Preface
Contents
Contributors
Part I: Abundance and Species Richness Calculation of Protists from Soil
Chapter 1: Soil Sampling, Processing, and Storage
1 Introduction
2 Materials
3 Methods
3.1 Soil Tracts Establishment
3.2 Soil Sample Collection
3.3 Soil Processing and Storage
4 Notes
References
Chapter 2: Initial Observation of Protist from Soil
1 Introduction
2 Materials
2.1 Media and Solutions
2.2 Other Requirements
3 Methods
3.1 Soil Sampling
3.2 Common Method
3.3 Observation of Naked Amoeba
3.4 Observation of Ciliates and Flagellates
4 Notes
References
Chapter 3: Abundance Calculation of Naked and Testate Amoebae from Soil
1 Introduction
2 Materials
2.1 Soil Sample Collection
2.2 Abundance Calculation of Naked Amoebae
2.3 Abundance Calculation of Testate Amoebae
2.4 Abundance Calculation of Testate Amoebae by Direct Count
3 Methods
3.1 Collection of Soil Sample
3.2 Abundance Calculation of Naked Amoebae
3.3 Abundance Calculation of Testate Amoebae
3.4 Abundance Calculation of Testate Amoebae by Direct Count
4 Notes
References
Chapter 4: Abundance Calculation of Mycophagous Amoebae
1 Introduction
2 Materials
2.1 Abundance Calculation of Mycophagous Amoebae by Culture Method
2.2 Abundance Calculation of Mycophagous Amoebae Using Microtiter Plates
3 Methods
3.1 Abundance Calculation of Mycophagous Amoebae by Culture Method
3.1.1 Observations
3.2 Abundance Calculation of Mycophagous Amoebae Using Microtiter Plates
4 Notes
References
Chapter 5: Abundance Calculation of Ciliates from Soil
1 Introduction
2 Materials
2.1 Abundance Calculation of Active Soil Ciliates
2.2 Abundance Calculation of Soil Ciliates: Active Forms and Cysts
3 Methods
3.1 Abundance Calculation of Active Soil Ciliates
3.2 Abundance Calculation of Soil Ciliates: Active Forms and Cysts
3.3 Calculation
4 Notes
References
Chapter 6: Abundance Calculation of Flagellates from Soil
1 Introduction
2 Materials
2.1 Direct Microscopic Counting [11]
2.2 Culture-Based Microtiter MNP Method [12]
3 Methods
3.1 Direct Microscopic Counting [11]
3.2 Culture-Based Microtiter MPN Method [12]
3.2.1 Ring Method
3.2.2 Darbyshire Et al. Modification
3.3 Calculation
References
Chapter 7: Methodology to Study Species Diversity of Naked Amoeba and Testate Amoeba
1 Introduction
2 Materials
2.1 Materials Required to Study Species Diversity of Naked Amoeba
2.1.1 Glass/Plasticwares
2.1.2 Media
2.2 Materials Required to Study Species Diversity of Testate Amoeba
2.2.1 Materials and Equipment
2.2.2 Solutions
3 Methods
3.1 Species Diversity of Naked Amoeba
3.2 Species Diversity of Testate Amoeba
4 Notes
References
Chapter 8: Non-flooded Petri Plate Method to Study the Species Diversity of Flagellates
1 Introduction
2 Materials
2.1 Requirements
2.2 Media
3 Method
4 Notes
References
Part II: Enumeration Techniques
Chapter 9: Enumeration of Testate Amoeba Through Direct Count from Soil
1 Introduction
2 Materials
2.1 Basic Requirements
2.2 Method 1 (Pre-treatment with Chemical)
2.3 Method 2 (Sieve and Stain Method)
3 Methods
3.1 Method 1(Pre-treatment with Chemical)
3.2 Method 2 (Sieve and Stain Method)
4 Notes
References
Chapter 10: Most Probable Number (MPN) Method for Enumeration of Soil Protists
1 Introduction
2 Materials
2.1 Amoeba Saline Solution (Modified NeffΒ΄s Amoeba Saline (See Note 1)
2.2 Food Bacteria
2.3 Flat-Bottom 96-Well Microplate (e.g., CAT No. 167008, Thermo Fisher Scientific)
2.4 Gas-Permeable Sealing Membrane for Microplates (e.g., Breathe-Easy Sealing Membrane, Diversified Biotech Inc., MA)
3 Methods
3.1 Preparation of a Soil Dilution Series
3.2 Incubation
3.3 Microscopic Observation
3.4 Estimation of MPN Count
4 Notes
References
Part III: Isolation of Protist from Soil
Chapter 11: Standard Solutions and Media Used for Isolation of Soil Protists
1 Introduction
2 Material
2.1 Basic Requirements
2.2 Media
3 Methods
4 Notes
References
Chapter 12: Isolation Techniques for Amoeba
1 Introduction
2 Materials
2.1 Amoeba Saline Solution (Modified NeffΒ΄s Amoeba Saline,) (See Note 1)
2.2 1% Agar Amoeba Saline Media
2.3 Food Bacteria Suspension
2.4 96-Well Microplate
2.5 Fine Capillary Pipette
2.6 Scalpel
3 Methods
3.1 Preparation of a Soil Dilution Series
3.2 Incubation
3.3 Subculturing Amoeba
3.4 Isolation of Amoeba
3.4.1 Migration Method
3.4.2 Pipette Technique
3.4.3 Dilution Technique
4 Notes
References
Chapter 13: Isolation of Ciliates and Flagellates from Soil
1 Introduction
2 Materials
3 Methods
3.1 Collection of Soil Sample
3.2 Incubation of Soil Sample
3.3 Enumeration of Ciliates and Flagellates
3.4 Calculations
3.5 Fixing and Staining
3.6 Identification
4 Notes
References
Chapter 14: Isolation and Enumeration of Mycophagous Protist
1 Introduction
2 Material
3 Methods
3.1 Isolation of Mycophagous Protists
3.2 Enumeration of Mycophagous Protists
4 Notes
References
Part IV: Enrichment of Soil Protists on Laboratory Media
Chapter 15: Media Used for Enrichment of Soil Protists
1 Introduction
2 Materials
2.1 Basic Requirements
2.2 Media
2.2.1 Proteose Peptone Medium
2.2.2 Protozoan Pellet Medium
2.2.3 Wheatgrass Medium (0.1% w/v)
2.2.4 Yeast-Beef Agar Medium (YBA)
2.2.5 Peptone Yeast Glucose (PYG) Agar Medium
3 Methods
3.1 Proteose Peptone Medium
3.2 Protozoan Pellet Medium
3.3 Wheatgrass Medium (0.1% w/v)
3.4 Yeast-Beef Agar Medium (YBA)
3.5 Peptone Yeast Glucose (PYG) Agar Medium
4 Notes
References
Chapter 16: Enrichment of Naked Amoebae Species
1 Introduction
2 Materials
2.1 Plasticwares and Other Requirements
2.2 Media
3 Methods
3.1 Preparation of Media and Inoculation
3.2 Enrichment of Naked Amoebae on Liquid Media
3.3 Enrichment of Naked Amoebae on Solid Agar Media
3.4 Enrichment of Naked Amoebae Formed as Cysts
4 Notes
References
Chapter 17: Enrichment of Ciliates and Flagellates
1 Introduction
2 Materials
2.1 Basic Requirements
2.2 Media
3 Methods
4 Notes
References
Chapter 18: Enrichment of Mycophagous Protists
1 Introduction
2 Materials
2.1 Basic Requirements
2.2 Media
3 Methods
4 Notes
References
Chapter 19: Mono-axenic Cultivation of Protists
1 Introduction
2 Materials
2.1 Glasswares and Equipment
2.2 Media
2.3 Cultures
3 Methods
3.1 Bacterial Culture Preparation
3.2 Protist Culture Preparation
3.3 Mono-axenic Culture Preparation
4 Notes
References
Chapter 20: Axenic Cultivation of Soil Protists
1 Introduction
2 Materials
2.1 Glass- and Plasticwares and Other Requirements
2.2 Media
3 Methods
3.1 Sample Collection and Isolation
3.2 Axenic Cultivation of Soil Protists
3.2.1 Using Antibiotics
3.2.2 Migration Method of Axenicization
4 Notes
References
Part V: Identification Techniques for Soil Protists
Chapter 21: Classical Wet Mount Method for Observing Live Protists
1 Introduction
2 Materials
3 Methods
4 Notes
Reference
Chapter 22: SEM: Sample Preparation, Fixation, and Staining of Protists
1 Introduction
2 Materials
2.1 Reagents
2.2 Chemicals
2.3 Plasticwares
3 Methods
3.1 Method 1
3.1.1 Sample Preparation
3.1.2 Fixation of the Sample
3.1.3 Dehydration
3.1.4 Mounting and Observation Using Scanning Electron Microscope
3.2 Alternative Method
3.2.1 Preparation of Ionic Liquid
3.2.2 Sample Preparation, Fixation, and Staining of Protists
3.2.3 Observation in Scanning Electron Microscope
4 Notes
References
Chapter 23: TEM: Sample Preparation, Fixation, and Staining of Protists
1 Introduction
2 Materials
2.1 Solutions and Chemicals
2.2 Plasticwares
3 Methods
3.1 Sampling
3.2 Fixation
3.3 Dehydration
3.4 Embedding
3.5 Shadow Casting
3.6 Staining and Observation
4 Notes
References
Chapter 24: Protargol Staining Technique for Identification of Soil Protists
1 Introduction
2 Materials
2.1 Fixatives
2.2 Solutions for Staining Procedure
3 Methods
3.1 Montagnes and LynnΒ΄s Filter Procedure
3.1.1 Fixation
3.1.2 Preparation of Filter
3.1.3 Staining Procedure
3.2 Staining Procedure 2
4 Notes
References
Chapter 25: FLUTAX Staining of Cilliates
1 Introduction
2 Materials
3 Method
3.1 FLUTAX 1 Staining
3.2 FLUTAX 2 Staining
4 Notes
References
Chapter 26: DNA Extraction from Trophozoites and Cysts Using Manual Protocol
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 27: DNA Isolation from Single Ciliate Isolate
1 Introduction
2 Materials
2.1 Reagents for DNA Extraction
2.2 Reagents for Agarose Gel Electrophoresis
3 Methods
3.1 Sampling and Single-Cell Isolation
3.2 Pretreatment
3.3 Isolation of Genomic DNA
3.4 Agarose Gel Electrophoresis
4 Notes
References
Chapter 28: Species Identification Through Sequencing
1 Introduction
2 Materials
2.1 Equipment and Other Requirements
2.2 PCR Amplification
2.3 Agarose Gel Electrophoresis
2.4 Sequencing
3 Methods
3.1 Sampling and Single-Cell Isolation
3.2 DNA Extraction
3.3 PCR Primers
3.4 PCR Amplification and Purification
3.5 Sequencing and Bioinformatic Analysis
3.6 DNA Barcoding for Ciliate Diversity Analysis
4 Notes
References
Chapter 29: DNA Barcoding Techniques for Protists
1 Introduction
2 Materials
2.1 DNA Extraction
2.2 PCR Amplification
2.3 Gel Electrophoresis and Purification
2.4 DNA Sequencing
2.5 Bioinformatics Analysis and Barcode Generation
3 Methods
3.1 DNA Extraction
3.2 PCR Amplification
3.3 Analyze PCR Products by Gel Electrophoresis
3.4 DNA Sequencing
3.5 Bioinformatics Analysis
4 Notes
References
Chapter 30: Unequivocal Identification Through Fluorescence In Situ Hybridization (FISH) Technique
1 Introduction
2 Materials
2.1 Basic Requirements
2.2 Solutions
2.3 Equipment
3 Methods
3.1 Preparation of Protist Cells
3.2 FISH Assay
4 Notes
References
Part VI: Other Protistan
Chapter 31: Isolation, DNA Extraction, Amplification, and Gel Electrophoresis of Single-Celled Nonmarine Foraminifera (Rhizari...
1 Introduction
2 Materials
2.1 Isolation
2.2 Guanidine Extraction
2.3 Amplification
2.4 Agarose Gel Electrophoresis
3 Methods
3.1 Isolation of Foraminifera from Samples
3.2 DNA Extraction with Guanidine Buffer
3.3 PCR Amplification
3.3.1 PCR Mix for One Tube (See Note 6)
3.3.2 Amplification Program
3.3.3 Reamplification Program
3.3.4 Amplification and Reamplification Primers
3.4 Agarose Gel Electrophoresis
4 Notes
References
Chapter 32: Isolation and Cultivation and Staining of Paramecium
1 Introduction
2 Material
2.1 Isolation
2.2 Cultivation
2.3 Staining Reagents
3 Methods
3.1 Isolation
3.2 Cultivation
3.3 Staining
4 Notes
References
Chapter 33: Isolation and Cultivation of Euglena
1 Introduction
2 Material
2.1 Sample
2.2 Enrichment Medium
2.3 Euglena Isolation Media
2.4 Euglena Agar Plates
2.5 Other Requirements
3 Method
4 Notes
References
Index