Laboratory Protocols in Fungal Biology: Current Methods in Fungal Biology

Contents
1: Various Methods of Long-Term Preservation of Fungal Cultures in All-Russian Collection of Microorganisms (VKM)
	1.1 Introduction
	1.2 Cryopreservation of Filamentous Fungi
	1.3 Freeze-Drying of Filamentous Fungi
	1.4 Drying in Sterile Soil of Filamentous Fungi
	1.5 Drying of Filamentous Fungi on Silica Gel
	1.6 Protocols
	1.7 Protocol of Drying on Silica Gel
		1.7.1 Preparation of Sterile Silica Gel and Ampoules
		1.7.2 Preparation of Cryoprotectant: 10% (v/v) Glycerol
		1.7.3 Preparation of Cultures
		1.7.4 Silica Gel Inoculation
		1.7.5 Filling of Vials
		1.7.6 Control of Viability
	Annexies
		Annex 1: Fields Attributes in the Table «Database Preservation Methods»
		Annex 2: Maximal Preservation Times for VKM Fungal Species
	References
Chapter 2: Sabouraud Agar and Other Fungal Growth Media
	2.1 Sabouraud Dextrose Agar
		2.1.1 History
		2.1.2 Theory
		2.1.3 Materials
		2.1.4 Method of Sabouraud Agar Preparation
			2.1.4.1 Standard Preparation
			2.1.4.2 Method of Sabouraud Agar, Emmons Modification Preparation
			2.1.4.3 Variations on Standard Sabouraud Agar
		2.1.5 Methods of Inoculation and Incubation
		2.1.6 Results
	2.2 Potato Dextrose Agar
		2.2.1 History
		2.2.2 Theory
		2.2.3 Methods of Preparation
		2.2.4 Methods of Inoculation and Incubation
			2.2.4.1 Quality Control
			2.2.4.2 Inoculation and Incubation
		2.2.5 Results
	2.3 Bird Seed Agar
		2.3.1 History
		2.3.2 Theory
		2.3.3 Methods of Preparation
			2.3.3.1 Preparation of Bird Seed Agar
		2.3.4 Method of Inoculation and Incubation
			2.3.4.1 Quality Control
			2.3.4.2 Inoculation and Incubation
		2.3.5 Results
	2.4 Dermatophyte Test Medium
		2.4.1 History
		2.4.2 Theory
		2.4.3 Method of Preparation
		2.4.4 Methods of Inoculation and Incubation
		2.4.5 Results
	2.5 Safety Notes
	2.6 Additional Fungal Growth Media
	References
Chapter 3: Fluorescence In Situ Hybridization of Uncultured Zoosporic Fungi
	3.1 Introduction
	3.2 Materials
	3.3 Methods
		3.3.1 Classical FISH Probing (See Note 3)
		3.3.2 CARD-FISH Probing (See Note 7)
	3.4 Notes
	References
Chapter 4: Technique for Identifying and Counting Infective Chytrid Sporangia Using the Chitinaceous Fluorochrome Calcofluor W...
	4.1 Introduction
	4.2 Materials
	4.3 Methods
		4.3.1 Concentrations of Cells (See Note 1)
		4.3.2 Preparation of Calcofluor Stock Solution
		4.3.3 Staining and Visualization
	4.4 Notes
	References
Chapter 5: Assessment of Host Immune Responses to Fungal Pathogens
	5.1 Introduction
	5.2 Methods
		5.2.1 Analysis of the Fungal Burden
			5.2.1.1 CFU
			5.2.1.2 qPCR13 Targeting the 18S rRNA Gene
		5.2.2 Isolation of the Immune System Organs, Infected Organs, and Immune Cells
			5.2.2.1 Thymus
			5.2.2.2 Bone Marrow
			5.2.2.3 Spleen
			5.2.2.4 Lymph Nodes
			5.2.2.5 Blood
			5.2.2.6 Bronchoalveolar Lavage Fluid (BALF)
			5.2.2.7 Infected Organs
			5.2.2.8 Separation of Immune Cells and Erythrocyte Lysis
			5.2.2.9 Histology
		5.2.3 Detection and Quantification of Immune Cells
			5.2.3.1 Cell Detection Using Flow Cytometry
			5.2.3.2 Cell Counting
		5.2.4 Detection and Quantification of Cytokines
			5.2.4.1 Intracellular Cytokine Detection Based on Flow Cytometry
			5.2.4.2 ELISA
			5.2.4.3 Other Methods
	5.3 Summary
	References
Chapter 6: Recent Advances in Applications of Support Vector Machines in Fungal Biology
	6.1 Introduction
		6.1.1 Support Vector Machine
	6.2 Domain Features
	6.3 Review of Some Recent Fungal Bioinformatics Applications Using SVM
		6.3.1 Haar Wavelets Features for Fungal Disease Detection in Maize Leaves
		6.3.2 Quality Detection of Pomegranate Fruit Infected with Fungal Disease
		6.3.3 Support Vector Machines Enabled Fungal Rust Disease Detection in Pea Plant (Pisam Sativam)
		6.3.4 Volatomic Visualization for Fungal Infection Detection on Storage Jasmine
		6.3.5 Hyperspectral Imaging Enabled Electronic Nose for Fungal Contamination Identification in Strawberries
		6.3.6 Rapid Discrimination of Fungal Species by the Colony Fingerprinting
		6.3.7 SVM Classifier Based Grape Leaf Disease Detection
		6.3.8 Hyphae Detection in Fungal Keratitis Images
		6.3.9 Plant Diseases Classification Using SVM and ANN (Artificial Neural Network)
		6.3.10 A Hybrid Combination of Multiple SVM Classifiers for Automatic Recognition of the Damages and Symptoms on Plant Leaves
		6.3.11 Machine Learning of Protein Interactions in Fungal Secretory Pathways
		6.3.12 Fungal Adhesins and Adhesin Like Proteins Predictions
		6.3.13 Computational Prediction of Antifungal Peptides
		6.3.14 A Novel Method of Annotation of Antifungal Peptides Based on Distributed Representation of Protein Sequences
		6.3.15 Identification of Antifungal Using Distributed Representation of Sequences
	6.4 Illustration of Use of SVM in Fungal Bioinformatics
	6.5 Concluding Remarks
	References
Chapter 7: Real-Time Quantitative PCR Assay for the Assessment of Uncultured Zoosporic Fungi
	7.1 Introduction
	7.2 Materials
	7.3 Methods
		7.3.1 DNA Extraction and Purification
		7.3.2 Real-Time qPCR Assays
	7.4 Notes
	References
Chapter 8: Assays for the Quantification of Antioxidant Enzymes in Fungi
	8.1 Introduction
	8.2 Materials and Methods
		8.2.1 Materials
	8.3 Methods
		8.3.1 Fungal Tissue Homogenization
	8.4 Protein Quantification
	8.5 Antioxidant Enzymes Related to the Decomposition of ROS
		8.5.1 Catalase (CAT) Activity
		8.5.2 Peroxidases (Px) Activity
		8.5.3 Superoxide Dismutase (SOD) Activity
	8.6 Thiol Redox State Related Enzymes
		8.6.1 Glutathione-S-Transferase (GST) Activity
		8.6.2 Glutathione Reductase (GR) Activity
		8.6.3 Glutathione Peroxidase (GP) Activity
		8.6.4 Glucose-6-Phosphate Dehydrogenase (G6PD) Activity
	8.7 Conclusions
	References
Chapter 9: Cellulomics of Live Yeast by Advanced and Correlative Microscopy
	9.1 Introduction
	9.2 Materials
	9.3 Experimental Methods
		9.3.1 Preparing Coverslips
			9.3.1.1 Cleaning Coverslips
			9.3.1.2 Coating Coverslips
				Poly-L-Lysine (PLL)
				Cell-Tak
		9.3.2 Sample Preparation
			9.3.2.1 Fixed Samples
			9.3.2.2 Live Samples
				Physical Immobilisation of Cells with PDMS Stamps
				Biochemical Trapping of Cells with Cell-Tak
		9.3.3 Pros and Cons of Immobilisation Techniques
	9.4 Imaging Modes
		9.4.1 Imaging Fixed Cells in QI Mode
		9.4.2 Imaging Live Cells in QI Mode
		9.4.3 Tracking Dynamic Processes in QI Mode
		9.4.4 Correlative AFM-QI-LSCM Live Cell Imaging
	9.5 AFM QI Mode Image/Data Processing
		9.5.1 AFM Image Processing and Overlay
	References
Chapter 10: Molecular Taxonomy and Multigene Phylogeny of Filamentous Fungi
	10.1 Introduction
	10.2 Fungal Genomic DNA Extraction
		10.2.1 Materials
		10.2.2 Method
	10.3 Agarose Gel Electrophoresis
		10.3.1 Material
		10.3.2 Method
	10.4 Selection of Gene Target and Their Amplification
		10.4.1 Materials
		10.4.2 Method
	10.5 PCR Product Purification and Quantification
	10.6 DNA Sequencing (Sanger Method)
		10.6.1 Materials
		10.6.2 Method
	10.7 Perform Sequencing PCR in Thermal Cycler Using Following Conditions
		10.7.1 Purification of Sequencing PCR Product
	10.8 Sequence Editing, Analysis, and Identification
		10.8.1 Basic Local Alignment Search Tool (BLAST)
		10.8.2 MycoBank Database Search
		10.8.3 UNITE Database Search
		10.8.4 Method
	10.9 Molecular Phylogeny and Phylogenetic Tree
		10.9.1 Phylogenetic Tree Construction
		10.9.2 Phylogenetic Tree Construction Using MEGA Software
	References
Chapter 11: Fluorochrome-Based Methods for Fungal Sample Examination
	11.1 Introduction
	11.2 Sample Preparation to Observe Fungi Nuclei and Chromosome
	11.3 DAPI Methods
		11.3.1 Materials
		11.3.2 Methods
		11.3.3 Culture Block on Inverted Microscope
		11.3.4 Slide and Coverslip Preparation
	11.4 Culture Block on Inverted Microscope and Propidium Iodide in Nuclei Marking
		11.4.1 Materials
		11.4.2 Method
	11.5 Sample Preparation to Observe Fungi Chromosome
		11.5.1 Materials
		11.5.2 Method
	11.6 Sample Preparation to Observe Fungi Cell-Wall
	11.7 Calcofluor White Staining
		11.7.1 Materials
	11.8 AlexaFluor 488 WGA Conjugate Staining
		11.8.1 Materials
		11.8.2 Method
	11.9 Reactive Oxygen Species (ROS) in Fungi
		11.9.1 Materials
		11.9.2 Methods
			11.9.2.1 Conidia Suspension
	11.10 Fungi Cellular Death Studies with Propidium Iodide
		11.10.1 Materials
		11.10.2 Methods
		11.10.3 Conidia or Yeast Suspension
	11.11 Live-Dead Test for Fungi Cells
		11.11.1 Materials
		11.11.2 Method
	11.12 Sample Preparation to Observe Fungi-Plant Interactions
		11.12.1 Materials
		11.12.2 Methods
	11.13 Important Plant Structures Defence Against Phytopathogenic Fungi
	11.14 Callose Deposition
		11.14.1 Materials
		11.14.2 Method
	11.15 Lignin Localization in Plant-Fungi Interactions
		11.15.1 Materials
		11.15.2 Method
	11.16 Sample Preparation to Observe Autofluorescent Fungi and Specific Structures
	11.17 Autofluorescent Rust Fungi
		11.17.1 Materials
		11.17.2 Method
	11.18 Autofluorescent Cercosporin
		11.18.1 Materials
		11.18.2 Method
	References
Chapter 12: Yeast Isolation Methods from Specialized Habitats
	12.1 Introduction
	12.2 Common Materials
	12.3 Methods
	12.4 Yeast Isolation Protocols from Natural Habitats
		12.4.1 Rumen Fluid/Digesta
			12.4.1.1 Materials
			12.4.1.2 Protocol
		12.4.2 Insect Gut (Termite/Beetle)
			12.4.2.1 Materials
			12.4.2.2 Protocol
		12.4.3 Hot Spring
			12.4.3.1 Materials
			12.4.3.2 Protocol
		12.4.4 Flower
			12.4.4.1 Materials
			12.4.4.2 Protocol
		12.4.5 Nectar
			12.4.5.1 Materials
			12.4.5.2 Protocol
		12.4.6 Rotten Wood
			12.4.6.1 Materials
			12.4.6.2 Protocol
	12.5 Yeast Isolation Protocols from Anthropogenic Habitats
		12.5.1 Compost
			12.5.1.1 Materials
			12.5.1.2 Protocol
		12.5.2 Molasses
			12.5.2.1 Materials
			12.5.2.2 Protocol
		12.5.3 Press Mud
			12.5.3.1 Materials
			12.5.3.2 Protocol
		12.5.4 Distillery Effluent/Spent Wash
			12.5.4.1 Materials
			12.5.4.2 Protocol
	References
Index