FunKey utilises a variety of characters that describe fungal fruit-bodies. Characters range from simple (such as the size and shape of the fruit-body) to complex (such as chemical reactions and microscopic features of the spores). Characters are either measurements (such as spore length) or multistate (with different character states, such as for spore print colour).

In the Lucid key window, characters and character states are listed in the Features Available panel. Help on each character state (choice) can be accessed by clicking on the icons in the bottom, right-hand corner of each character state thumbnail in the Features Available panel:
The left-hand icon displays the full image, while the right-hand icon opens the relevant Fact Sheet for more extensive information about that particular character state.

There is also a comprehensive Glossary of all terms used to describe characters and character states.

Characters are divided into macrocharacters (those visible to the naked eye or with a hand lens) and microcharacters (those visible only using a compound microscope at high magnification).

The summary below deals briefly with all 115 characters used for identification in FunKey, and the ways in which different structures relate to one another. In interpreting characters it is important to take into account variation caused by developmental and environmental factors.

Information is also provided on collecting agarics, and on equipment and techniques, both for macroscopic and microscopic examination, including microscopy.

In the following sections, the character names as used in the key are in bold, while other important terms are in bold italics. The order in which characters are introduced is very similar to their native order in the key.

The key uses a simplified set of states for a given character, with the aim of providing clear alternatives that are appropriate to identification at genus level. For some characters, such as textures of the pileus or stipe surface, a rich terminology is often used, describing subtle and complex variations. In the key, these terms have been aggregated into relatively few states. For example, terms for hairiness (such as pilose, villose, velvety, woolly, cottony, pubescent, tomentose, hirsute, strigose, hispid and so on) cover variations in hair length, stiffness and entanglement. For identification, we have found that simply using two states for hairiness (finely hairy and coarsely hairy) is sufficient; the slight extra power gained by having many states does not offset the difficulty in distinguishing between subtly different terms (and hence the likelihood of making an error in the identification).

Macrofungi are very variable in form due to developmental factors and the influence of the environment. This variation must be recognised and taken into account when identifying specimens.

Agaric fruit-bodies undergo considerable change during development, from an initial, unexpanded (button) stage through maturity to eventual decay and putrescence. Most are fleshy, and the fruit-bodies last for several days to several weeks. A few genera are tougher in texture and longer-lived. Macrocharacters should be assessed on mature fruit-bodies, unless otherwise specified (the simplest guide as to what constitutes a mature structure is that it will readily produce a dense spore print overnight). In the field, take the opportunity to observe a range of developmental stages, so as to ensure that mature material is collected. Where a partial veil is present, this will have broken to expose the lamellae, usually leaving an annulus or ring-zone on the stipe. Where a universal veil is present, this will have broken to leave a basal volva and/or patches of tissue on the pileus. The pileus margin is often quite incurved or inrolled in young fruit-bodies.

For Inky Caps (Coprinus, Coprinellus, Coprinopsis and Parasola) the fruit-body undergoes a dramatic developmental change at maturity. Enzymes autodigest the tissue of the pileus and lamellae, reducing it to an inky mass in a matter of hours.

Fleshy fruit-bodies are very susceptible to drying out, even in situ; many characters will be difficult to observe accurately on dry, withered specimens. Colour of the pileus can change significantly on drying (especially if the pileus is hygrophanous), and translucent striae will disappear. Similarly, a viscid or glutinous surface will be lost as the fruit-body begins to dry. As this happens the lamellae often become undulate when viewed edge-on, rather than straight as in fresh material. Avoid collecting waterlogged specimens, where adjacent lamellae often become stuck together. While some species smell unpleasant even when fresh, a putrid odour is a reasonable indicator of over-mature fruit-bodies.

When picking fruit-bodies, ensure that the base of the stipe (sometimes partially buried) is included, along with any pseudorhiza or attachment to a sclerotium. Collect a range of specimens from immature to mature, but avoid old and weathered or mouldy fruit-bodies. When transporting specimens, make sure they do not dry out. Specimens can be wrapped in waxed paper or placed directly in containers: margarine, yoghurt or ice-cream containers for larger specimens or fishing tackle boxes, with compartments, for smaller specimens. Fungi can be stored in a refrigerator for up to several days, but do not freeze. To make permanent collections, dry thoroughly in a food dehydrator or drying cabinet, and store dried collections in snap-lock bags. Check specimens several days after they have been sealed in the bag to ensure they are fully dried.

When using plastic containers to collect material in the field, putting a piece of white paper in the bottom of the container and placing a severed pileus lamellae downwards can yield a spore print by the time one has returned from the field. When making spore prints in this way, use a container or section of a tackle box that is just larger than the pileus, so that the pileus does not move around too much in transit, and stack containers so the pileus is horizontal.

The colour of the spore print is a very important character for identification; however, obtaining and then interpreting a spore print can be frustrating. A spore print is achieved by cutting off the stipe at the apex, and placing the pileus with the lamellae downwards on a piece of paper. Cover the pileus with a glass or container to prevent it drying out.

Use pure white paper so that you can distinguish white from pale cream or pale pink prints. Placing the pileus mid-way on overlapping sheets of black and white paper can also assist in observing white or very pale prints. Draw a line around the pileus so you can see where the spore print is deposited. A dense spore print is produced overnight, but within a couple of hours a faint print may appear and give an indication of the colour. Set up the spore print as soon as possible after collecting so that the fruit-bodies are fresh and a spore print can obtained prior to commencing identification. Viewing the paper at an angle will assist in confirming the presence of a print.

If a spore print cannot be obtained, indications of the spore print colour can sometimes be found by examining the apex of the stipe, especially where it is not strictly vertical, and the upper surface of the annulus, or even the fibres of a ring zone (as in Cortinarius). Where one pileus sits over another, a spore deposit can also be present on the lower pileus. In the field, careful observation of vegetation and soil below the pileus will sometimes reveal a spore deposit.

Spore print colour varies from white, pale cream or pink, through a range of browns, to black; on rare occasions, it is pale lilac or greenish. Spore print colour can be compared against colour charts such as Royal Botanic Gardens Edinburgh (1969), Rayner (1970) and Kornerup & Wanscher (1978) for discrimination of specific shades such as clay brown, rust brown or chocolate brown.

The exact spore print colour can be difficult to establish, particularly if the print is light, or if colour is being inferred from a spore deposit on the pileus, stipe or underlying vegetation. As with any character, if you have difficulty in selecting the most appropriate state, you should always choose more than one.

An agaric fruit-body consists of a pileus (cap) beneath which hang the lamellae (gills). In most agarics the pileus is supported by a stipe (stem or stalk). The interior substance of the fruit-body (the flesh) is called the context.

The colour of the pileus, lamellae and stipe is an important feature. Genera with strong, bright colours (such as yellow, red, purple, blue or green) will key out more readily than the many pale or brown genera. The pileus can be concentrically zoned in more or less contrasting colours.

The dimensions of the pileus (diameter) and stipe (length and diameter) are diagnostic for some genera, especially in combination with other characters. Measure the stipe diameter at half the distance between base and apex.

In the brief descriptions that are accessible in the key through the taxon fact sheets, pileus diameter is indicated as follows: small (less than 25 mm), medium (25-75 mm), large (75-200 mm) and very large (greater than 200 mm).

The consistency of the pileus and stipe is typically fleshy, but it varies from quite fragile to hard and woody.

The young fruit-body in some genera is covered initially by a universal veil that ruptures to leave a basal volva, and often patches of residual tissue on the pileus surface. A partial veil, when present, initially joins the pileus edge to the stipe, covering the lamellae. The partial veil can be membranous, or made up of fine fibres like cobweb (in which case it is called a cortina).

As the fruit-body matures, the partial veil ruptures, sometimes leaving an annulus (ring) on the stipe, or merely a zone on the stipe surface. The annulus can be moveable, have ridges or scales on the undersurface, or it can be striate above.

The overall pileus shape and the shape of the centre of the pileus varies, as does the surface texture of the pileus. Surface texture variation is described in two ways. The character surface texture (cracks, pits or wrinkles) describes modifications to the surface of the pileus itself. The character pileus surface (hairs or scales) describes hairs (fine or coarse), fibres, scales or patches of tissue that project above the pileus surface; when these are absent the pileus is described as glabrous. Some surface features, particularly squamules and patches of tissue, are often remnants of a universal veil. In addition, the surface of the pileus can be viscid or glutinous (different degrees of sliminess) or, alternatively, dry or moist. The edge of the pileus can be striate (striped), either by the lamellae showing through the pileus tissue (translucent-striate) or by grooves or ridges on the surface or pleats at the pileus margin (grooved, ribbed or pleated). Rarely, the pileus splits through the lamellae trama (the interior flesh of the lamellae). As the pileus dries, it can change colour significantly (hygrophanous). When young, the pileus margin is usually inrolled or at least incurved, but in genera such as Mycena the immature pileus edge is more or less parallel to the stipe. The pileus edge can be smooth, or appendiculate, with scales or fibres hanging from the edge (these are usually remnants of a partial veil). A potassium hydroxide (KOH) solution applied to the pileus surface will sometimes cause a distinct colour change.

The lamellae attachment to the stipe ranges from free to deeply decurrent. The thickness of lamellae varies, as does their spacing (crowded together to distant). The edge colour of the lamellae can be the same as or different to that of the lamellae faces. The edge texture can be even or serrate (toothed), or the edge can have a gelatinous thread. The edge of lamellae can be split lengthwise. The faces of the lamellae can be mottled or uniformly coloured. In most genera, short lamellae (lamellulae) are also present. Lamellae can be regularly forked or they can have distinct anastomoses and cross-walls (intervenose) such that adjacent lamellae have frequent connections. In genera such as Coprinus the mature lamellae autodigest and liquefy (deliquescent). When pieces of lamellae of certain genera are mounted in KOH, coloured pigment can be seen to diffuse into the surrounding fluid.

Stipe position can vary from central to lateral, or a stipe can be lacking entirely (with the pileus attached directly to the substrate). The overall stipe shape varies, as does the stipe base shape. The stipe surface texture can be smooth to squamulose or pruinose, and it can be viscid or not. A pseudorhiza (rooting base) or sclerotium (sterile tuber-like structure) can be present at the base. The stipe can arise directly from substrates such as fallen leaves (insititious) without any basal mycelium or, alternatively, the basal tomentum can be white or variously coloured. Rhizomorphs are thin, ropy aggregations of hyphae arising at the stipe base; they are often black and shiny and resemble horse hair. Sterile criniform stipes, topped by a tiny aborted pileus, are rarely produced.

In some genera the surface of the pileus or stipe, or their context can change colour on cutting or bruising. Latex (milky juice) can ooze from cuts to the fruit-body. Fruit-bodies of a few genera are luminous.

The odour of some agarics is highly distinctive, with smells ranging from quite pleasant to very offensive! Odour is included in the key, but to take account of the varying olfactory ability of different people, all genera are coded as having no distinctive odour, in addition to any distinctive odours that can be detected by those with sensitive noses.

Agarics grow on a range of substrates, and the distinction between wood, litter, dung and soil is an important one. The substrate is sometimes difficult to determine, especially with species that grow on buried wood, but FunKey accommodates misinterpretations of this character. The growth habit of fruit-bodies varies from solitary to densely clustered. Associated plants can assist identification, because some fungi grow preferentially in lawns, others only with exotic trees. A limited range of genera grow in urban habitats or in gardens.

There are two main modes of nutrition. Saprotrophs gain nutrition from the breakdown of organic substrates, and grow on these substrates (wood, dung, litter) or on the ground. Mycorrhizal agarics form a mutualistic relationship with plants, and always grow on the ground. A few agarics (such as Armillaria) are parasitic on living plants. However, agarics growing directly on wood are mostly saprotrophs, while those on the ground can be saprotrophs or mycorrhizal. The nutritional mode tends to be uniform within a genus but is not directly discernable from the fruit-body, and thus this characteristic is not used in the key.

Distribution is given by States and Territories. For this purpose, the Australian Capital Territory is included in New South Wales.

A hand lens (or dissecting microscope) is essential in order to observe certain macrocharacters. We have distinguished states of characters, such as pileus and stipe surface and lamellae edge colour and texture, based on what can be seen at a magnification of ×5 with a hand lens. Of course, more detail can be observed at higher magnifications, but such detail should not be used as the sole basis for choosing between character states. It is possible to view many macrocharacters of the fruit-body in situ. A small mirror (such as a long-handled dentist's mirror) can be useful to inspect the lamellae.

A median longitudinal section of the fruit-body (slice it lengthwise in half through the stipe) assists determination of lamellae attachment, and is also necessary to see colour changes on exposing the stipe and pileus context.

Microcharacters are of great importance in identifying fungi, since many genera are similar macroscopically, but have distinctive spores and other microscopic characters. Microscopic characters must be observed under high magnification using a compound microscope (see microscopy).

Agaric fruit-bodies are predominantly made up of hyphae, which are typically long, thin, cylindrical structures with septa (cross-walls). Compartments of hyphae or any modifications of hyphae (such as globose structures) are not referred to as cells, but as elements.

The hymenium (spore-bearing layer) covers the outer surface of each face of the lamella, and sometimes also the edge. Spores are produced from specialized elements of the hymenium called basidia. Each spore arises from a small curved projection at the apex of the basidium, called a sterigma (plural sterigmata), to which it is attached by a minute projection, the hilar appendage (apiculus). Immature basidia are called basidioles: they are similar in shape and size to basidia but lack sterigmata.

Spores are three-dimensional objects. The width (distance between sides in side view) and breadth (distance between sides in face view) can differ. In side view (profile) the hilar appendage is visible at the base to one side; in face view (front or frontal view) the hilar appendage is visible at the centre of one end of the spore. Thus, in the diagram below, spores a and c are in side view, and spore b is in face view.

In end view (polar view) the spore is viewed with the long axis pointing directly at the observer.

In side view, the side facing the long axis of the basidium when the spore is attached to the sterigma (which is the side the hilar appendage is on) is the adaxial (dorsal) side, the opposite is the abaxial (ventral) side.

Spores are minute, mostly between 5 and 20 micrometres long, and vary in length and width. Spores also vary considerably in shape, both in terms of the overall ratio of length to width, and the general outline (whether rounded, angular, pear-shaped and so on) in side or end view. Spores are rarely medially constricted. The colour of spores under the microscope ranges from hyaline (colourless) through shades of brown to very dark (in a manner similar to the spore print, but not always predictable from the spore print colour). The reaction of spores in Melzer's Reagent is highly characteristic of each genus. Spore surface ornamentation can be absent (i.e. the spores are smooth), or spores can have a warty, spiny or ridged surface, with the ornamentation height being variable between species and genera. Ornamented spores sometimes have a bare area (plage), or they can be partially or completely covered by a thin outer membrane (perispore). The spore wall can be thickened. The apex sometimes has a germ pore, and then is often flattened. Spores in agarics are usually unicellular; only rarely are they septate.

Cystidia are differentiated structures that, when present, are often important for microscopic identification. They are sterile, and occur in the hymenium on the edge or face of lamellae, or at the terminal ends of hyphae on the pileus or stipe surface. Cystidia are named according to their position: pleurocystidia occur on the face of the lamellae, cheilocystidia on the edge of the lamellae, pileocystidia on the surface of the pileus and caulocystidia on the surface of the stipe. Each type of cystidium varies in overall shape, apex shape and branching.

Special types of cystidia occurring in the hymenium of some genera include chrysocystidia (with yellow contents in KOH) and gloeosphex cystidia (with an hour-glass-shaped tip covered by a droplet). Hymenial cystidia can have thick walls or be capped with crystalline material.

On each lamella, the hymenium (spore-bearing layer) comprises mature and immature basidia, sometimes intermingled with sterile elements (cystidia, see above). The length and width of basidia can vary between genera. Occasionally, the size of basidia is markedly polymorphic, with two or more size classes present within a hymenium. The number of spores produced on each basidium is usually four, but sometimes there are regularly only one or two and, on rare occasions, as many as eight spores per basidium.

In cross-section the outer layer of the pileus (the pileipellis or cuticle) can consist of cylindrical hyphae that are parallel to the surface (a cutis), or more or less vertical in a palisade (hymeniderm), or in some other arrangement. The hyphae of the pileipellis can be gelatinised or they can be branched or have intracellular or encrusting pigment. The terminal elements of the pileipellis vary in shape and surface features. The layer immediately beneath the pileipellis (the hypodermium) can consist of hyphae whose shape is similar to or different from those of the pileipellis, and the hyphae can be gelatinised. The trama (flesh) of the pileus is usually a hyaline colour in KOH. The tramal hyphae can be uniform in appearance (monomitic), or comprise two different kinds (dimitic); these are described under the hyphal system. Where the hyphal system is dimitic (comprising generative and skeletal hyphae), the skeletal hyphae shape is diagnostic. The generative hyphae diameter can vary, and the presence of thick-walled hyphae in the trama is also diagnostic. Distinctive, smooth or ornamented swollen elements can occasionally occur in the pileal trama.

The lamellar trama (the thin layer of flesh between the two faces) is usually made up of descending, more or less parallel hyphae in a pattern that is said to be regular. Alternatively, the hyphae can be irregular or slanted on each side towards or away from the edge. On rare occasions the direction of the trama is radiate rather than descending. The hyphae of the lamellar trama are sometimes dextrinoid (reddish brown in Melzer's Reagent). Occasionally, there are protruding bundles of hyphae on the faces of the lamellae that form hyphal pegs.

The trama (interior flesh) of the stipe usually consists of parallel hyphae of similar diameter; however, there is sometimes a mixture of broader and narrower hyphae (sarcodimitic).

Clamp connections are specialised structures sometimes formed at the septum (wall) between adjacent compartments of hyphae, and at the base of basidia and cystidia.

When studying microscopic characters, avoid recording details from spores and other structures of contaminating microfungi. Fruit-bodies usually have at least some pollen grains or spores of other species adhering to their surface. In addition, a wide range of microfungi grow on or in the fruit-bodies of macrofungi. Contaminating spores can sometimes be present in large quantities, in which case there will usually be some sign of infection at the macroscopic level, such as powdery or furry patches. The mould Sepedonium infects Boletaceae such as Phylloporus, starting as white cottony patches that eventually engulf the whole fruit-body. Old agaric fruit-bodies are often hosts to pin moulds, such as Spinellus, which form a fluffy covering, while small agarics can support the gregarious, yellow perithecia of Barya.

When using FunKey with macroscopic characters only, you will often find that an identification cannot discriminate between a number of genera, and microscopic characters are needed to continue the identification to a single genus.

Microscopic characters require more preparation and work than macroscopic characters, but they are essential for properly identifying some genera, and they are always useful for confirming identifications. If you are able to invest in a compound microscope and some simple equipment and stains, you will find that accurate identification of fungi becomes much more achievable. Most of the techniques described below are quite straightforward, and can be readily mastered with a little practice.

Fresh material can be examined directly in water or in Stains and reagents, either as a Squash preparation or after cutting Sections.

Dried material needs to be rehydrated before it can be observed under the microscope. Some material can be rehydrated simply by placing directly in water, but dried fungal tissue often does not rehydrate well in this way. A weak solution of potassium hydroxide (KOH) usually assists rehydration, and also helps to free up hyphae that have become stuck together on drying. Well-dried material, particularly thin sections, will rehydrate within minutes when placed in several drops of KOH on a slide. Otherwise, place small chunks of tissue in KOH solution in small wells or vials (covered so as to not dry out) for several hours or overnight as necessary. Once rehydrated, sections can be cut and the material viewed directly in KOH, or transferred to other solutions. Potassium hydroxide is also useful for examining fresh tissue of fungi with tough context such as those in the Polyporales.

Remove fragments or sections of tissue from the sample with fine forceps or a blade, place in a drop of mounting medium on a slide, and gently add a cover slip. Small pieces of tissue are better than large chunks. For thin sections, the mounting medium will spread under the cover slip. For larger tissue fragments which are to be observed from a squash preparation, squash the sample by pressing gently at this stage.

Place the tissue fragment in a small drop of mounting medium and then add cover slip

Remove excess mounting medium by placing the slide and cover slip face down on absorbent paper and pressing very gently. Be careful not to get mounting medium on the centre of the cover slip. Alternatively, hold the slide more at less at right angles to the bench surface and then tap the long edge of the slide on to absorbent paper lying flat. This action will often move the cover slip to the edge. The slide can be held against the paper and excess mounting medium will bleed onto the paper. Push the cover slip back to the centre of the slide with forceps or a needle. If large air spaces form beneath the cover slip, too much mounting medium has been removed, and the piece of tissue is probably too thick. Specimens from sandy soils often have sand grains in the tissue, which can be larger than the thickness of the tissue being examined, in which case the cover slip will rock on the sand grain. Remove sand grains by dragging them away with forceps.

A full examination of all significant microcharacters includes inspection of tissue from the lamellar edge (for cheilocystidia), a cross-section of a lamella (for pleurocystidia and the orientation and staining of the lamellae trama), a radial cross-section of the pileus (for pileipellis, hypoderm and pileus trama structure) and a peel of the stipe surface (for caulocystidia). In addition, some tissue arrangements can only be confirmed by a cross-section of the stipe (for sarcodimitic tissue) and a longitudinal section of a lamella (for radial lamellar trama). Nevertheless, many diagnostic characters can be seen from just a few simple Squash preparations or Peels and scalps rather than cutting Sections.

Sections must be as thin as possible. It is quite possible to cut thin sections by hand, but some practice is required. The tissue structure of fresh material is often clearer than in rehydrated material, but fresh material can be quite watery and difficult to cut without becoming compressed. With dried material, it is often easier to cut sections first and then rehydrate.

A dissecting microscope can be used to view tissue that is being cut for sections. Use new, sharp razor blades. Double sided blades can be snapped in two. Take care when cutting sections not to cut your fingers!

When preparing a cross-section of the lamella, cut out a piece of a single lamella that includes most of the distance between top (where attached to the pileus) and edge, and is about 1 to 2 cm long. Place the piece flat on a slide. If the lamella is deep (longer than 2 cm from top to edge), cut the piece in half parallel to the edge, and discard the half that does not include the edge. With a razor blade in one hand, place the first finger of your other hand firmly on the piece of the lamella, arching the finger slightly. The lamella piece should be oriented so that your finger is at right angles to the axis of the lamella which runs from top to edge. Place the blade against your finger at right angles to the tip of your finger. Cut straight down with the blade. You will need to experiment to get the right balance between downward pressure and a sideways, sawing motion. Roll your fingertip back and forth slightly to adjust the thickness of the next section that will be cut. If the direction of the cut is not quite parallel to the fresh edge of the previous cut, the section will be thicker at one end than the other, and thus just the right thickness in the middle!

The lamella cross-section will be like a narrow slice of pizza, triangular with a very small angle at the apex. The apex of the triangle is at the lamella edge, with a row of basidia along each of the two long sides. Between the rows of basidia is the lamellar trama, in which the orientation of the hyphae should be clearly visible. If the section is too thick, it is likely to sit so that you look at the hymenium in top view (with the apices of numerous basidia and basidioles visible) rather than the cross-section.

To prepare a radial cross-section of the lamella, cut out a piece of a single lamella as for the cross-section, but make the first cut in the middle parallel to the edge of the lamella, and then take sections from the fresh edge, cutting parallel to the edge.

When sectioning the pileus, a radial cross-section will best show the pileipellis structure and the structures of the underlying hypoderm (if present) and trama. Start with a block of tissue that includes the pileus surface and some underlying context. This block can be removed from a medial longitudinal section of the whole fruit-body, by cutting from the exposed upper pileus context. Alternatively, when the pileus is convex, start with a scalp section. The block or scalp should come from about half way between the edge and the centre of the pileus. Be careful to keep track of the orientation of the scalp or block so that you know which is the upper surface and which is the radial axis. Making the scalp or block rectangular in top view with the longest side parallel to the radial axis can help, as can making it relatively thin. To cut the cross-section, use the same technique as for sectioning lamellae, initially placing your finger on the pileus tissue so that the razor blade cuts along the radial axis.

Orientate the section by looking for spores and particles of debris that indicate the pileus surface (although in a very thick section, which includes the upper part of lamellae, the hymenium, with spores, will be visible on the other side of the section to the surface). The surface layer is the pileipellis and below this is the pileus trama. A differentiated hypoderm can lie between the pileipellis and the trama. If the section is too thick, it is likely to sit so that you look at the top of the pileipellis, and not at the cross-section.

When sectioning the stipe, cut an elongated rectangle from the stipe surface along the long axis of the stipe. This piece can be cut in longitudinal section to observe the presence of caulocystidia. In order to observe the stipe trama for sarcodimitic tissue, prepare a cross-section of the stipe by cutting the stipe in half, and then cutting thin sections at right angles to the long axis (like slicing carrot).

Another technique for sectioning lamellae, pileus or stipe is to immobilise the piece of tissue being sectioned on a slide with clear nail varnish. Let the nail varnish set, and then cut cross-sections.

After sectioning, the thin section will either adhere to the blade or to the slide. If the former, tap the blade gently into a drop of mounting medium on a slide, if the latter, forceps or a fine brush can be used to pick up the section and transfer it to the drop of mounting medium. Use a small drop of mounting medium so that the section stays under the cover slip when that is applied. Sections can easily roll over from the intended view or become twisted. Place the cover slip gently on the section so as not to squash it or tip it over. Slight pressure on the cover slip can assist in removing air bubbles and flattening sections, but care must be taken not to disrupt the original orientation of hyphae until this has been established. To avoid twisted or rolled over sections Young (1998b) suggests placing the section on a slide and covering with a cover slip straight away. The orientation of the section can then be checked under the dissecting microscope before adding mounting medium at the edge of the cover slip.

Whether examining a piece of tissue or a section, once an initial inspection of a slide has been made, gentle pressure can be applied to the cover slip. This spreads out the tissue, which does mean that if a section is being examined, the relative positions and arrangement of elements and hyphae is disrupted, but it becomes easier to observe individual elements (such as basidia and any cystidia) and hyphae (such as in the pileus trama). Pressure can be applied to the cover slip by tapping with the eraser at the blunt end of a pencil. However, once immersion oil has been applied, the best method is to use a needle or the tip of forceps (but not very fine forceps, the tips of which readily break or bend). The slide is inspected, the objective swung out, the cover slip tapped, and the objective swung back. Inspect again to see if the elements and hyphae are sufficiently spread out. Repeat until the elements and hyphae are clearly visible. Mounting medium can be pushed out from under the cover slip and need to be removed between tapping. Some experimentation is required to get the right balance between moving the elements and hyphae apart sufficiently, and pulverising the tissue.

For the pileus and stipe surface, a scalp or peel can yield useful information.

A scalp section can be cut where the surface of the structure is curved, such as in a convex pileus, or a cylindrical stipe. Cut more or less parallel to the surface, using the curvature of the surface to end up with a very thin sliver of tissue that includes the surface and some underlying context. For the pileus, the scalp is very shallowly dome-shaped, thinner around the edges.

A peel is obtained with fine forceps. Push one tip of the forceps just below the surface, then close the forceps and pull gently so that a strip of tissue snaps off to one side of the tips of the forceps, and then peel off on the other side a piece of the surface. If the peel remains connected at the other end to that held by the forceps, use a razor blade to separate the other end from the surface. When placing the peel in mounting medium, take care that the outer surface is uppermost.

There are several simple microscopic preparations that can yield useful information on spores, basidia, cystidia and hyphae, without the need to cut sections.

To examine lamellae structures, break off a small rectangular fragment from the edge of a lamella with fine forceps. Make the fragment a little longer than wide, and make the long axis more or less parallel with the edge of the lamella (so that it is easy to locate the edge under the microscope). The edge will be straighter than the opposite side of the fragment. The fragment only needs to be a couple of millimetres long. Examine intact before applying any pressure. If the specimen is mature, basidia with apical sterigmata will be visible along with spores, some immature and still attached to basidia, and also numerous free spores, which will readily drift off into the mounting medium. If there are cheilocystidia, they will usually be quite apparent along the edge of the lamella. Cheilocystidia differ from basidia and basidioles (immature basidia) in their shape and/or size, and can have thickened walls or coloured contents. There can be a continuous row of cheilocystidia, or they can be mixed with basidia and basidioles. On the face of the lamella, the tops of basidia can be seen, and any pleurocystidia are also visible. A small fragment gouged from higher on the lamella can be observed to confirm the presence of pleurocystidia.

For the pileus surface a peel or scalp will indicate of the presence of pileocystidia and can assist in the interpretation of the pileipellis structure (although a radial cross-section is desirable for this also).

For the stipe surface, the peel is along the long axis of the stipe, and will indicate the presence of caulocystidia.

Spores on lamellae are a mixture of immature and mature. Mature spores can be examined from a spore print, or from stipe or pileus scalps or peels or from the upper surface of an annulus (where present). Spores on the stipe have fallen from the lamellae, those on the pileus drift there through air currents, or are deposited from nearby fruit-bodies (such as in a caespitose cluster).

Hyphae and spores of different genera are differently coloured in some stains and reagents, the most important of which is Melzer's Reagent (containing iodine). When assessing any colour changes in spores or hyphae it is important to compare the colour in water mounts with that caused by the application of reagent. Water can be used to examine fragments of tissue or sections, but hyphae are often hyaline, and easier to visualise when stained, such as by Congo Red, which is a good general-purpose mounting medium.

When using chemicals, always follow instructions about safe use and consult Safety Data Sheets for hazardous chemicals.

Use a relatively weak solution (3-5%) for rehydrating tissue of dried specimens, and as a mounting agent for fresh material. A weak KOH solution can also be used to detect microchemical reactions such as for chrysocystidia or the pileus trama hyphae. A stronger solution (10-15%) is applied to the pileus or stipe surface to induce macrochemical reactions (colour changes).

An essential stain for studying spores of fungi, and also useful for some tissues. There might be no reaction, an amyloid (blue-black) or a dextrinoid (reddish brown) reaction. In addition to the different colour reactions in Melzer's Reagent, spore ornamentation of hyaline spores can be more readily visible due to the optical properties of the reagent.

Make up 1% solution of Congo Red dye in water and add a drop of this solution to material mounted in 3% KOH.

This is a good general purpose stain for studying fungal tissues. The stain is taken up by the walls of hyphae and spores.

A compound microscope is essential for viewing microcharacters. Binocular rather than monocular microscopes are more comfortable to use. Low power objectives are employed to position the specimen being viewed, and these do not need to be of highest quality. A ×100 (oil immersion) objective is ideal, but most structures can be seen with a reasonable quality ×40 objective. However, fine detail of spore ornamentation might not be visible with the ×40 objective. Normal bright field illumination is suitable for viewing all the characters used in FunKey. Phase contrast or differential interference contrast objectives provides extra detail, particularly of spore ornamentation, and they can also make tissue structure easier to visualise.

Advice and experience with suitable microscopes can be sought from field naturalists clubs, which sometimes have microscopical groups, or fungal studies groups. A list of contacts for such groups is provided in the Fungimap Newsletter.

Observe tissue fragments or sections at low power to locate them in the field of view. Once material is centred, higher magnification objectives can be swung into place. With the ×40 objective the overall structure of tissues, such as the orientation of the lamellar trama or the type of pileipellis, can be observed, as can many details of cystidia and spore shape and ornamentation.

If making measurement with the ×40 objective, the eyepiece scale will usually be equivalent to about 2.5 micrometres for each division, so for smaller structures such as spores less than 10 micrometres long, measure at least to half a division of the scale so that the measurements are accurate to about the nearest micrometre.

If using a ×100 objective, observe first with the ×40 objective to orientate the tissue (such as locating the edge of a lamellar fragment, or the surface of a pileipellis cross-section), then swing out the ×40 objective, add a small drop of immersion oil, and swing in the ×100 objective. If the material is focussed with the ×40 objective, it should be in focus with the ×100 objective. If not, use only the fine focus to slightly move the objective up or down until in focus. Be very careful not to drive the objective into the cover slip. If in doubt, look at the objective side-on while focussing, and lower the objective until the smallest of gaps is apparent between cover slip and objective, and then looking through the eyepieces, use the fine focus to raise the objective until the material is in focus.

When using oil immersion, use as little oil as possible. Be very careful not to get oil on the other objectives. Once oil has been added to the cover slip, do not move from the ×100 to the ×40 objective, otherwise oil may get on to the ×40 objective, and then must be removed with lens cleaning fluid.

Correct set up of the lighting and alignment of the microscope is essential. Light is controlled by a basal diaphragm (the field diaphragm), and there is a sub-stage condenser which has its own diaphragm. The sub-stage condenser can be lowered or raised and also moved from side to side. Manipulation of the position of the sub-stage condenser and the amount of closure of the diaphragms makes a big difference to the clarity of the field of view. Koehler illumination is a method for set up of the microscope according to the following steps: (1) turn on light source, (2) close down the field diaphragm, (3) move the sub-stage condenser up or down so that the edge of the diaphragm is in sharp focus. At this point, the sub-stage condenser may need to be centred (there are two adjustment screws) so that the circle of light is in the centre of the field of view, (4) open the field diaphragm so that it is just outside of the field of view, (5) adjust the sub-stage diaphragm so that when the field of view is observed by taking out one of the eyepieces, the circle of light is about two-thirds to three-quarters of the area of the field of view. Follow these steps for optimum set up; however, moving the condenser or the sub-stage diaphragm from these optimal positions can sometimes enhance details, so experiment! The intensity of the light source should also be adjusted to provide sufficient illumination that does not strain the eyes.