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The general objective of these laboratory exercises is for students to experience the investigative process, to acquire specific skills, and be ready to demonstrate these by presenting their findings to the class. Every student must maintain good records in a permanent lab book (please do not use a ring binder). Marks (10%) will be assigned based on the accuracy, clarity, and originality of presentations as recorded in the lab book. A feature of some labs will be a quiz based on material from previous labs – such quizzes + general participation (attendance) will amount to 10% of the final grade.

Lab. # 1. Introduction to Eukaryotic Microbiology

Lab # 2. Chromistan fungi, Chytrids and Zygomycetes.

Lab # 3. Ascomycetes

Lab # 4. Basidiomycetes

Lab # 5 Fungal Identification

Lab # 6. Genetics ( Computer based)

Lab # 7. Nematode destroying Fungi.

The study of microorganisms requires the use of certain special techniques aimed at seeing, culturing, enumerating, or sampling microbial species in different environments. The most frequently used techniques vary depending on the type of organisms. In this first exercise, you will acquire expertise in the use of the microscope to see microorganisms. You will learn to use the dilution plate count technique to enumerate yeast, the streak-plate technique for the isolation of yeasts, and the spot inoculation technique to isolate and purify moulds.

One of the fundamental skills that you, as a microbiologist, must master is the ability to use a compound microscope with accuracy and ease. You have had some experience using a microscope in Bio 022 / 023, Bio 290a and perhaps other courses. Below follows a quick review of good technique.

A diagram of the major parts of the microscope will be discussed. The compound microscope has two sets of lenses: the ocular (eyepiece) and the objectives (stage lenses). The oculars are standard at 10X magnification. The objectives are mounted in a revolving nosepiece and consist of 4, 10, 40 and 100 power lenses. The three lower magnification lenses are "dry" lenses, but oil must be used (only) with the 100X objective. Oil is used to increase the numerical aperture of the lens. The numerical aperture describes the light gathering ability of a lens. Lenses with high magnification or power require considerable light and the immersion oil is used to direct the refracted light path into the objective. Most of your microscopes are parfocal, meaning that it is not necessary to make major focusing adjustments when switching from one objective to another.

Adjustment of the light will be important for all lens combinations. In general, use a sufficient light to give the specimen good clear contrast but not enough to be dazzling, causing possible eye strain. Most importantly, learn and practice the use of every appropriate control knob on your microscope. A good microscopist constantly adjusts the fine focus as observations are made.

Handle all microscopes with extreme care! They are delicate optical instruments subject to permanent damage if they are pounded around. Carry your scope with both hands and deposit it gently on the bench. Place the microscope on the edge of the bench so that your can use it comfortably. Our Olympus microscopes have rheostats to control the brightness of the light itself.

General Procedure.

1. Put a slide with specimen on the stage, secured under the clips. With microscope light on, use the low power objective and bring the specimen into focus.
2. Focus on an object of your choice, and adjust the focus with the fine control. Remember, a good microscopist constantly adjusts the fine focus as observations are made.
3. Switch the objective to higher power ones in sequence, making sure that the objective does not touch the specimen and readjusting the focus as needed each time.
4. Be very careful focusing when you are using the higher power objectives (x40, x100) as these lens are very close to the slide and it is easy to ‘crash’ the objective into the slide causing damage to the lens.
5. Before changing the specimen, switch back to low power objective and start again.

 

2. Calibration of the microscope

To calibrate the diameter of the field of view for each of the 4 objectives, use the following procedure.

1. Select the 10X objective lens and place on the stage a stage micrometer. The stage micrometer is an etched 1 mm line divided into 100 parts. Each division corresponds to 10 µm. Adjust the slide such that it traverses the broadest part of your view and determine the number of divisions required to cross the field.

2. Repeat this procedure for each objective.

3. Record the size of the field of view for each magnification. This table will be very useful for many exercises during this term.

3. Preparation of a wet mount

EXERCISE 1. Basic microscopy and observation/drawing skills.

Using the fungal material provided, examine under the microscope and draw what you see.

1. Take a clean microscope slide.

2. Place a drop of water, or other liquid such as lactophenol, on a slide and suspend some material likely to contain microorganisms.

3. Hold a coverslip at a 45 degree angle to your slide. Lower the coverslip gently onto the drop. This reduces the capture of air bubbles.

4. Transfer the slide to the microscope stage and observe under low power to scan the preparation. You may find that organisms of interest are too small and further magnification is needed, in which case you should increase the magnification. At each step try to estimate the size of some cells.

5. Watch for special features within the cells.

6. Record your observations in the form of a careful drawing.

1. Important: the labels must refer to morphology. An optical microscope normally does not allow you to identify the chemical composition of cellular material or the ploidy level of a cell. It is therefore not correct to label something "cellulose wall", or "haploid nucleus" unless you can justify this on the basis of your observations.

Because of the small size of microorganisms, much microbiological work starts with preparing cultures, so that a significant amount of material is available for subsequent manipulations. Before you proceed, you must first get acquainted with some fundamentals on the aseptic handling of microorganisms.

WORK IN PAIRS.

The success of yeast as a major object of study in genetics, biochemistry, and molecular biology is owed to a large part to the ease with which yeast can be manipulated in the laboratory. Essentially, most culture techniques that apply to heterotrophic bacteria (eg. E. coli) can also be used for yeast. To illustrate that point, we shall use the dilution plate count technique to estimate the number of viable cells in a grain of active dry yeast. The principle of this method is that under certain conditions a small number of viable unicellular microorganisms spread onto the surface of a Petri plate will grow into an equivalent number of colonies or clones.

1. Plate 0.10 mL of suspension from each tube to the appropriate plate, starting from tube 6 upward.
2. Spread the plated suspensions evenly across the surface of the plates using an alcohol-sterilized bent glass rod (hockey stick). Again, you may use the same rod provided you proceed upward, from tube 6 to tube 1.

A plate containing a suitable number of colonies (30-300) must be counted, and the number of colonies multiplied by the appropriate dilution factor to obtain an estimate of the number of colony-forming-units (CFU) per mL of the original suspension. In the present case, this will be converted to estimate the number of viable CFUs per gram of active dry yeast. The reasons for counting only the plate with more than 30 but less than 300 colonies are important. They have to do with sampling error and possible confluent growth. In reporting your results, avoid excessive significant digits and use exponential notation.

Lab # 2. Chromistan fungi, Chytrids and Zygomycetes.

The array of living forms generally discussed under the heading "Mycology" is changing gradually. Formerly, any organism that did not clearly fit the definition of an alga, a protozoan, a bacterium, a plant, or an animal was considered a fungus. This included the Actinomycetes, which are in fact filamentous bacteria and, until recently, the slime protists, which are separate organisms related to the protozoa. Recent studies of rRNA have helped separate species which have traditionally been considered fungi into 2 Kingdoms.

We shall follow the broad classification used in the recommended text by Kendrick (Fig.1). This views fungi and fungal-like organisms as being divided amongst 2 kingdoms:-

1. Kingdom Chromista - containing some 'fungus-like' organisms:-

i) several kinds of algae including the giant brown kelps

ii) non-fungal groups such as slime moulds

iii) motile, zoospore forming fungus-like organisms in the 2 main phyla:-

Hyphochytriomycota and Oomycota.:

2. Kingdom Eumycota – True Fungi. Mostly lacking motile cells and divided into 4 phyla:-

1. Chytridiomycota, exception – do have motile cells
2. Zygomycota - mostly coenocytic cells i.e no septae.

 

iii) Glomeromycota - specialized fungi forming mycorrhizae -recently split off from the Zygomycota - usually no sexual stages.

3. Dikaryomycota- divided by septae into cells with subphyla:-

Ascomycotina - sexual spores inside an ascus

Basidiomycotina - sexual spores borne on outside of a basidium

Saprolegnia propagates by virtue of zoospores produced in the tip of specialized hyphae, the zoosporangia. Primary zoospores encyst and the cysts germinate into secondary zoospores. A zoospore that succeeds in finding food (dead seeds) gives rise to a new mycelium.

Saprolegnia may be monoecious (having seperate male and female structures but borne on same individual) or dioecious (having seperate male and female structures borne on different individuals). In monoecious forms, some hyphal branches undergo meiosis as their develop into antheridia (male gametangia), whereas other branches differentiate, also with meiosis, into oogonia (females).

A. Phylum Chytridiomycetes, fungi which form zoospores with one posterior whiplash flagellum

Several groups can be recognized within the Chytridiomycetes, from very simple to very complex. Our study will focus on a well-studied example, Allomyces.

Allomyces macrogynus is a free-living, eucarpic fungus in which the alternation of the haploid and the diploid phases is so distinct. Four different spore-bearing structures may be observed. The gametothallus bears, in pairs, orange-coloured gametangia (male), and colourless female gametangia. The smaller male gametes, responsible for the orange colour, may swim and fuse with female gametes, which are larger and slower swimmers. The resulting zygotes lose their flagella (which causes them to sink!), and germinate into sporothalli with basal rhizoids.

Spores of Allomyces are produced on other individuals (the sporothalli). Pale, thin walled zoosporangia (mitosporangia) produce large, clear zoospores (mitospores), and darker meiosporangia are usually observed in a state of dormancy.

B. Phylum Zygomycotina, coenocytic fungi with asexual sporangia and sexual zygospores

Zygomycetes, like many Chromistan fungi are coenocytic (multinucleate - hyphae not divided into cells by partitions or septae). Most species form a mycelium, but some can differentiate into unicellular (yeast) forms. Asexual reproduction is by formation of sporangia, which may or may not contain separate spores. Sexuality is usually the result of gametangial fusion resulting in the formation of a persistent, multinucleate structure called zygospore (or zygosporangium).

This order contains the most common Zygomycetes. It includes many moulds which participate in the decay of plant materials in nature. Members of the Mucorales have much industrial importance, being used for the production of fumaric, lactic, citric, succinic, and oxalic acids, and cortisone. A few species are parasitic. Some saprophytic species are common food and laboratory contaminants, or are used directly in the production of certain fermented soybean foods such as Tempeh. Mucorales produce extensive mycelium, and some grow unicellularly as yeast cells. Some make complex specialized growth structures.

The common bread mould, Rhizopus stolonifer (R. nigricans), forms black sporangiospores able to germinate into highly invasive hyphae. The hyphae rapidly invade their substrate. Some hyphae differentiate into aerial hyphae and clinging rhizoids. Others thicken and darken to become sporangiophores. The sporangia are dark and spherical.

1. Mount a closed plate upright on your microscope stage and examine that various forms of growth in Rhizopus directly through the lid of the plate. Record the results. Do not attempt to find anything of significance deeper in the plate. Most structures of interest are stuck to the lid itself.

Phycomyces blakesleeanus is remarkable in many ways. Its sporangiophores can reach enormous proportions and exhibit a phototactic response. Blakeslee, who investigated sexual compatibility in this fungus nearly 90 years ago, discovered an interesting pattern known as heterothallism.

1. Sub-phylum : Ascomycotina, fungi with a restricted dikaryophase and ascospores borne inside asci.

Ascomycotina grow vegetatively as septate mycelium or single-celled forms, or yeasts. Hyphal septa may be incomplete, with the result that some compartments may contain indeterminate numbers of nuclei. Yeast forms are uninucleate. Asexual spores are conidia (externally delineated spores). Sexual reproduction is by formation of intracellular meiotic spores in an ascus. Some species occur only asexually and are classified in the form-subdivision Deuteromycotina.

Most ascomycetes other than the yeasts form asci in containers (ascocarps) developed from the ascoma.. Of these,

- the cleistothecium is spheroidal with no opening,

- the perithecium and the pseudothecium are pear-shaped with an apical opening called an ostiole, and

- the apothecium is open like a cup or a dish.

Perithecia are either free or imbedded in the cortex of a stroma (fruit body), and apothecia occasionally close upon themselves to form a spheroidal hypogeous (underground) fruit body.

A cleistothecium has no opening so spore release depends on the cleistothecial wall deliquescing at maturity.

Eurotium chevalieri is the teleomorph or sexual state of the anamorph or asexual state of the mould Aspergillus amstelodamii. Hyphae give rise to club-shaped conidiophores, at the end of which flask-shaped cells, the phialides, produce chains of conidia. A typical foot cell is found at the base. Asci are formed inside cleistothecia. Each ascus generally contains 6 to 8 ornate ascospores. Conidia of various Aspergillus species are often intensely pigmented in green, yellow, black, purple, or other colours. Aspergilli are frequent contaminants, the agents of various mildews, or the cause of food spoilage. Some species produce potent mycotoxins. Much of the citric acid used in soft drinks is produced from Aspergillus fermentations.

1. Scrape some green material from an appropriate plate, using a stiff loop. The yellow areas contain only cleistothecia, whereas the green areas contain both cleistothecia and sporangiophores.
2. Mount some of this material in a drop of lactofuchsin, and lightly press a coverslip on top.
3. At low power, locate conidiophores and cleistothecia. Compare their size and appearance. Record your observations in sketches

Some penicillia have a known sexual state, but they are most frequently isolated as anamorphs. The vegetative growth is similar to that of Aspergillus, but the conidiophores are penicillate, or paintbrush-shaped, and lack a foot cell. Conidium pigmentation is usually blue. Penicillium species are important in the ripening of cheeses such as Camembert or Roquefort and their numerous relatives (Brie, St-André, Blue, Stilton, Gorgonzola, etc.). The first antibiotic, penicllin, is extracted from Penicillium . Many other fungal antibiotics, produced by the fungi to fight off bacterial attack, have been isolated and are now in use..

1. Using the prepared slides provided, examine/draw phialides and conidia at high power

Sordaria was used in the Biology 022/023 to demonstrate first and second division segregation at meiosis. A related fungus, Neurospora played a major role in the development of modern genetics, partly because of the ease with which the ascospores of this type of organism can be dissected and crossed. A film on the life cycle of Sordaria will be presented.

Some fungi group their ascocarps in clusters in a body called a stroma

Xylaria polymorpha (dead man's fingers), Dibotryon morbosum (black knot), and Claviceps purpurea (Ergot fungus) have completely different life histories, but share the common characteristics of being plant pathogens and of forming their asci at the base of perithecia grouped together just under the surface of a stroma or fruit body.

Fungi with compound perithecia in stroma.

Apothecia are open cup-shaped ascocarps. They may be produced individually or grouped together on a complex fruit body. View the Demo material noting particularly:-

1. Peziza
2. Morchella

Morchella species are among the most prized mushrooms for gastronomists. Dried and fresh morels are sold to fine restaurants for considerable amounts of money. Attempts to cultivate fruit bodies of morels have generally been unsuccessful up until very recently. Examine preserved fruit bodies of Morchella. Where are the apothecia located?

Tuber, the truffle, is a hypogeous fungus i.e. it forms its fruit bodies underground.. Like the morel, it establishes mycorrhizal relationships with certain trees. Good truffles, which sell for unimaginable prices, are used as seasonings in paté or other delicacies.

1. Examine apothecia of Tuber. From prepared slides sketch and record the location of the hymenium ( the fertile surface)

Over 500 of the nearly 800 known yeast species are Ascomycetes – often grouped together (Hemiascomycetes). Widely distributed wherever plants and animals interact, most yeasts serve as food enrichment for insects that feed on plant parts that are otherwise nutritionally poor. Some species, for example Candida albicans, are associated with mammals, including humans, causing various infections of the skin, or the respiratory or genito-urinary tract.

V. Lichens

Lichens are fungi (mostly Ascomycetes, occasionally Basidiomycetes) that have formed an associations with either green algae (Chrysophyceae) or occasionally blue-green algae (Cyanobacteria) (or sometimes with both types of algae). The Lichens are 95% fungal in biomass and are named from their fungal component. Over 40% of Ascomycetes no longer exist by themselves, only in their lichenized form.

Most lichens therefore produce typical Ascomycete sexual structures or ascoma ( e.g apothecia) which generate typical asci and ascospores. No mechanism has developed to include the algal component in these sexual spores so whenever an ascospore germinates, its only chance of long term survival is to find exactly the right kind of alga to associate with. – something that is made even more difficult by the fact that forming the association only seems to occur when the fungus is conditioned to do so by starvation ( at least this is the experience in lab attempts to create the association). Thus it is likely in most circumstances that lichen reproduction is primarily by the release of asexual structures- either simple fragmentation of the lichen, or release of small balls of cells (soredia) or projecting pegs ( isedia) – all of which involve both algal and fungal components.

1. Examine the lichen material provided and find any sexual structures.
2. Draw the lichen(s) showing the location and appearance of these sexual structures
3. Make a thin section of the lichen with a razor blade and examine it under the microscope. Draw and label the section, making clear the location of any algal cells, the details of the sexual structure ( is it an apothecium, cleistothecium or perithecium?) etc.

2. Sub-phylum : Basidiomycotina, fungi with an extensive dikaryophase and basidiospores borne externally on basidia.

Basidiomycetes are best known for the great diversity of their fruit bodies. Basidiomycetes differ from ascomycetes in 3 important ways. First, their septa are complete, because the central pore, called a dolipore, is covered by a membranous network that controls cytoplasmic flow. Second, the dikaryotic phase, which is transitory at best in the Ascomycotina, represents a major part of the life cycle in Basidiomycotina. Third, the sexual (meiotic) spores of basidiomycetes are delimited externally on a cell called a basidium. Two major classes are:-.

1. Holobasidiomycetes incl. mushrooms and toadstools. These form basidia (not divided into compartments) directly from dikaryotic cells. The basidia may be formed (1) freely, (2) in an exposed layer, (hymenium) or (3) internally, in a gleba.

2. Teliomycetes, which includes the rusts, the smuts, and a few yeasts, produce no fruiting boidies and are characterized by the formation of dormant teliospores which give rise, by meiosis, to basidium-like structures that are subdivided into 4 compartments.

1. Class : Holobabasidiomycetes, fungi which form single-celled basidia)

1. The Hymenomycetes which actively ‘shoot’ their basidiospores,
2. The Gastromycetes which don’t shoot out the spores.

Agaricales are a major group of Basidiomycetes, which produce the familiar ‘mushroom’ type fruiting body which on its undersurface is divided into many radial gills. A film describing the complete life cycle of Flammulina velutipes will be presented. The concepts of dikaryotic mycelium, clamp connection, basidium, and basidiospore will be introduced. Here the hymenium is at the surface of gills held up in a cap or pileus. Other important examples are shown as preserved specimens in the laboratory.

1. Examine specimens of Agaricus, Armillaria, Amanita, and Coprinus.
2. Observe and note important morphological features important in recognizing these fungi, namely the ring or annulus, the cup or volva, and the phenomenon of gill deliquescence.
3. Why is it important to be able to recognize Amanita?
4. Visit the departmental display cabinet (end of corridor between rooms 234 and 235) featuring Armillaria and study/make notes
5. What are the major types of ‘wood-rotters’ and in what way do they differ in their ability to produce enzymes ?
6. Why are fungi important in forests ?

The Agaricales as we just saw produce their basidia on hymenial surfaces borne on radial gills. However, other basidiomycetes exhibit an great variety of ways in which they form a hymenium. A number of preserved specimens are used to illustrate this. The basidia may be formed on surface folds, smooth surfaces, inside cylinders, on the surface of teeth, or in pores.

1. Learn to recognize chanterelles (Cantharellus), coral fungi (Clavaria) and bracket fungi (Polyporus, Ganoderma).
2. Compare the location and arrangement of the hymenium in these fungi.
3. How do these factors affect the dispersal of spores in these fungi?

Some basidiomycetous macrofungi do not form a hymenium, but instead, produce amorphous masses of spores, the gleba. In this case, spore dispersal is highly dependent on the intervention of animals.

1. Examine and learn to recognize stinkhorns (Phallus, Mutinus) and puff-balls (Lycoperdon, Calvatia).
2. Compare the location and arrangement of the gleba in these fungi.
3. How do these factors affect the dispersal of spores in these fungi?

This group of fungi encompasses many plant pathogens termed rusts and smuts, as well as saprophytic yeasts with similar life cycles. One of the more famous of these is Ustilago maydis, better known here as corn smut. Although corn smut is considered a disease to most corn growers, it is treated as a delicacy in Mexico. Huitlacoche, as it is known, is used as a tasty food component in a way similar to truffles. It is delicious. The characteristic of this class is the formation of a teliospore from the fusion of the two nuclei in a dikaryotic mycelial cell. After a period of dormancy, the teliospore germinates meiotically into a tetranucleate germ tube (promycelium) which produces haploid basidiospores by repeated budding.

There are 2 major orders: i) Uredinales (Rusts); ii) Ustilaginales (Smuts)

1. UREDINALES (Rusts).
   Rust fungi are among the most important crop pathogens causing millions of $$ damage each year. They often have complex life cycles (macrocyclic) involving up to 5 different kinds of spores. Some species are hetereocious meaning that they alternate between two quite different kinds of host plant .eg Puccinia graminis is an extremely important pathogen of wheat , barley etc causing black stem rust. However, in order to complete its life cycle it must at a certain stage attack the Barberry plant (which is not important economically as a crop). Other species are autoecious meaning they don’t alternate between species and/or produce fewer than 5 spore types (microcyclic)
2. RUSTS – Demonstration material

2. USTILAGINALES (Smut Fungi).

Smut Fungi get their name from the very dark teliospores that they form in big masses – looking often like soot. Unlike rust fungi, smut fungi can

1. be grown in artificial culture (basidiospores behave like yeasts and can bud repeatedly forming sporidia – usually haploid, but diploid strains can be created)
2. are never heteroecious
3. have no specialized male and female cells – sexual fusion occurs between identical basidiospores or sporidia (remember that rusts produce spermatia (‘male’) and receptive hyphae (‘female’)

Smut Fungi usually develop systemically in the host plant (i.e. spread throughout the plant rather than restricted to a local spot and often only form their diploid overwintering teliospores in the flowers (stamens, ovaries) of the plant.

1. The Smut fungus – Microbotryum violaceum (Anther smut of Carnations) – previously known as Ustilago violacea.

This fungus has interesting effects on the host including causing a sex change !

The life cycle (see Front cover of lab manual) will be discussed in class.

1. Each bench has been provided with a Petri plate on which spores of M. violaceum have been allowed to germinate. Cut out a small square of agar, place it on a slide and overlay with a coverslip. Examine at 40 x and look for germinated spores. Draw the basidium (sometimes called promycelium) and basidiospores budding off it . See if you can see that the basidiospores keep budding in a yeast-like way to produce sporidia.

SMUTS – Demonstration material Examine, make notes on and draw the specimens on the demonstration bench

  Gill structure.

1. Fungus Foray - bring in your own fungal samples (mushrooms, bracket fungi, moulds etc).

http://www.botany.utoronto.ca/ResearchLabs/MallochLab/Malloch/Moulds/Contents.html

Preparation of slides

An interesting technique used by some mycologists for preparing microscope mounts involves sticking the mould to a bit of cellophane tape. The tape is pressed lightly against the colony so that some of the hyphae and spores stick to it. This is then placed sticky side up in a drop of mounting fluid (see below) on a microscope slide and covered with a cover slip. This technique is not restricted to colonies in Petri dishes; it works well on naturally occurring colonies in most habitats. The cellophane tape technique is commonly used for sampling moulds in indoor environments.

Slide cultures

Mounting media

1.Water + wetting agent

2.KOH + phloxine

3.Lacto-fuchsin

4. Melzer's Solution

5. Shear's mounting medium

IDENTIFICATION OF MOULDS

Use of dichotomous keys

Keys to sixty common genera of moulds

Two approaches are taken to identification in this section: a set of dichotomous keys and a set of picture keys. Which you choose depends upon your individual preference. Some people are verbal in nature and do best when everything is written out; these people usually prefer dichotomous or other types of textual keys. Others are visual and prefer to match what they see to an image. You may even find you do best with a combination of the two.

KEYS TO SOME COMMON GENERA OF MOULDS

GROUP I

1. Spores 1-celled 2

1. Spores with more than one cell 12

2. (1) Colonies, spores, and other tissues colourless or brightly coloured 3

2. Colonies, spores, and/or other tissues dark coloured 8

3. (2) Spores produced in chains 4

Often chains of spores break apart thoroughly when placed in a water mount. In many species of Aspergillus and Penicillium a few spores will remain together in a group, so that you can assume that they were originally in chains. On the other hand, species of Cladosporium produce chains of spores that completely disassociate on contact and leave no clue about their original orientation. The easiest way to check this is to examine the colony under the 10X objective of a compound microscope, being careful not to get spores on the lens.

3. Spores not produced in chains 6

4. (3) Conidiophores with a swollen head or vesicle bearing bottle-shaped phialides

Aspergillus

4. Conidiophores not swollen at apex 5

5. (4) Spores in unbranched chains, borne from clusters of cylindrical to bottle-shaped phialides; colonies usually green

Penicillium

Compare with Paecilomyces (group II), Gliocladium (group III), and Scopulariopsis (group III).

Chrysonilia

6. (3) Spores borne in a sporangium with a columella; often with only the columella evident as a swollen hyphal tip; hyphae not septate

Mucor

Compare Rhizopus (group I), Mortierella (group II), Absidia, Circinella (group V), and Zygorhynchus

6. Spores produced externally; hyphae septate 7

7. (6) Conidiophores well-developed and usually with a central axis; very fast growing and with conidiophores usually produced in small cushions of hyphae; often green

Trichoderma

Compare with Verticillium (group II) and Gliocladium (group III)

Acremonium

Compare with Verticillium (group II), Sporothrix (group IV), and Phialophora (group IV)

8. (2) Spores in chains, produced externally 9

8. Spores not in chains, produced inside sporangia or fruiting bodies (pycnidia) 10

9. (8) Conidiophores with a swollen head or vesicle bearing bottle-shaped phialides; conidial chains unbranched

Aspergillus

Cladosporium

10. (8) Spores produced inside a fruiting body (pycnidium) with a cellular wall; hyphae septate

Phoma

Compare Pyrenochaeta (group IV) and Microsphaeropsis (group IV) and also be sure that asci are not present at a very early stage

10.Spores produced within a sporangium with a columella, often with only the columella evident as a swollen hyphal tip; hyphae not septate 11

11. (10) Sporangiophores with rhizoids (branched "roots") at base

Rhizopus

Compare with Absidia (figure 2B)

Mucor

Compare with Mortierella (group II), Absidia (figure 2B), Circinella (group V), and Zygorhynchus (figure 2C)

12. (1) Spores with transverse septa only 13

12. Spores with both transverse and vertical septa 14

13. (12) Spores dark, produced in branched chains

Cladosporium

Fusarium

Compare Cylindrocarpon (not treated here), Candelabrella, Monacrosporium (all group III), and Trichophyton (group V)

14. (12) Spores usually in chains, usually club-shaped; colonies grey to brown

Alternaria

Compare Ulocladium and Stemphylium (group II)

Epicoccum

Compare with Stemphylium (group II)

KEYS TO SOME COMMON GENERA OF MOULDS

GROUP II

1. Colonies composed of hyphae, or at least with some hyphae present 2

1. Colonies lacking hyphae; short chains of "budding" cells may be produced 16

2. (1) Spores 1-celled 3

2. Spores with more than one cell 14

3. (2) Spores and hyphae colourless or brightly coloured 4

3. Spores and/or hyphae dark coloured 10

4. (3) Spores produced in chains 5

4. Spores not produced in chains 7

5. (4) Spores produced from small clusters of tapering phialides, often rather pointed at the ends

Paecilomyces

Compare with Penicillium (group I) and Verticillium (group II)

5. Spores produced by the simple fragmentation of hyphal segments into individual cells 6

6. (5) Colonies very slow growing (slower than 5 mm/week), often grey, often with a strong earthy odour; hyphae usually less than 1 µ in diameter

Streptomyces

Geotrichum

Compare with Geomyces (group IV)

7. (4) Spores produced in sporangia, with sporangia often broken and represented only by simple blunt sporangiophores (no swollen columella); colonies often velvety in texture and pink to brown

Mortierella

Compare with Mucor (group I) and Absidia (figure 2B)

7. Spores produced externally 8

8. (7) Spores produced in large numbers and completely covering the surface of large terminal cells; cells of conidiophores often flattening in alternating planes as they dry; colonies often producing black stony sclerotia

Botrytis

8. Spores produced at the tips of terminal cells and never covering them; cells of the conidiophore not flattening characteristically upon drying 9

9. (8) Conidia produced in small round masses at the tips of phialides; phialides in whorls, tapering gradually to a very narrow tip

Verticillium

Compare with Acremonium (group I)

Chrysosporium

Sepedonium and Trichophyton (both group V) and Geomyces (group IV) are similar

10. (3) Spores produced in sporangia or in fruiting bodies 11

10. Spores produced externally 12

11. (10) Spores produced within densely hairy fruiting bodies (perithecia), very dark; asci present when young

Chaetomium

11. Spores produced in sporangia (Go back to 7)

12. (10) Conidiophores united to form large synnemata that have a sterile base and a spore-bearing upper part, often accompanied by spores of Echinobotryum (group V)

Doratomyces

Compare with Trichurus and Graphium (both group III)

12. Conidiophores never united to form such structures 13

13. (12) Spores arising in dense masses directly from swellings on the vegetative mycelium; colonies usually rather flat and moist

Aureobasidium

Botrytis

14. (2) Spores with transverse walls only, colourless; colonies often pink; often associated with eelworms

Arthrobotrys

Compare with Trichothecium (group V), Candelabrella and Geniculifera (both group III)

14. Spores with transverse and vertical walls, dark brown 15

15. (14) Conidiophores more or less straight because of their elongation directly through the scar of the previous spore, bearing only one spore at a time

Stemphylium

Compare with Pithomyces (group IV)

Ulocladium

Compare with Pithomyces (group IV) and Curvularia (not treated here)

16. (1) Cells very small, seldom more than 1-2 µ in diameter, dividing by simple fission into two equal-sized daughter cells, sometimes containing a single internal spore

Bacteria

Yeasts

Compare Aureobasidium (group II), Candida (group III), and Exophiala