and Entoloma species) and very occasionally (eg some Sistotrema species) 6 or 8. Each spore is raised on a projection called a sterigma (plural sterigmata) and it is usually necessary to focus up and down on the basidia to check how many spores are present. The apiculus is the name given to the short projection at one end of the spore where it was attached to the sterigma.

Spores are a very useful aid to identification. Their colour can be seen without the use of a microscope but other features can only be seen with a microscope. A magnification of at least x 400 is recommended for looking at spores.

Spore features:

  • Size – a calibrated graticule in one of the eyepieces is necessary to measure spores. It is usual to measure both the length and the width of a spore using micrometres or microns (ie 1000th of a millimetre!) often written μ. 

  • Shape – spores vary in shape from globose to ellipsoid, from limoniform (lemon shaped) to reniform (kidney shaped) or lacrymoid (tear shaped), they can be strangulated in the centre or shaped like a bullet. Most good field guides include illustrations of different spore shapes; Fungi of Switerland (Breitenbach and Kranzlin, Vols 1 – 6) have good illustrations of spore shape alongside each included species.

  • Ornamentation – range from spines to warts of various sizes as well as networks of ridges. Sometimes these stain in Melzer’s. It should be noted that many spores are completely smooth.

  • Guttules – the presence of one or more droplets within the spore

  • Loosening perispore and spore walls – in some species the outer wall of the spore (perispore) loosens and detatches itself giving the spore a characteristic outline (Galerina calyptrata, some Coprinus species). Other spores may be thick walled.

  • Germ pores – present or absent. This is an area where the wall of the spore shows a distinct thinning and where germinating hyphae will emerge. This is usually at the end of the spore opposite the apiculus (the apiculus is where the spore was attached to the sterigma)..

'Sometimes the spores can immediately place the fungus into a genus'

Make sure that you are familiar with as many of the following spore shapes as possible:

  • Russula and Lactarius spores are variously ornamented with a network of ridges, spines and warts –staining blue black in Melzer’s solution.

  • Entoloma spores are pink and angular, sometimes almost cuboid or stellate.

  • Laccaria – round or ovate spiny, white spores

  • Inocybe – many Inocybe species have dull brown nodulose spores

  • Lepiota cristata and close relatives – spores bullet shaped.


Cystidia

Cystidia are sterile cells that appear on the outer surfaces of many fungi. They are distinguished on the basis of which surface they appear on, how deeply they originate in the gill trama and also on their contents. The nomenclature varies between different texts and only those cystidia that you are most likely to come across during the early stages of microscopy are described below.

Name of cystidia

Distinguishing character

Cheilocystidia

Found on the gill edge

Pleurocystidia

Found on the gill face

Caulocystidia

Found on the stipe surface

Pileocystidia 

Found on pileus (cap) surface

Chrysocystidia

Have contents that go yellow in ammonia or KOH useful when working with, for example, the genera Stropharia and Hypholoma

Cheilocystidia and pleurocystidia are found on the gills and their presence or absence are usually easy to note when you make a gill section (see ‘Fungal microscopy techniques’ below). It is also possible to decide whether or not they are thick walled and whether there are any encrustations on the top (metuloid) from a gill section but to get a clear idea of the overall shape of the cystidia it is necessary to make a ‘squash’ preparation (see ‘Fungal microscopy techniques’ below). Gathering information about the gill cystidia is particularly important for the genera Pluteus, Mycena, Melanoleuca and Inocybe and you should try and look at material from each of these to familiarise yourself with their cystidia. Macrocystidia cucumis also has very distinctive cystidia.

Caulocystidia are found on the stipes of some fungi and are of particular importance in Inocybe, Galerina and Conocybe. They are sometimes visible to the naked eye as a pruinosity and careful collection (ie don’t hold them by the stipe!) of specimens is always important to avoid damaging these characters. They can be observed under the microscope by taking a scalp but their presence or absence can often be determined in small fruit bodies by simply placing them onto a slide and looking under the lowest magnification of the compound microscope.



Fungal microscopy techniques:

The techniques described below are particularly designed for the agarics and boletes but can be adapted for use when searching for the microscopic features of ascomycetes and other groups of basidiomycetes. Be aware that you need to set up your microscope properly to get the best results. Some advice on doing this is available on the British Mycological Society website.

Viewing under low magnification

When dealing with small fungi in particular, a great deal of information can be obtained by placing the fungus onto a microscope slide without any other preparation. Use the lowest magnification on a compound microscope and move the stage so that the entire fungus passes through the field of view. You will need to focus up and down according to the size of the subject in view.

It is possible to use the same process to look at the number of spores on the collection’s basidia. Remove a complete gill from the fungus and lay it flat onto a slide. If the spores are mostly 4 to a basidium then they will be visible in clusters of 4. If the spores are mostly 2 or 3, then they will appear as a random pattern. This works particularly well with dark spored species.


Taking sections

A common mistake when starting out is to make the section too thick. Indeed this applies generally to microscopy – you only need a very small amount of material on the slide. If available, do your cutting using a dissection microscope – it will help with this problem.

Carefully cut out an entire gill, keeping note of which edge is the cut edge and which the natural gill edge. Place it flat and without water, onto a slide. Take a sharp razor blade and cut the gill into very thin slices. The cuts should run from the top of the gill where it was attached to the cap, straight down to the natural edge. They should be so thin that they flop over onto the glass and it is possible to clearly see the filamentous cells (hyphae) that make up the flesh (trama). Mount these using water and cover slip and check under low magnification to see whether some of the slices in your preparation are thin enough to see the gill trama. This method is also useful for checking whether your fungus has pleurocystidia and / or cheilocystidia and whether the basidia are 2 or 4 spored.


Scalp preparation

This is a useful technique for looking at the structure of the cap cuticle and also for looking at hairs or cystidia on the stipe. It takes a little practice and there are other ways to take a scalp, so if this really doesn’t work for you, find out about other methods.

Use a sharp razor blade and remove a very thin scalp from the skin of the cap, usually at a mid point between the edge and the centre of the cap. This section should then be mounted in the normal way and checked using a microscope. Remember to keep aware of which way up the scalp is – you need to see the top of the section, not the underside. Again the scalp needs to be thin enough to be able to clearly see individual cells rather than an impenetrable mass of flesh!


Squash preparation

Squashing disrupts the fungal cells and allows you to see the shape of particular cells (eg cystidia) more clearly as they should be floating in the mount away from connecting tissues. Place a small piece of the fungus containing the structures that you wish to see onto a slide and mount in a drop of water. Place a cover slip on top. Gently tap the specimen with a pencil mounted rubber. Check down the microscope to see whether you have achieved the required effect. Remember that you will not be able to tell, for example, whether you are looking at cheilocystidia or pleurocystidia unless you have carefully cut a small section to exclude one or the other. Squashes of small quantities of flesh can demonstrate the presence of either a heteromerous or homoiomerous trama and can be useful if you are looking for clamps.


Looking at spores

For the most reliable results always take spores from a spore print as these will be fully mature. Size and ornamentation can vary between immature and mature spores and this must be taken into account if you are taking information about spores from a preparation of the gill edge only. Spores that have fallen onto the top of the stipe will tend to be more mature than those from the gill edge.

Place a small amount of water onto the spores (or Melzer’s if the spore print is white and you need to determine whether the spores are amyloid or dextrinoid) and place a cover slip on top. The spores will be moving around initially so allow them to settle down before attempting to measure them! Too much water will tend to make the spores more mobile.


Using oil immersion

The most detailed field of view possible with a compound microscope is usually a magnification of x1000. Mount the preparation in water (or other as appropriate) and add the cover slip as usual. Using the x400 position the slide on the stage where the area of interest is visible. Rack the stage well down and place a small blob of immersion oil on top of the cover slip and over the area of interest. Swing the immersion lens into place. Carefully rack back up using coarse focus until the lens just touches the blob of oil – look at the stage rather than down the optics to achieve this. Transfer to using the microscope optics and continue to rack carefully up using the fine focus until the field comes into focus. You will probably need more light to view at this magnification. Remember that at this magnification a little goes a long way and it is easy to focus too quickly, pass the point of focus and break the cover slip!

Once you have finished with oil immersion, the lens should be cleaned using a lens cleaning tissue or, if available, xylene. If you are planning to reuse the cover slip, remove excess oil with a tissue. The cover slip should then be washed in detergent and rinsed before drying.


General hints and tips for microscopy

Dissection or Stereo Microscopes

This type of microscope is relatively low powered, rarely more than x 40. There is however, space to work between the 'stage' and the lenses which means many fungi can be placed onto the stage and closely examined without having to take sections or mount on glass slides. This can open up an incredibly beautiful world, particular when looking at small ascomycetes (and as a bonus, accompanying mosses, liverworts, lichens and invertebrates!).

A stereo microscope is also very useful for cutting sections and generally making preparations for the compound microscope. Whilst stereo microscopes are not as expensive as a compounds, it is cheaper and almost as effective to use a stamp collector's visor that also leaves you with two free hands.

Compound Microscopes

These are the higher powered microscopes that will enable you to look at the microscopic features described above. Having an oil immersion feature will enable magnification of up to 1000 times. A more three dimensional effect can be gained by having phase contrast lenses but these are not essential when starting out as most features can be clearly seen in a good preparation without. 

A binocular microscope is recommended however, as is a mechanical stage and built in lighting. A monocular microscope can be very tiring to use initially as you have to learn how to look down it using only one eye (not at all impossible and it's what all the early mycologists used and achieved amazing results with!). A mechanical stage, where the slide is fixed to the stage and the stage itself moved by a couple of knobs on the side, as opposed to move the slide by hand, makes it so much easier to move the preparation around. The more expensive the microscope, the smoother the mechanical stage is. Without a built in light, a secondary light source has to be carefully positioned to get the correct effect. Most modern microscopes come with a built in light source which is far more convenient. It is still necessary to correctly set the sub stage condenser to optimize what can be seen.

Magnification

Most compound microscopes have at least three different lens magnifications for example, x10 might be the lowest for looking at general features, x 40 the one that you will use most for microscopic features and x 100 ‘oil immersion’ which is used to look carefully at particular structures. NB all of these lenses are magnified again by the eyepieces, usually by x 10 thus what you see will either be x 100, x 400 or x 1000 its actual size; it is these latter magnifications that are referred to in texts. It is sensible to start with a low magnification to centre your preparation on the microscope stage and learn how to focus. Then move up to a higher magnification if you need further detail.

Keep the microscopes covered when not in use to prevent excess build up of dust.

Mounting the preparation

For examining fresh fungal material, it is recommended that you mount your preparation on a glass slide using a small amount of water (other reagents can be used as you progress), covering the specimen and the water with a cover-slip. A correctly prepared slide will have the fluid just meeting the edge of the cover-slip all the way around. Too little fluid will result in air bubbles or a large air space under the cover-slip. Too much fluid will spill out around the cover-slip and can even end up on top of it. Remove excess water by applying the corner of a clean tissue or blotting paper to the edge of the excess to draw it up.

It is important to remove any particles of sand or grit from the preparation before placing the cover slip. They will distort what you are seeing and break the cover slip, particularly in a squash preparation (see above).

Air bubbles in the preparation

Air bubbles can cause endless distraction to beginners trying to decide if they are in any way connected with what they are hoping to see! Air trapped in the preparation beneath the cover slip can appear as crazy paving patterns, black tyre like structures or even perfectly round ‘spores’. They often hide the structures that you are trying to view.

To reduce the risk of air bubbles, hold the edges of a cover slip between thumb and forefinger and bring the bottom edge up to touch the bottom of the fluid on the slide. Gently lower the cover slip down supporting it with a pair of forceps or a mounted needle.

When is it in focus?

This sounds like a simple question but often causes early difficulties, usually quickly resolved by practice and experience. The trick is to keep moving the fine focus up and down as you look around the preparation; because of the depth of the section and the high degree of magnification, different structures in your section will come in and out of focus. This is particularly important, for example, when checking the number of spores present on the basidia. You will need to practice moving the stage with one hand and focusing up and down with the other.



Chemical tests

Please read the relevant health and safety notes before using any chemicals.  Wearing disposable gloves is recommended.

  • Fe (iron) crystal or solution on the stipe of Russula species can be a useful aid to identification in this genus.

  • If you have a white spore print, a drop of Melzer’s solution onto the dry spore print will tell you whether or not the spores are inamyloid, amyloid or dextrinoid.

  • There are many other stains and chemicals that you might come across in the keys. Most are not essential for identification purposes.



Useful references for micro features:

Marriott J.V.R. 1994 Guides for the amateur mycologist 2. Guide to identification with a microscope. British Mycological Society

Largent D., Johnson D. & Watling R 1977. How to identify mushrooms to genus III: microscopic features. Mad River Press

Watling R. 1974 Identification of the larger fungi. Hulton Educational Publications (out of print)



Buying a microscope

As with most things in life, microscopes can be relatively cheap or extremely expensive. Generally speaking you will get what you pay for but there are several perfectly adequate compound microscopes that are quite suitable for starting out. Reputable companies include Brunel Microscopes and Hampshire Micro. Recommended also and providing good quality and competitively priced microscopes are Micro Insturments Ltd., 18, Hanborough Park, Long Hanborough, Witney, Oxon, OX29 8LH  Tel: (0)1993 883595 Fax: (0)1993 883616 Web: http://www.m-i.co.uk

Additional equipment including glass slides, cover slips, eye piece graticules and calibrators can also be bought from these companies. 

 



Regular gill trama
Irregular gill trama
divergent gill trama

inverse gill trama

cellular cap cuticle

filamentous cap cuticle









































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