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.6.Microscopy, DEFTPettipher G.J., Mansell R., McKinnon C.H.& Cousins, CM.(1980) Rapid membrane filtrationepifluorescent technique for direct inumeration of bacteria in raw milk.Appl Environ Microbiol,39,423-429.Denyer S.P.& Ward K.H.(1983) A rapid method for the detection of bacterial contaminants inintravenous fluids using membrane filtration and epifluorescent microscopy.J Parental Sci Technol,37, 156-158.Flow cytometryShapiro H.M.(1990) Flow cytometry in laboratory microbiology: new directions.Am Soc MicrobiolNews, 56, 584-586.MicrocalorimetryBeezer A.E.(1980) Biological Microcalorimetry.London: Academic Press.ImpedanceSilley P.& Forsythe S.(1996) Impedance microbiology a rapid change for microbiologists.J Bacterial80, 233-243.BioluminescenceStanley P.E., McCarthy B.J.& Smither R.(eds) (1989) ATP Luminescence: Rapid Methods inMicrobiology.Society of Applied Bacteriology Technical Series No.26.Oxford: Blackwell ScientificPublications.Stewart G.S.A.B., Loessner M.J.& Scherer S.(1996) The bacterial lux gene bioluminescent biosensorrevisited.Am Soc Microbiol News, 62, 297-301.General referenceStannard C.J., Petit S.B.& Skinner F.A.(1989) Rapid Microbiological Methods for Foods, Beveragesand Pharmaceuticals.Society of Applied Bacteriology Technical Series No.25.Oxford: BlackwellScientific Publications. Yeasts and moulds21 Introduction 4 Cryptococcus neoformans2 Saccharomyces cerevisiae 5 Neurospora crassa2.1 The life cycle2.2 Metabolism and physiology 6 Penicillium and Aspergillus2.3 Cell wall7 Epidermophyton, Microsporum and3 Candida albicans Trichophyton3.1 Pharmaceutical and clinical significance3.2 Alternative morphologies 8 ReferencesIntroductionYeasts and moulds are members of the fungi.Yeasts are characterized as being essentiallyunicellular, whereas moulds are composed of filaments which en masse frequentlyappear fuzzy or powdery.The familar budding yeast Saccharomyces cerevisiae, alsoknown as Baker's or Brewer's yeast, is usually thought of as the typical yeast.Thegreen mould Penicillium digitatum, a frequent spoiler of fruits such as apples or oranges,and the bread mould Neurospora crassa will also be well-known to many.These lattertwo organisms are properly considered as typical moulds.As is usually the case, however,life is not completely straightforward for there are a considerable number of so-called'dimorphic fungi' which can alternate between yeast-like and filamentous forms.Onesuch organisms is Candida albicans.To make matters more complicated, it has beenrediscovered that the would-be typical yeast S.cerevisiae can also form filamentsunder a variety of different conditions (Gimeno et al.1992).All of these fungi havepharmaceutical and medical significance.The precise nature of this significance isdifferent in each case.For example, S.cerevisiae is generally regarded as a totally safeorganism suitable for use in human food and drink; the reason for its importance isbecause it is by far the best understood eukaryotic organism on the planet.In contrast,Cryptococcus neoformans has a variety of ways by which it can evade defencemechanisms of the immune system, but is relatively little studied.In between thesetwo extremes are many yeasts and moulds, which are omnipresent in the environment,in or on our foods, or a part of the normal flora of humans, but all of which canopportunistically contaminate pharmaceutical preparations or cause post-operativedisease.All fungi pose a threat to immunocompromised individuals.This knowledgeshould be weighed against a background of a general lack of suitable antifungal agents(see Chapter 5).The approach of this chapter will be to first describe S.cerevisiae inconsiderable detail because so much is known about it.Then, other yeasts and mouldswill be considered in turn, pointing out (where appropriate) significant differencesfrom S.cerevisiae or from each other.Yeasts and moulds 35 Saccharomyces cerevisiaeSaccharomyces cerevisiae has a predominant place in the realms of cell biology andmolecular biology where it has become accepted as the universal model eukaryote.The main reason for this is its genetic tractability.Traditionally, for reasons associatedwith its importance to the food and drink industry, a great deal was known about thebiochemistry and physiology of this yeast.Later, with the advent of yeast genetics, avast range of well-characterized mutants became available.In turn, because S.cerevisiaecan be transformed and is readily amenable to genetic manipulation, this permittedthe isolation and characterization of many yeast genes.Ultimately, in mid 1996the nucleotide sequence of the entire genome of the organism was reported.Thisachievement is still only a far-off dream for molecular biologists studying most othereukaryotic organisms.Nevertheless, it is possible to identify genes from other organismsby means of genetic complementation in S.cerevisiae.Explained briefly, only onepiece of DNA from another organism will be able to substitute for a mutation in aknown gene in S.cerevisiae this is a segment of DNA which carries the homologousgene (i.e.codes for the same function) in the other organism.The availability of well-defined mutants in S.cerevisiae combined with the facility of genetic manipulationand this yeast's short generation time, make this a very rapid way to identify heterologous(i.e.belonging to another organism) genes.Many of the latest concepts in cell andmolecular biology (e.g.concerning control of the cell cycle) have been developed andtested in this organism.Naturally then, since it is the prime model eukaryote, it is alsothe best understood fungus.The life cycleThe life cycle of S.cerevisiae is shown in Fig.2.1.It can exist both as a haploid (onecopy of each chromosome per cell) or as a diploid (two copies of each chromosome percell).Haploids exists as one of two sexes referred to as mating type a and mating typea.When two haploid cells come close together they cause each other to arrest in theGl phase of the cell cycle.Each subsequently produces a special protuberance enablinggrowth towards the mating partner.These somewhat abnormal looking cells are termed'schmoos'.A haploid will only mate with another haploid of the opposite mating type.This is achieved by the expression of specific oligopeptide mating pheromones(hormones with brings about behavioual change in cells of the opposite sex) and thepossession of surface receptors only for the opposite pheromone (hence, mating type astrains produce only a-factor and have receptors for a-factor, whilst mating type astrains produce only cu-factor and have receptors for a-factor).The resulting diploid,like the haploids from which it arose, is capable of repeated rounds of vegetativereproduction.The vegetative cell cycle of S.cerevisiae has received extensive attention.Thereare many justifications for this.Firstly, the cell cycle in this organism has manyconvenient 'landmarks' (Hartwell 1974, 1978; Pringle 1978) which make it very easyto identify the exact point in the cell cycle at which a cell happens to be.Examples ofthese landmark events include bud emergence, the size of the bud, mitosis (nucleardivision takes place through the neck between the 'mother' cell and the bud), and cell Fig.2.1 The life cycle of Saccharomyces cerevisiae.separation.Other markers of cell cycle progress are also apparent to the more experiencedobserver (Fig.2.2).The reader will notice from Fig.2.2 that the 'daughter' which isformed is smaller than the mother cell from which it arose
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