Editor: Dr Kevin Kavanagh, Department of Biology, National University of Ireland, Maynooth, Co. Kildare, Ireland

"There is no other current book that gives the details on experimental procedures that this one does…a very worthwhile acquisition for investigators working with fungi." (Doody's Health Services) "Will be very useful to many students and bench scientists wishing to expand their repertoire of practical skills in medical mycology." (Microbiology Today, March 2008)Medical Mycology is a useful guide for molecular, immunological, and cytological techniques that will provide useful to researchers and students alike. (The Yale Journal of Biology and Medicine, June 2007)

• Preface xiiiList of Contributors xv1 Diagnosis of Candida Infection in Tissue by Immunohistochemistry 1Malcolm D. Richardson, Riina Rautemaa and Jarkko Hietanen1.1 Introduction 11.2 Specificity of monoclonal antibody 3H8 for C. albicans 3Protocol 1.1 Testing of specificity of monoclonal antibody 3H8 41.3 Evaluation of monoclonal antibody 3H8 for the detection of C. albicans morphological forms 6Protocol 1.2 Evaluation of monoclonal antibody 3H8 for the detection of C. albicans morphological forms 61.4 Application of immunohistochemistry in the diagnosis of Candida periodontal disease 7Protocol 1.3 Use of monoclonal antibody 3H8 in the detection of C. albicans in tissue 81.5 References 112 Transmission Electron Microscopy of Pathogenic Fungi 13Guy Tronchin and Jean-Philippe Bouchara2.1 Introduction 132.2 Glutaraldehyde-potassium-permanganate or glutaraldehyde-osmiumtetroxide fixation for ultrastructural analysis 16Protocol 2.1 Glutaraldehyde-osmium tetroxide (#) or glutaraldehyde-potassium permanganate (*) fixation for ultrastructural analysis 172.3 Identification of the different compartments of the secretory pathway in yeasts 18Protocol 2.2 Identification of the different compartments of the secretory pathway in yeasts 192.4 Cytochemical localization of acid phosphatase in yeasts 20Protocol 2.3 Cytochemical localization of acid phosphatase in yeasts 212.5 Detection of anionic sites 23Protocol 2.4 Detection of anionic sites 232.6 Detection of glycoconjugates by the periodic acid-thiocarbohydrazide-silver proteinate technique (PATAg) 25Protocol 2.5 Detection of glycoconjugates by the periodic acid-thiocarbohydrazide-silver proteinate technique (PATAg) 262.7 Enzyme-gold approach for the detection of polysaccharides in the cell wall 28Protocol 2.6 Enzyme-gold approach for the detection of polysaccharides in the cell wall 292.8 Detection of glycoconjugates by the lectin-gold technique 30Protocol 2.7 Detection of glycoconjugates by the lectin-gold technique 312.9 Immunogold detection of antigens on ultrathin sections of acrylic resin 33Protocol 2.8 Immunogold detection of antigens on ultrathin sections of acrylic resin 342.10 Cryofixation and freeze substitution for ultrastructural analysis and immunodetection 36Protocol 2.9 Cryofixation and freeze substitution for ultrastructural analysis and immunodetection 372.11 Overview 382.12 References 393 Evaluation of Molecular Responses and Antifungal Activity of Phagocytes to Opportunistic Fungi 43Maria Simitsopoulou and Emmanuel Roilides3.1 Introduction 433.2 Preparation of conidia and hyphae of opportunistic fungi 45Protocol 3.1 Preparation of conidia and hyphae of opportunistic fungi 45Protocol 3.2 Preparation of hyphal fragments 473.3 Isolation of human monocytes from whole blood 48Protocol 3.3 Isolation of human MNCs from whole blood 483.4 Analysis of human MNC function in response to fungal infection 51Protocol 3.4 XTT microassay 52Protocol 3.5 Superoxide anion assay in 96-well plate 53Protocol 3.6 Hydrogen peroxide-rhodamine assay 55Protocol 3.7 Phagocytosis assay 553.5 Evaluation of immunomodulators in response to fungal infection 57Protocol 3.8 Analysis of gene expression by RT-PCR 58Protocol 3.9 Analysis of pathway-specific gene expression by microarray technology 63Protocol 3.10 Assessment of cytokines and chemokines by ELISA 663.6 Overview 673.7 References 684 Determination of the Virulence Factors of Candida Albicans and Related Yeast Species 69Khaled H. Abu-Elteen and Mawieh Hamad4.1 Introduction 694.2 Measurement of Candida species adhesion in vitro 70Protocol 4.1 The visual assessment of candidal adhesion to BECs 70Protocol 4.2 The radiometric measurement of candidal adhesion 734.3 Adhesion to inanimate surfaces 75Protocol 4.3 Assessment of candidal adhesion to denture acrylic material 75Protocol 4.4 Adherence of Candida to plastic catheter surfaces 764.4 C. albicans strain differentiation 77Protocol 4.5 Resistogram typing 77Protocol 4.6 The slide agglutination technique 79Protocol 4.7 Serotyping of C. albicans by flow cytomerty 804.5 Phenotypic switching in C. albicans 81Protocol 4.8 Evaluation of phenotype switching in C. albicans 824.6 Extracellular enzymes secreted by C. albicans 83Protocol 4.9 Measurement of extracellular proteinase production by C. albicans 85Protocol 4.10 Measurement of extracellular proteinase produced by C. albicans (staib method) 86Protocol 4.11 Measurement of extracellular phospholipases of C. albicans 874.7 Germ-tube formation in C. albicans 88Protocol 4.12 Germ-tube formation assay 884.8 References 895 Analysis of Drug Resistance in Pathogenic Fungi 93Gary P. Moran, Emmanuelle Pinjon, David C. Coleman and Derek J. Sullivan5.1 Introduction 935.2 Method for the determination of minimum inhibitory concentrations (MICs) of antifungal agents for yeasts 96Protocol 5.1 Method for the determination of minimum inhibitory concentrations (MICs) of antifungal agents for yeasts 975.3 Measurement of Rhodamine 6G uptake and glucose-induced efflux by ABC transporters 102Protocol 5.2 Measurement of rhodamine 6G uptake and glucose-induced efflux 1025.4 Analysis of expression of multidrug transporters in pathogenic fungi 1055.5 Analysis of point mutations in genes encoding cytochrome P- 450 lanosterol demethylase 1065.6 Qualitative detection of alterations in membrane sterol contents 108Protocol 5.3 Qualitative detection of alterations in membrane sterol contents 1095.7 Overview 1105.8 References 1106 Animal Models for Evaluation of Antifungal Efficacy Against Filamentous Fungi 115Eric Dannaoui6.1 Introduction 1156.2 Disseminated zygomycosis in non-immunosuppressed mice 118Protocol 6.1 Disseminated zygomycosis in non-immunosuppressed mice 1186.3 Animal model of disseminated aspergillosis 125Protocol 6.2 Disseminated aspergillosis in neutropoenic mice 1266.4 Study design for evaluation of antifungal combinations therapy in animal models 130Protocol 6.3 Study design for the evaluation of combination therapy in animal models 1316.5 References 1337 Proteomic Analysis of Pathogenic Fungi 137Alan Murphy7.1 Introduction 137Protocol 7.1 2D SDS-PAGE of protein samples 1397.2 Protein digestion in preparation for mass spectrometry by MALDI-TOF 140Protocol 7.2 Peptide mass fingerprinting (PMF) by MALDI-TOF mass spectrometry 141Protocol 7.2a In-gel digestion 142Protocol 7.2b In-solution digestion 1437.3 MALDI-TOF mass spectrometry 145Protocol 7.3 Preparation of matrix for MALDI-TOF 1477.4 Peptide mass fingerprinting (PMF) 149Protocol 7.4 Post-source decay (PSD) and chemically assisted fragmentation (CAF) 1507.5 Interpreting MALDI-TOF result spectra 1527.6 Overview 1567.7 References 1578 Extraction and Detection of DNA and RNA from Yeast 159Patrick Geraghty and Kevin Kavanagh8.1 Introduction 1598.2 The extraction of yeast DNA with the aid of phenol: chloroform 161Protocol 8.1 Whole-cell DNA extraction from C. albicans using phenol: chloroform 161Protocol 8.2 Rapid extraction of DNA from C. albicans colonies for PCR 1648.3 Detection of yeast DNA using radio-labelled probes 165Protocol 8.3 DNA detection by Southern blotting 1658.4 Extraction of whole-cell RNA using two different protocols 169Protocol 8.4 The extraction of whole-cell RNA from yeast using phenol: chloroform 170Protocol 8.5 Rapid extraction of whole-yeast-cell RNA 1728.5 Detection and expression levels of specific genes by the examination of mRNA in yeast 174Protocol 8.6 Examining mRNA content as a means of investigating gene-expression profile by northern blot analysis 175Protocol 8.7 Examining mRNA content as a means of investigating gene-expression profile by RT-PCR analysis 1768.6 References 1799 Microarrays for Studying Pathogenicity in Candida Albicans 181André Nantel, Tracey Rigby, Hervé Hogues and Malcolm Whiteway9.1 Introduction 1819.2 DNA microarrays 1829.3 Building a second-generation 2-colour long oligonucleotide microarray for C. albicans 183Protocol 9.1 Isolation of C. albicans RNA 185Protocol 9.2 Isolation of total RNA using the hot phenol method 186Protocol 9.3 Isolation of Poly-A+ mRNA 187Protocol 9.4 Determination of the efficiency of incorporation of the probe 192Protocol 9.5 Hybridization to DNA microarrays 1949.4 Experiment design 1969.5 Microarray-based studies in C. albicans 2009.6 Conclusion 2059.7 References 20510 Molecular Techniques for Application with Aspergillus Fumigatus 211Nir Osherov and Jacob Romano10.1 Introduction 21110.2 Preparation of knockout vectors for gene disruption and deletion in A. fumigatus 212Protocol 10.1 Preparation of knockout vectors for gene disruption and deletion in A. fumigatus 21310.3 Transformation of A. fumigatus 217Protocol 10.2 Chemical transformation of A. fumigatus 21810.4 Molecular verification of correct single integration (PCR-based) 220Protocol 10.3 Molecular verification of correct integration by PCR 22110.5 General strategies for the phenotypic characterization of A. fumigatus mutant strains 223Protocol 10.4 General strategies for the phenotypic characterization of mutants 22310.6 References 22911 Promoter Analysis and Generation of Knock-out Mutants in Aspergillus Fumigatus 231Matthias Brock, Alexander Gehrke, Venelina Sugareva and Axel A. Brakhage11.1 Introduction 23111.2 Site-directed mutagenesis of promoter elements 233Protocol 11.1 Site-directed mutation of promoter elements 23311.3 lacZ as a reporter gene 236Protocol 11.2 lacZ as a reporter gene: discontinuous determination of b-galactosidase activity 237Protocol 11.3 lacZ as a reporter gene: continuous determination of b-galactosidase activity 23911.4 Transformation of A. fumigatus 241Protocol 11.4 Transformation of A. fumigatus 24211.5 Hygromycin B as a selection marker for transformation 246Protocol 11.5 Hygromycin B as a selection marker for transformation 24711.6 pyrG as a selection marker for transformation 249Protocol 11.6 pyrG as a selection marker for transformation 24911.7 URA-blaster (A. niger pyrG) as a reusable selection marker system for gene deletions/disruptions 251Protocol 11.7 URA-blaster (A. niger pyrG) as a reusable selection marker system for gene deletions/disruptions 25311.8 References 25512 Microarray Technology for Studying the Virulence of Aspergillus Fumigatus 257Darius Armstrong-James and Thomas Rogers12.1 Introduction 25712.2 Isolation of RNA from A. fumigatus 259Protocol 12.1 Isolation of total RNA from A. fumigatus 26012.3 Reverse transcription of RNA and fluorescent labelling of cDNA 263Protocol 12.2 Indirect labelling of cDNA with fluorescent dyes 26412.4 Hybridization of fluorescent probes to DNA microarrays and post-hybridization washing 266Protocol 12.3 Hybridization of fluorescent probes to DNA microarrays and post-hybridization washing 26712.5 Image acquisition from hybridized microarrays 269Protocol 12.4 Image acquisition from hybridized microarrays 26912.6 Microarray image analysis 270Protocol 12.5 Microarray image analysis 27112.7 References 27213 Techniques and Strategies for Studying Virulence Factors in Cryptococcus Neoformans 275Nancy Lee and Guilhem Janbon13.1 Introduction 27513.2 Construction of C. neoformans gene-disruption cassettes 278Protocol 13.1 Construction of C. neoformans gene-disruption cassettes 27813.3 Genetic transformation of C. neoformans 283Protocol 13.2 Biolistic transformation of C. neoformans 283Protocol 13.3 Transformation via electroporation 28613.4 Extraction of genomic DNA from C. neoformans 287Protocol 13.4 DNA for use in library construction and hybridization analysis 288Protocol 13.5 DNA for use in PCR 29113.5 Screening and identification of deletion strains 292Protocol 13.6 Screening and identification of deletion strains 29213.6 Measuring capsule size in C. neoformans 297Protocol 13.7 Measuring capsule size in C. neoformans 29813.7 Purification of glucuronoxylomannan (GXM) 298Protocol 13.8 Purification of glucuronoxylomannan (GXM) 29913.8 References 30114 Genetic Manipulation of Zygomycetes 305Ashraf S. Ibrahim and Christopher D. Skory14.1 Introduction 30514.2 Genetic tools to manipulate mucorales 30614.3 Selectable markers used with mucorales fungi 30714.4 Introduction of DNA used for transformation 308Protocol 14.1 Protoplasting of R. oryzae 309Protocol 14.2 Biolistic delivery system transformation of R. oryzae 313Protocol 14.3 A. tumefaciens-mediated transformation 31614.5 Molecular analysis of transformants 31914.6 Overview 32214.7 References 323Index 327

Editor: Dr Kevin Kavanagh, Department of Biology, National University of Ireland, Maynooth, Co. Kildare, Ireland

"There is no other current book that gives the details on experimental procedures that this one does…a very worthwhile acquisition for investigators working with fungi." (Doody's Health Services) "Will be very useful to many students and bench scientists wishing to expand their repertoire of practical skills in medical mycology." (Microbiology Today, March 2008)Medical Mycology is a useful guide for molecular, immunological, and cytological techniques that will provide useful to researchers and students alike. (The Yale Journal of Biology and Medicine, June 2007)

• Preface xiiiList of Contributors xv1 Diagnosis of Candida Infection in Tissue by Immunohistochemistry 1Malcolm D. Richardson, Riina Rautemaa and Jarkko Hietanen1.1 Introduction 11.2 Specificity of monoclonal antibody 3H8 for C. albicans 3Protocol 1.1 Testing of specificity of monoclonal antibody 3H8 41.3 Evaluation of monoclonal antibody 3H8 for the detection of C. albicans morphological forms 6Protocol 1.2 Evaluation of monoclonal antibody 3H8 for the detection of C. albicans morphological forms 61.4 Application of immunohistochemistry in the diagnosis of Candida periodontal disease 7Protocol 1.3 Use of monoclonal antibody 3H8 in the detection of C. albicans in tissue 81.5 References 112 Transmission Electron Microscopy of Pathogenic Fungi 13Guy Tronchin and Jean-Philippe Bouchara2.1 Introduction 132.2 Glutaraldehyde-potassium-permanganate or glutaraldehyde-osmiumtetroxide fixation for ultrastructural analysis 16Protocol 2.1 Glutaraldehyde-osmium tetroxide (#) or glutaraldehyde-potassium permanganate (*) fixation for ultrastructural analysis 172.3 Identification of the different compartments of the secretory pathway in yeasts 18Protocol 2.2 Identification of the different compartments of the secretory pathway in yeasts 192.4 Cytochemical localization of acid phosphatase in yeasts 20Protocol 2.3 Cytochemical localization of acid phosphatase in yeasts 212.5 Detection of anionic sites 23Protocol 2.4 Detection of anionic sites 232.6 Detection of glycoconjugates by the periodic acid-thiocarbohydrazide-silver proteinate technique (PATAg) 25Protocol 2.5 Detection of glycoconjugates by the periodic acid-thiocarbohydrazide-silver proteinate technique (PATAg) 262.7 Enzyme-gold approach for the detection of polysaccharides in the cell wall 28Protocol 2.6 Enzyme-gold approach for the detection of polysaccharides in the cell wall 292.8 Detection of glycoconjugates by the lectin-gold technique 30Protocol 2.7 Detection of glycoconjugates by the lectin-gold technique 312.9 Immunogold detection of antigens on ultrathin sections of acrylic resin 33Protocol 2.8 Immunogold detection of antigens on ultrathin sections of acrylic resin 342.10 Cryofixation and freeze substitution for ultrastructural analysis and immunodetection 36Protocol 2.9 Cryofixation and freeze substitution for ultrastructural analysis and immunodetection 372.11 Overview 382.12 References 393 Evaluation of Molecular Responses and Antifungal Activity of Phagocytes to Opportunistic Fungi 43Maria Simitsopoulou and Emmanuel Roilides3.1 Introduction 433.2 Preparation of conidia and hyphae of opportunistic fungi 45Protocol 3.1 Preparation of conidia and hyphae of opportunistic fungi 45Protocol 3.2 Preparation of hyphal fragments 473.3 Isolation of human monocytes from whole blood 48Protocol 3.3 Isolation of human MNCs from whole blood 483.4 Analysis of human MNC function in response to fungal infection 51Protocol 3.4 XTT microassay 52Protocol 3.5 Superoxide anion assay in 96-well plate 53Protocol 3.6 Hydrogen peroxide-rhodamine assay 55Protocol 3.7 Phagocytosis assay 553.5 Evaluation of immunomodulators in response to fungal infection 57Protocol 3.8 Analysis of gene expression by RT-PCR 58Protocol 3.9 Analysis of pathway-specific gene expression by microarray technology 63Protocol 3.10 Assessment of cytokines and chemokines by ELISA 663.6 Overview 673.7 References 684 Determination of the Virulence Factors of Candida Albicans and Related Yeast Species 69Khaled H. Abu-Elteen and Mawieh Hamad4.1 Introduction 694.2 Measurement of Candida species adhesion in vitro 70Protocol 4.1 The visual assessment of candidal adhesion to BECs 70Protocol 4.2 The radiometric measurement of candidal adhesion 734.3 Adhesion to inanimate surfaces 75Protocol 4.3 Assessment of candidal adhesion to denture acrylic material 75Protocol 4.4 Adherence of Candida to plastic catheter surfaces 764.4 C. albicans strain differentiation 77Protocol 4.5 Resistogram typing 77Protocol 4.6 The slide agglutination technique 79Protocol 4.7 Serotyping of C. albicans by flow cytomerty 804.5 Phenotypic switching in C. albicans 81Protocol 4.8 Evaluation of phenotype switching in C. albicans 824.6 Extracellular enzymes secreted by C. albicans 83Protocol 4.9 Measurement of extracellular proteinase production by C. albicans 85Protocol 4.10 Measurement of extracellular proteinase produced by C. albicans (staib method) 86Protocol 4.11 Measurement of extracellular phospholipases of C. albicans 874.7 Germ-tube formation in C. albicans 88Protocol 4.12 Germ-tube formation assay 884.8 References 895 Analysis of Drug Resistance in Pathogenic Fungi 93Gary P. Moran, Emmanuelle Pinjon, David C. Coleman and Derek J. Sullivan5.1 Introduction 935.2 Method for the determination of minimum inhibitory concentrations (MICs) of antifungal agents for yeasts 96Protocol 5.1 Method for the determination of minimum inhibitory concentrations (MICs) of antifungal agents for yeasts 975.3 Measurement of Rhodamine 6G uptake and glucose-induced efflux by ABC transporters 102Protocol 5.2 Measurement of rhodamine 6G uptake and glucose-induced efflux 1025.4 Analysis of expression of multidrug transporters in pathogenic fungi 1055.5 Analysis of point mutations in genes encoding cytochrome P- 450 lanosterol demethylase 1065.6 Qualitative detection of alterations in membrane sterol contents 108Protocol 5.3 Qualitative detection of alterations in membrane sterol contents 1095.7 Overview 1105.8 References 1106 Animal Models for Evaluation of Antifungal Efficacy Against Filamentous Fungi 115Eric Dannaoui6.1 Introduction 1156.2 Disseminated zygomycosis in non-immunosuppressed mice 118Protocol 6.1 Disseminated zygomycosis in non-immunosuppressed mice 1186.3 Animal model of disseminated aspergillosis 125Protocol 6.2 Disseminated aspergillosis in neutropoenic mice 1266.4 Study design for evaluation of antifungal combinations therapy in animal models 130Protocol 6.3 Study design for the evaluation of combination therapy in animal models 1316.5 References 1337 Proteomic Analysis of Pathogenic Fungi 137Alan Murphy7.1 Introduction 137Protocol 7.1 2D SDS-PAGE of protein samples 1397.2 Protein digestion in preparation for mass spectrometry by MALDI-TOF 140Protocol 7.2 Peptide mass fingerprinting (PMF) by MALDI-TOF mass spectrometry 141Protocol 7.2a In-gel digestion 142Protocol 7.2b In-solution digestion 1437.3 MALDI-TOF mass spectrometry 145Protocol 7.3 Preparation of matrix for MALDI-TOF 1477.4 Peptide mass fingerprinting (PMF) 149Protocol 7.4 Post-source decay (PSD) and chemically assisted fragmentation (CAF) 1507.5 Interpreting MALDI-TOF result spectra 1527.6 Overview 1567.7 References 1578 Extraction and Detection of DNA and RNA from Yeast 159Patrick Geraghty and Kevin Kavanagh8.1 Introduction 1598.2 The extraction of yeast DNA with the aid of phenol: chloroform 161Protocol 8.1 Whole-cell DNA extraction from C. albicans using phenol: chloroform 161Protocol 8.2 Rapid extraction of DNA from C. albicans colonies for PCR 1648.3 Detection of yeast DNA using radio-labelled probes 165Protocol 8.3 DNA detection by Southern blotting 1658.4 Extraction of whole-cell RNA using two different protocols 169Protocol 8.4 The extraction of whole-cell RNA from yeast using phenol: chloroform 170Protocol 8.5 Rapid extraction of whole-yeast-cell RNA 1728.5 Detection and expression levels of specific genes by the examination of mRNA in yeast 174Protocol 8.6 Examining mRNA content as a means of investigating gene-expression profile by northern blot analysis 175Protocol 8.7 Examining mRNA content as a means of investigating gene-expression profile by RT-PCR analysis 1768.6 References 1799 Microarrays for Studying Pathogenicity in Candida Albicans 181André Nantel, Tracey Rigby, Hervé Hogues and Malcolm Whiteway9.1 Introduction 1819.2 DNA microarrays 1829.3 Building a second-generation 2-colour long oligonucleotide microarray for C. albicans 183Protocol 9.1 Isolation of C. albicans RNA 185Protocol 9.2 Isolation of total RNA using the hot phenol method 186Protocol 9.3 Isolation of Poly-A+ mRNA 187Protocol 9.4 Determination of the efficiency of incorporation of the probe 192Protocol 9.5 Hybridization to DNA microarrays 1949.4 Experiment design 1969.5 Microarray-based studies in C. albicans 2009.6 Conclusion 2059.7 References 20510 Molecular Techniques for Application with Aspergillus Fumigatus 211Nir Osherov and Jacob Romano10.1 Introduction 21110.2 Preparation of knockout vectors for gene disruption and deletion in A. fumigatus 212Protocol 10.1 Preparation of knockout vectors for gene disruption and deletion in A. fumigatus 21310.3 Transformation of A. fumigatus 217Protocol 10.2 Chemical transformation of A. fumigatus 21810.4 Molecular verification of correct single integration (PCR-based) 220Protocol 10.3 Molecular verification of correct integration by PCR 22110.5 General strategies for the phenotypic characterization of A. fumigatus mutant strains 223Protocol 10.4 General strategies for the phenotypic characterization of mutants 22310.6 References 22911 Promoter Analysis and Generation of Knock-out Mutants in Aspergillus Fumigatus 231Matthias Brock, Alexander Gehrke, Venelina Sugareva and Axel A. Brakhage11.1 Introduction 23111.2 Site-directed mutagenesis of promoter elements 233Protocol 11.1 Site-directed mutation of promoter elements 23311.3 lacZ as a reporter gene 236Protocol 11.2 lacZ as a reporter gene: discontinuous determination of b-galactosidase activity 237Protocol 11.3 lacZ as a reporter gene: continuous determination of b-galactosidase activity 23911.4 Transformation of A. fumigatus 241Protocol 11.4 Transformation of A. fumigatus 24211.5 Hygromycin B as a selection marker for transformation 246Protocol 11.5 Hygromycin B as a selection marker for transformation 24711.6 pyrG as a selection marker for transformation 249Protocol 11.6 pyrG as a selection marker for transformation 24911.7 URA-blaster (A. niger pyrG) as a reusable selection marker system for gene deletions/disruptions 251Protocol 11.7 URA-blaster (A. niger pyrG) as a reusable selection marker system for gene deletions/disruptions 25311.8 References 25512 Microarray Technology for Studying the Virulence of Aspergillus Fumigatus 257Darius Armstrong-James and Thomas Rogers12.1 Introduction 25712.2 Isolation of RNA from A. fumigatus 259Protocol 12.1 Isolation of total RNA from A. fumigatus 26012.3 Reverse transcription of RNA and fluorescent labelling of cDNA 263Protocol 12.2 Indirect labelling of cDNA with fluorescent dyes 26412.4 Hybridization of fluorescent probes to DNA microarrays and post-hybridization washing 266Protocol 12.3 Hybridization of fluorescent probes to DNA microarrays and post-hybridization washing 26712.5 Image acquisition from hybridized microarrays 269Protocol 12.4 Image acquisition from hybridized microarrays 26912.6 Microarray image analysis 270Protocol 12.5 Microarray image analysis 27112.7 References 27213 Techniques and Strategies for Studying Virulence Factors in Cryptococcus Neoformans 275Nancy Lee and Guilhem Janbon13.1 Introduction 27513.2 Construction of C. neoformans gene-disruption cassettes 278Protocol 13.1 Construction of C. neoformans gene-disruption cassettes 27813.3 Genetic transformation of C. neoformans 283Protocol 13.2 Biolistic transformation of C. neoformans 283Protocol 13.3 Transformation via electroporation 28613.4 Extraction of genomic DNA from C. neoformans 287Protocol 13.4 DNA for use in library construction and hybridization analysis 288Protocol 13.5 DNA for use in PCR 29113.5 Screening and identification of deletion strains 292Protocol 13.6 Screening and identification of deletion strains 29213.6 Measuring capsule size in C. neoformans 297Protocol 13.7 Measuring capsule size in C. neoformans 29813.7 Purification of glucuronoxylomannan (GXM) 298Protocol 13.8 Purification of glucuronoxylomannan (GXM) 29913.8 References 30114 Genetic Manipulation of Zygomycetes 305Ashraf S. Ibrahim and Christopher D. Skory14.1 Introduction 30514.2 Genetic tools to manipulate mucorales 30614.3 Selectable markers used with mucorales fungi 30714.4 Introduction of DNA used for transformation 308Protocol 14.1 Protoplasting of R. oryzae 309Protocol 14.2 Biolistic delivery system transformation of R. oryzae 313Protocol 14.3 A. tumefaciens-mediated transformation 31614.5 Molecular analysis of transformants 31914.6 Overview 32214.7 References 323Index 327

Editor: Dr Kevin Kavanagh, Department of Biology, National University of Ireland, Maynooth, Co. Kildare, Ireland

"There is no other current book that gives the details on experimental procedures that this one does…a very worthwhile acquisition for investigators working with fungi." (Doody's Health Services) "Will be very useful to many students and bench scientists wishing to expand their repertoire of practical skills in medical mycology." (Microbiology Today, March 2008)Medical Mycology is a useful guide for molecular, immunological, and cytological techniques that will provide useful to researchers and students alike. (The Yale Journal of Biology and Medicine, June 2007)

• Preface xiiiList of Contributors xv1 Diagnosis of Candida Infection in Tissue by Immunohistochemistry 1Malcolm D. Richardson, Riina Rautemaa and Jarkko Hietanen1.1 Introduction 11.2 Specificity of monoclonal antibody 3H8 for C. albicans 3Protocol 1.1 Testing of specificity of monoclonal antibody 3H8 41.3 Evaluation of monoclonal antibody 3H8 for the detection of C. albicans morphological forms 6Protocol 1.2 Evaluation of monoclonal antibody 3H8 for the detection of C. albicans morphological forms 61.4 Application of immunohistochemistry in the diagnosis of Candida periodontal disease 7Protocol 1.3 Use of monoclonal antibody 3H8 in the detection of C. albicans in tissue 81.5 References 112 Transmission Electron Microscopy of Pathogenic Fungi 13Guy Tronchin and Jean-Philippe Bouchara2.1 Introduction 132.2 Glutaraldehyde-potassium-permanganate or glutaraldehyde-osmiumtetroxide fixation for ultrastructural analysis 16Protocol 2.1 Glutaraldehyde-osmium tetroxide (#) or glutaraldehyde-potassium permanganate (*) fixation for ultrastructural analysis 172.3 Identification of the different compartments of the secretory pathway in yeasts 18Protocol 2.2 Identification of the different compartments of the secretory pathway in yeasts 192.4 Cytochemical localization of acid phosphatase in yeasts 20Protocol 2.3 Cytochemical localization of acid phosphatase in yeasts 212.5 Detection of anionic sites 23Protocol 2.4 Detection of anionic sites 232.6 Detection of glycoconjugates by the periodic acid-thiocarbohydrazide-silver proteinate technique (PATAg) 25Protocol 2.5 Detection of glycoconjugates by the periodic acid-thiocarbohydrazide-silver proteinate technique (PATAg) 262.7 Enzyme-gold approach for the detection of polysaccharides in the cell wall 28Protocol 2.6 Enzyme-gold approach for the detection of polysaccharides in the cell wall 292.8 Detection of glycoconjugates by the lectin-gold technique 30Protocol 2.7 Detection of glycoconjugates by the lectin-gold technique 312.9 Immunogold detection of antigens on ultrathin sections of acrylic resin 33Protocol 2.8 Immunogold detection of antigens on ultrathin sections of acrylic resin 342.10 Cryofixation and freeze substitution for ultrastructural analysis and immunodetection 36Protocol 2.9 Cryofixation and freeze substitution for ultrastructural analysis and immunodetection 372.11 Overview 382.12 References 393 Evaluation of Molecular Responses and Antifungal Activity of Phagocytes to Opportunistic Fungi 43Maria Simitsopoulou and Emmanuel Roilides3.1 Introduction 433.2 Preparation of conidia and hyphae of opportunistic fungi 45Protocol 3.1 Preparation of conidia and hyphae of opportunistic fungi 45Protocol 3.2 Preparation of hyphal fragments 473.3 Isolation of human monocytes from whole blood 48Protocol 3.3 Isolation of human MNCs from whole blood 483.4 Analysis of human MNC function in response to fungal infection 51Protocol 3.4 XTT microassay 52Protocol 3.5 Superoxide anion assay in 96-well plate 53Protocol 3.6 Hydrogen peroxide-rhodamine assay 55Protocol 3.7 Phagocytosis assay 553.5 Evaluation of immunomodulators in response to fungal infection 57Protocol 3.8 Analysis of gene expression by RT-PCR 58Protocol 3.9 Analysis of pathway-specific gene expression by microarray technology 63Protocol 3.10 Assessment of cytokines and chemokines by ELISA 663.6 Overview 673.7 References 684 Determination of the Virulence Factors of Candida Albicans and Related Yeast Species 69Khaled H. Abu-Elteen and Mawieh Hamad4.1 Introduction 694.2 Measurement of Candida species adhesion in vitro 70Protocol 4.1 The visual assessment of candidal adhesion to BECs 70Protocol 4.2 The radiometric measurement of candidal adhesion 734.3 Adhesion to inanimate surfaces 75Protocol 4.3 Assessment of candidal adhesion to denture acrylic material 75Protocol 4.4 Adherence of Candida to plastic catheter surfaces 764.4 C. albicans strain differentiation 77Protocol 4.5 Resistogram typing 77Protocol 4.6 The slide agglutination technique 79Protocol 4.7 Serotyping of C. albicans by flow cytomerty 804.5 Phenotypic switching in C. albicans 81Protocol 4.8 Evaluation of phenotype switching in C. albicans 824.6 Extracellular enzymes secreted by C. albicans 83Protocol 4.9 Measurement of extracellular proteinase production by C. albicans 85Protocol 4.10 Measurement of extracellular proteinase produced by C. albicans (staib method) 86Protocol 4.11 Measurement of extracellular phospholipases of C. albicans 874.7 Germ-tube formation in C. albicans 88Protocol 4.12 Germ-tube formation assay 884.8 References 895 Analysis of Drug Resistance in Pathogenic Fungi 93Gary P. Moran, Emmanuelle Pinjon, David C. Coleman and Derek J. Sullivan5.1 Introduction 935.2 Method for the determination of minimum inhibitory concentrations (MICs) of antifungal agents for yeasts 96Protocol 5.1 Method for the determination of minimum inhibitory concentrations (MICs) of antifungal agents for yeasts 975.3 Measurement of Rhodamine 6G uptake and glucose-induced efflux by ABC transporters 102Protocol 5.2 Measurement of rhodamine 6G uptake and glucose-induced efflux 1025.4 Analysis of expression of multidrug transporters in pathogenic fungi 1055.5 Analysis of point mutations in genes encoding cytochrome P- 450 lanosterol demethylase 1065.6 Qualitative detection of alterations in membrane sterol contents 108Protocol 5.3 Qualitative detection of alterations in membrane sterol contents 1095.7 Overview 1105.8 References 1106 Animal Models for Evaluation of Antifungal Efficacy Against Filamentous Fungi 115Eric Dannaoui6.1 Introduction 1156.2 Disseminated zygomycosis in non-immunosuppressed mice 118Protocol 6.1 Disseminated zygomycosis in non-immunosuppressed mice 1186.3 Animal model of disseminated aspergillosis 125Protocol 6.2 Disseminated aspergillosis in neutropoenic mice 1266.4 Study design for evaluation of antifungal combinations therapy in animal models 130Protocol 6.3 Study design for the evaluation of combination therapy in animal models 1316.5 References 1337 Proteomic Analysis of Pathogenic Fungi 137Alan Murphy7.1 Introduction 137Protocol 7.1 2D SDS-PAGE of protein samples 1397.2 Protein digestion in preparation for mass spectrometry by MALDI-TOF 140Protocol 7.2 Peptide mass fingerprinting (PMF) by MALDI-TOF mass spectrometry 141Protocol 7.2a In-gel digestion 142Protocol 7.2b In-solution digestion 1437.3 MALDI-TOF mass spectrometry 145Protocol 7.3 Preparation of matrix for MALDI-TOF 1477.4 Peptide mass fingerprinting (PMF) 149Protocol 7.4 Post-source decay (PSD) and chemically assisted fragmentation (CAF) 1507.5 Interpreting MALDI-TOF result spectra 1527.6 Overview 1567.7 References 1578 Extraction and Detection of DNA and RNA from Yeast 159Patrick Geraghty and Kevin Kavanagh8.1 Introduction 1598.2 The extraction of yeast DNA with the aid of phenol: chloroform 161Protocol 8.1 Whole-cell DNA extraction from C. albicans using phenol: chloroform 161Protocol 8.2 Rapid extraction of DNA from C. albicans colonies for PCR 1648.3 Detection of yeast DNA using radio-labelled probes 165Protocol 8.3 DNA detection by Southern blotting 1658.4 Extraction of whole-cell RNA using two different protocols 169Protocol 8.4 The extraction of whole-cell RNA from yeast using phenol: chloroform 170Protocol 8.5 Rapid extraction of whole-yeast-cell RNA 1728.5 Detection and expression levels of specific genes by the examination of mRNA in yeast 174Protocol 8.6 Examining mRNA content as a means of investigating gene-expression profile by northern blot analysis 175Protocol 8.7 Examining mRNA content as a means of investigating gene-expression profile by RT-PCR analysis 1768.6 References 1799 Microarrays for Studying Pathogenicity in Candida Albicans 181André Nantel, Tracey Rigby, Hervé Hogues and Malcolm Whiteway9.1 Introduction 1819.2 DNA microarrays 1829.3 Building a second-generation 2-colour long oligonucleotide microarray for C. albicans 183Protocol 9.1 Isolation of C. albicans RNA 185Protocol 9.2 Isolation of total RNA using the hot phenol method 186Protocol 9.3 Isolation of Poly-A+ mRNA 187Protocol 9.4 Determination of the efficiency of incorporation of the probe 192Protocol 9.5 Hybridization to DNA microarrays 1949.4 Experiment design 1969.5 Microarray-based studies in C. albicans 2009.6 Conclusion 2059.7 References 20510 Molecular Techniques for Application with Aspergillus Fumigatus 211Nir Osherov and Jacob Romano10.1 Introduction 21110.2 Preparation of knockout vectors for gene disruption and deletion in A. fumigatus 212Protocol 10.1 Preparation of knockout vectors for gene disruption and deletion in A. fumigatus 21310.3 Transformation of A. fumigatus 217Protocol 10.2 Chemical transformation of A. fumigatus 21810.4 Molecular verification of correct single integration (PCR-based) 220Protocol 10.3 Molecular verification of correct integration by PCR 22110.5 General strategies for the phenotypic characterization of A. fumigatus mutant strains 223Protocol 10.4 General strategies for the phenotypic characterization of mutants 22310.6 References 22911 Promoter Analysis and Generation of Knock-out Mutants in Aspergillus Fumigatus 231Matthias Brock, Alexander Gehrke, Venelina Sugareva and Axel A. Brakhage11.1 Introduction 23111.2 Site-directed mutagenesis of promoter elements 233Protocol 11.1 Site-directed mutation of promoter elements 23311.3 lacZ as a reporter gene 236Protocol 11.2 lacZ as a reporter gene: discontinuous determination of b-galactosidase activity 237Protocol 11.3 lacZ as a reporter gene: continuous determination of b-galactosidase activity 23911.4 Transformation of A. fumigatus 241Protocol 11.4 Transformation of A. fumigatus 24211.5 Hygromycin B as a selection marker for transformation 246Protocol 11.5 Hygromycin B as a selection marker for transformation 24711.6 pyrG as a selection marker for transformation 249Protocol 11.6 pyrG as a selection marker for transformation 24911.7 URA-blaster (A. niger pyrG) as a reusable selection marker system for gene deletions/disruptions 251Protocol 11.7 URA-blaster (A. niger pyrG) as a reusable selection marker system for gene deletions/disruptions 25311.8 References 25512 Microarray Technology for Studying the Virulence of Aspergillus Fumigatus 257Darius Armstrong-James and Thomas Rogers12.1 Introduction 25712.2 Isolation of RNA from A. fumigatus 259Protocol 12.1 Isolation of total RNA from A. fumigatus 26012.3 Reverse transcription of RNA and fluorescent labelling of cDNA 263Protocol 12.2 Indirect labelling of cDNA with fluorescent dyes 26412.4 Hybridization of fluorescent probes to DNA microarrays and post-hybridization washing 266Protocol 12.3 Hybridization of fluorescent probes to DNA microarrays and post-hybridization washing 26712.5 Image acquisition from hybridized microarrays 269Protocol 12.4 Image acquisition from hybridized microarrays 26912.6 Microarray image analysis 270Protocol 12.5 Microarray image analysis 27112.7 References 27213 Techniques and Strategies for Studying Virulence Factors in Cryptococcus Neoformans 275Nancy Lee and Guilhem Janbon13.1 Introduction 27513.2 Construction of C. neoformans gene-disruption cassettes 278Protocol 13.1 Construction of C. neoformans gene-disruption cassettes 27813.3 Genetic transformation of C. neoformans 283Protocol 13.2 Biolistic transformation of C. neoformans 283Protocol 13.3 Transformation via electroporation 28613.4 Extraction of genomic DNA from C. neoformans 287Protocol 13.4 DNA for use in library construction and hybridization analysis 288Protocol 13.5 DNA for use in PCR 29113.5 Screening and identification of deletion strains 292Protocol 13.6 Screening and identification of deletion strains 29213.6 Measuring capsule size in C. neoformans 297Protocol 13.7 Measuring capsule size in C. neoformans 29813.7 Purification of glucuronoxylomannan (GXM) 298Protocol 13.8 Purification of glucuronoxylomannan (GXM) 29913.8 References 30114 Genetic Manipulation of Zygomycetes 305Ashraf S. Ibrahim and Christopher D. Skory14.1 Introduction 30514.2 Genetic tools to manipulate mucorales 30614.3 Selectable markers used with mucorales fungi 30714.4 Introduction of DNA used for transformation 308Protocol 14.1 Protoplasting of R. oryzae 309Protocol 14.2 Biolistic delivery system transformation of R. oryzae 313Protocol 14.3 A. tumefaciens-mediated transformation 31614.5 Molecular analysis of transformants 31914.6 Overview 32214.7 References 323Index 327