Methods of Soil Analysis, Part 2: Microbiological and Biochemical Properties: 12 (SSSA Book Series)

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	Title Page
	Copyright Page
	Table of Contents
	Foreword
	Preface
	Contributors
	Conversion Factors for SI and non-SI Units
1
	Chapter 1 Soil Sampling for Microbiological Analysis
		1-1 Principles
			1-1.1 Judgement Samples
			1-1.2 Simple Random Samples
			1-1.3 Stratified Random Samples
			1-1.4 Systematic Samples
			1-1.5 Composite Samples
			1-1.6 Number of Samples to Take
		1-2 Methods
			1-2.1 Bulk Samples for Isolation
			1-2.2 Characterization Studies
			1-2.3 Processing Samples for Microbiological Studies
		1-3 Sources of Error
		1-4 Concluding Remarks
		References
2
	Chapter 2 Statistical Treatment of Microbial Data
		2-1 Characteristics of the Lognormal Distribution
		2-2 Diagnosing Lognormality
			2-2.1 Sample Statistics as Indicators of Asymmetry
			2-2.2 Graphical Methods
			2-2.3 Goodness-of-Fit Tests
		2-3 Estimating Population Parameters from Sample Data
			2-3.1 General Considerations
			2-3.2 The Uniformly Minimum Variance Unbiased Estimators
			2-3.3 Application of the UMVU Estimators
			2-3.4 Confidence Intervals
		2-4 Selecting the Appropriate Location Parameter
			2-4.1 The Mean vs. the Median: General Considerations
			2-4.2 Effects of Sample Volume on the Mean and Median
			2-4.3 The Median as the Location Parameter of Choice
			2-4.4 The Mean as the Location Parameter of Choice
		2-5 Hypothesis Testing
			2-5.1 General Considerations
			2-5.2 Efficacy of Hypothesis Testing Procedures
			2-5.3 Recommendations
		2-6 Sample Number Requirements
			2-6.1 Estimating the Mean
			2-6.2 Compositing
			2-6.3 Power of Statistical Tests
		2-7 Concluding Remarks
		Appendix 1: Statistical Tables
		Appendix 2: Computer Programs
		References
3
	Chapter 3 Soil Sterilization
		3-1 Principles
		3-2 Moist Heat
			3-2.1 Materials
			3-2.2 Procedures
			3-2.3 Comments
		3-3 Dry Heat
			3-3.1 Materials
			3-3.2 Procedures
			3-3.3 Comments
		3-4 Gamma Irradiation
			3-4.1 Materials
			3-4.2 Procedures
			3-4.3 Comments
		3-5 Microwave Irradiation
		3-6 Gaseous Compounds
			3-6.1 Materials
			3-6.2 Procedures
			3-6.3 Comments
		3-7 Nongaseous Compounds
			3-7.1 Mercuric Chloride
			3-7.2 Azide
		3-8 Conclusions
		References
4
	Chapter 4 Soil Water Potential
		4-1 Principles
		4-2 Materials
		4-3 Procedure
		4-4 Comments
		References
5
	Chapter 5 Most Probable Number Counts
		5-1 Principles
			5-1.1 Theoretical Assumptions
			5-1.2 The Mathematical Solution of the Most Probable Number
			5-1.3 Confidence Limits and Population Estimate Separation
			5-1.4 Reliability of Experimental Results and Tests of Technique
			5-1.5 Calibration of MPN Technique
		5-2 Methodology
			5-2.1 Experimental Design
			5-2.2 Materials
			5-2.3 Soil Sampling, Preparation and Storage Prior to Dilution
			5-2.4 Preparation of the Dilution Series and Culture of Inoculated Test Units
			5-2.5 Recording Experimental Results
			5-2.6 Assigning Tabular Population Estimates to Results
			5-2.7 Correcting for Initial Dilution and Inoculant Volume
			5-2.8 Constructing Confidence Limits
			5-2.9 Sample Calculations
		5-3 Comments
		Acknowledgment
		References
6
	Chapter 6 Light Microscopic Methods for Studying Soil Microorganisms
		6-1 Sampling of Soil for Microscopic Observation
			6-1.1 Time of Sampling
			6-1.2 Depth of Sampling
			6-1.3 Rhizosphere vs. Nonrhizosphere Soil
		6-2 Microscopic Enumeration of Total Bacteria in Soil
			6-2.1 Separation of Bacteria from Soil by Dispersion, Dilution, and Selective Filtration
			6-2.2 Enumeration of Specific Bacterial Types by Immunofluorescence Microscopy
			6-2.3 Separation of Bacteria from Soil by Extraction and Flocculation
			6-2.4 Separation of Bacteria from Soil by Density Gradient Centrifugation
			6-2.5 Determining the Efficiency of Recovering Bacteria from Soil for Microscopic Enumeration
		6-3 Determining the Proportion of Viable Soil Bacteria Using a Cell Elongation Assay
			6-3.1 Principle
			6-3.2 Procedure
			6-3.3 Comments
		6-4 Determining the Proportion of Viable Soil Bacteria by Following the Reduction of Tetrazolium Dyes to Formazan
			6-4.1 Principle
			6-4.2 Procedure
			6-4.3 Staining Procedure (from Schmidt & Paul, 1982
			6-4.4 Comments
		6-5 Microscopic Determination of the Mycelial Length of Soil Fungi
			6-5.1 Agar Film Technique
			6-5.2 Membrane Filtration Method for Determining the Mycelial Length
		6-6 Determining the Proportion of Metabolically Active Fungal Mycelia by Following the Hydrolysis of Fluorescein Diacetate
			6-6.1 Procedure
			6-6.2 Comments
		6-7 Determining the Proportion of Metabolically Active Fungal Mycelia by Following the Reduction of Tetrazolium Dyes to Formazan
			6-7.1 Procedure
			6-7.2 Comments
		6-8 Determining the Weight of Soil Biomass from Microscopic Estimates of Biovolume
			6-8.1 Influence of Cellular Water Content on a Dry Weight: Biovolume Conversion Factor
			6-8.2 Comments
			6-8.3 Calculation of Bacterial Biomass (WB)
			6-8.4 Calculation of Fungal Biomass (WF)
			6-8.5 Comments
		6-9 Microscopic Enumeration of Bacteria with Fluorescently Labeled Oligonucleotides Directed at Specific Regions of 16S Ribosomal RNA
			6-9.1 Procedure (from Amann et al., 1990)
			6-9.2 Procedure (from Tsien et al., 1990)
		Acknowledgment
		References
7
	Chapter 7 Viruses
		7-1 General Principles of Analysis
		7-2 Phages
			7-2.1 Direct Counts
			7-2.2 Enrichment Procedures
			7-2.3 Purification and Storage
		7-3 Enteric Viruses
			7-3.1 Direct Counts
		References
8
	Chapter 8 Recovery and Enumeration of Viable Bacteria
		8-1 Principles of Enumerating Soil Bacteria
			8-1.1 Limitations of Plate Counts
		8-2 Materials and Equipment
			8-2.1 Field
			8-2.2 Laboratory
		8-3 Collection and Preparation of Soil Samples
		8-4 Release of Bacteria from Soils
		8-5 Diluents Used in Recovery and Enumeration of Soil Bacteria
		8-6 Preparation of Serial Dilutions
			8-6.1 Procedure-Ten-Fold Dilution Series
			8-6.2 Comments
		8-7 Plating Techniques
			8-7.1 The Pour Plate Technique
			8-7.2 The Spread Plate Technique
			8-7.3 The Drop Plate Method
		8-8 Media for Enumeration of Soil Bacteria
			8-8.1 Addition of Inhibitors to Culture Media
			8-8.2 Some Examples of Selective Media
			8-8.3 Comments
			8-8.4 "Strength" of Culture Media for Soil Bacteria
		8-9 Analysis and Presentation of Plate Count Data
			8-9.1 The "30 to 300 Rule"
		8-10 Conclusion
		References
9
	Chapter 9 Coliform Bacteria
		9-1 Recovery and Enumeration of Fecal Coliforms from Soil
			9-1.1 Collection of Samples-Soil
			9-1.2 Enumeration of Bacteria
		9-2 Detection and Enumeration of Total Coliforms
			9-2.1 Multiple Tube Fermentation
			9-2.2 Confirmation of Fecal Coliforms
		9-3 Rapid Test for Detection of E. Coli in Soil
			9-3.1 Introduction
			9-3.2 Materials
			9-3.3 Supplies
			9-3.4 Procedure
			9-3.5 Comments
		9-4 Direct Methods for Detection of E. Coli in Soil
		Acknowledgment
		References
10
	Chapter 10 Autotrophic Nitrifying Bacteria
		10-1 Enumeration by Most Probable Number
			10-1.1 Ammonium Oxidizers by Most Probable Number
			10-1.2 Nitrite Oxidizers by Most Probable Number
		10-2 Diversity of Nitrifiers
			10-2.1 Introduction
			10-2.2 Materials
			10-2.3 Procedure
			10-2.4 Comments
		10-3 Immunofluorescence Examination
			10-3.1 Introduction
			10-3.2 Materials
			10-3.3 Procedure
		10-4 Isolation of Nitrifiers
			10-4.1 Introduction
			10-4.2 Materials
			10-4.3 Procedure
		10-5 Maintenance of Pure Cultures
			10-5.1 Introduction
			10-5.2 Materials
			10-5.3 Procedure
		10-6 Nitrifying Activity in Soils
			10-6.1 Nitrifying Potential of Soil
			10-6.2 Short-Term Nitrifying Activity
		References
11
	Chapter 11 Free-Living Dinitrogen-Fixing Bacteria
		11-1 The Acetylene Reduction Assay
		11-2 Methods for Dinitrogen Fixers in General
			11-2.1 Principles
			11-2.2 Classification into Major Genera
		11-3 Methods for Azotobacteraceae
			11-3.1 Principles
			11-3.2 Method for Azotobacter and Azomonas
			11-3.3 Method for Beijerinckia
		11-4 Method for Methanotrophs
			11-4.1 Principles
			11-4.2 Plate Count Method for Methanotrophs
			11-4.3 Most Probable Number Method for Methanotrophs
		11-5 Method for Hydrogen-Using Dinitrogen Fixers
			11-5.1 Principles
			11-5.2 Prepared Materials
			11-5.3 Procedure
		11-6 Method for Cyanobacteria
			11-6.1 Principles
			11-6.2 Prepared Materials
			11-6.3 Procedure
		11-7 Method for Photosynthetic Purple Nonsulfur Bacteria
			11-7.1 Principles
			11-7.2 Prepared Materials
			11-7.3 Procedure
		11-8 Method for Clostridia
			11-8.1 Principles
			11-8.2 Prepared Materials
			11-8.3 Procedure
			11-8.4 Comments
		11-9 Method for Sulfate-Reducing Bacteria
			11-9.1 Principles
			11-9.2 Prepared Materials
			11-9.3 Procedure
		References
12
	Chapter 12 Legume Nodule Symbionts
		12-1 Nodule Collection and the Isolation of Symbionts
			12-1.1 Principles
			12-1.2 Materials
			12-1.3 Procedure
			12-1.4 Comments
		12-2 Cultivation of Nodule Symbionts
			12-2.1 Principles
			12-2.2 Materials
			12-2.3 Procedure
		12-3 Maintenance of Cultures
			12-3.1 Principles
			12-3.2 Materials
			12-3.3 Procedures
			12-3.4 Comments
		12-4 Enumeration of Nodule Symbionts in Soil and Inoculants
			12-4.1 Principles
			12-4.2 Materials
			12-4.3 Procedure
			12-4.4 Comments
		12-5 Inoculants for Field Experimentation
			12-5.1 Principles
			12-5.2 Materials
			12-5.3 Procedure
			12-5.4 Comments
		12-6 Inoculation of Seed
			12-6.1 Principles
			12-6.2 Materials
			12-6.3 Procedure
		12-7 Field Experimentation Involving Inoculation
			12-7.1 Principles
		12-8 Growth-Pouch Infection Assays
			12-8.1 Principles
			12-8.2 Materials
			12-8.3 Procedure
			12-8.3 Comments
		References
13
	Chapter 13 Anaerobic Bacteria and Processes
		13-1 Principles
		13-2 Methods for Removal of Oxygen
			13-2.1 Removal of Oxygen from Gas Lines and Filter-Sterilization of Gases (Martin, 1971; Zehnder, 1976)
			13-2.2 Cold Catalytic Oxygen Removal with Hydrogen
			13-2.3 Other Methods
		13-3 Methods for Reduction of Media
			13-3.1 Materials
			13-3.2 Procedure
			13-3.3 Comments
		13-4 Redox Indicators
			13-4.1 Methylene Blue (Skinner, 1971)
			13-4.2 Resazurin
			13-4.3 Phenosafranine (Bryant, 1963)
			13-4.4 Comments
		13-5 Culture Methods
			13-5.1 Conventional Anaerobic Techniques
			13-5.2 Strict Anaerobic Techniques
		13-6 Enumeration Methods
			13-6.1 General Comments
			13-6.2 Heterotrophs (Molongoski & Klug, 1976)
			13-6.3 Clostridia (Gibbs & Freame, 1965)
			13-6.4 Sulfate Reducers (Pankhurst, 1971; also see Postgate, 1984)
			13-6.5 Carbon Dioxide Reducers (Braun et al., 1979)
		13-7 Simple Method to Carry Out Anaerobic Incubations of Soil
			13-7.1 Materials
			13-7.2 Procedure
			13-7.2 Comments
		Acknowledgments
		References
14
	Chapter 14 Denitrifiers
		14-1 Nitrate Reducing Processes
			14-1.1 Assimilatory Nitrate Reduction
			14-1.2 Respiratory Denitrification
			14-1.3 Dissimilatory Nitrate Reduction to Ammonium
			14-1.4 Nitrate Respiration
			14-1.5 Nonrespiratory Denitrification
			14-1.6 Chemodenitrification
		14-2 Key Physiological and Ecological Features of Respiratory Denitrifiers
		14-3 Enumeration of Denitrifiers
			14-3.1 Principle
			14-3.2 Materials
			14-3.3 Procedures
			14-3.4 Comments
		14-4 Enumeration of Dissimilatory Nitrate to Ammonium Reducers
			14-4.1 Principle
			14-4.2 Materials and Procedure
			14-4.3 Comments
		14-5 Denitrifier Enzyme Activity
			14-5.1 Principle
			14-5.2 Materials
			14-5.3 Procedure
			14-5.4 Comments
		14-6 Isolation of Denitrifiers
			14-6.1 Principle
			14-6.2 Materials
			14-6.3 Procedure
			14-6.4 Comments
		14-7 Confirmation of Respiratory Denitrification
			14-7.1 Principles
			14-7.2 Materials
			14-7.3 Procedure
			14-7.4 Comments
			14-7.5 Further Characterization and Confirmation of Respiratory Denitrification
		14-8 Taxonomic Identification
		Acknowledgment
		References
15
	Chapter 15 Actinomycetes
		15-1 Enumeration, Enrichment, and Isolation
			15-1.1 Principles
			15-1.2 Direct Methods for Observation and Enumeration
			15-1.3 Methods for Isolation and Enumeration
			15-1.4 Comments
		15-2 Isolation of Physiological Groups
			15-2.1 Principles
			15-2.2 Isolation of Autotrophs
			15-2.3 Isolation and Enumeration of Thermophils
			15-2.4 Isolation of Acidophilic and Alkalophilic Actinomycetes
			15-2.5 Acidophilic Actinomycetes
			15-2.6 Isolation and Enumeration of Microaerophilic Actinomycetes
			15-2.7 Isolation and Detection of Actinomycetes with Biodegradative Activity
			15-2.8 Detection of Antimicrobial Activity
		15-3 Grouping and Identification of Actinomycetes
			15-3.1 Principles
			15-3.2 Cell Wall Analysis
			15-3.3 Maintenance of Isolates
			15-3.4 Morphological Examination
			15-3.5 Comments
		References
16
	Chapter 16 Frankia and the Actinorhizal Symbiosis
		16-1 Characteristics of Frankia
			16-1.1 Attributes of Actinorhizae
		16-2 Isolation, Culturing, and Maintenance of Frankia Strains
			16-2.1 Isolation of Frankia Strains
			16-2.2 Cultivation and Maintenance
			16-2.3 Strain Registry
			16-2.4 Taxonomy
		16-3 Quantification and Differentiation of Frankia Strains
			16-3.1 Quantification
			16-3.2 Strain Differentiation
		16-4 Characterization of Frankia in Symbiosis
			16-4.1 Host Specificity
			16-4.2 Nodule Metabolism (Nitrogen Fixation)
			16-4.3 Nodule Morphology (Sporulation)
			16-4.4 Nodule DNA/RNA Assays
		16-5 Quantification in Soil
			16-5.1 Plants as a Bioassay System
			16-5.2 Fluorescent Antibodies
			16-5.3 DNA/RNA Probes
		16-6 Conclusion
		Acknowledgments
		References
17
	Chapter 17 Filamentous Fungi
		17-1 Qualitative Studies: Isolation Methods
			17-1.1 Choice of Appropriate Isolating Media
			17-1.2 General Soil Studies
			17-1.3 Special Substrates
			17-1.4 Selective Methods
		17-2 Quantitative Methods
			17-2.1 Introduction
			17-2.2 Direct Observation Methods
			17-2.3 Chemical Methods
			17-2.4 Physiological Methods
		References
18
	Chapter 18 Vesicular-Arbuscular Mycorrhizal Fungi
		18-1 Quantification of Vesicular-Arbuscular Mycorrhizal Propagules in Soil
			18-1.1 Introduction
			18-1.2 Most Probable Number Assay
			18-1.3 Infectivity Assays
		18-2 Quantification of Vesicular-Arbuscular Mycorrhizal Colonization in Roots
			18-2.1 Visualizing Vesicular-Arbuscular Mycorrhizal Fungi in Roots
			18-2.2 Estimation of Colonized Root Length
			18-2.3 Chemical Determinations
		18-3 Quantification of Vesicular-Arbuscular Mycorrhizal External Hyphae
			18-3.1 Introduction
			18-3.2 Indirect Methods for Total Hyphae
			18-3.3 Direct Methods for Total Hyphae
			18-3.4 Detection of Active Hyphae
		18-4 Recovery of Vesicular-Arbuscular Mycorrhizal Fungal Spores
			18-4.1 Wet Sieving and Decanting/Density Gradient Centrifugation
		18-5 Identification of Vesicular-Arbuscular Mycorrhizal Fungi
		18-6 Assessment of Growth Response and Selection of Effective Isolates
			18-6.1 Phosphorus-Response Curves and Mycorrhizal Dependency
			18-6.2 Screening for Effective Isolates
		18-7 Production and Use of Vesicular-Arbuscular Mycorrhizal Inocula
			18-7.1 Soil-Based Pot Cultures
			18-7.2 Soil-Less Media
			18-7.3 Nutrient Flow and Aeroponic Systems
			18-7.4 Storage of Inoculum
			18-7.5 Application of Vesicular-Arbuscular Mycorrhizal Inocula
		18-8 Monoxenic Cultures for Basic Research
		References
19
	Chapter 19 Isolation of Microorganisms Producing Antibiotics
		19-1 General Principles
			19-1.1 Preface
			19-1.2 Sampling Strategies
			19-1.3 Selection of Isolation Method
			19-1.4 Selection of Indicator Organisms
			19-1.5 Assay Standardization
		19-2 Microbiological Media
			19-2.1 General Comments
			19-2.2 Common Media for Antibiosis Assays
		19-3 Preparation of Inocula
			19-3.1 General Comments
			19-3.2 Bacterial Inocula
			19-3.3 Fungal Inocula
		19-4 Dual Culture Detection Methods
			19-4.1 Introduction
			19-4.2 Microbial Lawn Technique
			19-4.3 Fungal Disk Technique
			19-4.4 Cross Streak Technique
		19-5 Culture Filtrate Methods
			19-5.1 Introduction
			19-5.2 Preparation of Culture Filtrates
			19-5.3 Paper Disk Technique
			19-5.4 Agar Well Technique
			19-5.5 Radial Growth Technique
			19-5.6 Biomass Technique
		19-6 Screening Methods
			19-6.1 Introduction
			19-6.2 Single Agar Layer Technique
			19-6.3 Multiple Agar Layer Techniques
		19-7 Methods for Selected Classes of Compounds
			19-7.1 Introduction
			19-7.2 Bacteriocins
			19-7.3 Siderophores
			19-7.4 Mycolytic Enzymes
			19-7.5 Volatile Compounds
		19-8 Concluding Comments
		References
20
	Chapter 20 Microbiological Procedures for Biodegradation Research
		20-1 The Enrichment Culture
			20-1.1 Principles
			20-1.2 Materials
			20-1.3 Procedure
		20-2 Isolation of Pure Cultures
			20-2.1 Principles
			20-2.2 Materials
			20-2.3 Procedures
		20-3 Maintenance of Cultures
			20-3.1 Principles
			20-3.2 Materials
			20-3.3 Procedure
		20-4 Growth in Liquid Cultures
			20-4.1 Principles
			20-4.2 Materials
			20-4.3 Procedure
		20-5 Preparation of Washed Cell Suspensions
			20-5.1 Principles
			20-5.2 Materials
			20-5.3 Procedure
		20-6 Preparation and Use of Cell-Free Extracts
			20-6.1 Principles
			20-6.2 Materials
			20-6.3 Procedure
		20-7 Oxygen Consumption
			20-7.1 Principles
			20-7.2 Materials
			20-7.3 Procedure
		20-8 Chloride Determination
			20-8.2 Materials
			20-8.2 Materials
			20-8.3 Procedure
		20-9 Conclusion
		References
21
	Chapter 21 Algae and Cyanobacteria
		21-1 Identification of Soil Algae and Cyanobacteria
			21-1.1 Principles
			21-1.2 Materials and Procedure
		21-2 Direct Methods for Enumeration
			21-2.1 Principles
			21-2.2 Materials and Procedure for Collecting Soil Veneers
			21-2.3 Microscopy and Enumeration by Cell Counting
			21-2.4 Enumeration by Chlorophyll Autofluorescence
			21-2.5 Implanted Slide Method
			21-2.6 Method for Diatom Frustules
			21-2.7 Methods for Cyanobacteria in Rice Fields
			21-2.8 Procedure for Endolithic Microalgae
		21-3 Indirect Methods for Enumeration
			21-3.1 Enumeration of Colony-Forming Units on Solid Media
			21-3.2 Most Probable Number Method for Enumeration
			21-3.3 Chlorophyll Extraction and Quantification
		21-4 Methods for Isolation and Purification of Microalgal Cultures
			21-4.1 Procedure for Preparing Fine Capillary Pipets
			21-4.2 Isolation and Purification by Repeated Washing
			21-4.3 Isolation and Purification by the Streak Plate Method
			21-4.4 Purification by the Centrifugation Technique
			21-4.5 Purification by the Zoospore Technique
		21-5 Methods for Growth and Storage of Microalgal Cultures
			21-5.1 Culture Methods and Growth Media
			21-5.2 Storage and Preservation Methods
		21-6 Methods for Estimating Photosynthesis
		21-7 Methods for Measuring Cyanobacterial Dinitrogen Fixation
		21-8 Methods for Studying Endosymbiotic Cyanobacteria in Cycad Roots
			21-8.1 Principles
			21-8.2 Procedure for Coralloid Roots and Root Sections
			21-8.3 Procedure for Isolating Endosymbiotic Cyanobacteria
		Acknowledgment
		References
22
	Chapter 22 Nematodes
		22-1 Nematode Sampling
			22-1.1 Nematode Distribution
			22-1.2 Sample Collection
			22-1.3 Sampling Pattern
			22-1.4 Timing of Sampling Collections
			22-1.5 Sample Size
			22-1.6 Nematode Sampling for Ecological Studies
		22-2 Extraction of Nematodes from Soil
			22-2.1 Cobb Sieving and Decanting (Wet Sieving) (Cobb, 1918)
			22-2.2 Modified Baermann Funnel Method
			22-2.3 Density (Sucrose) Centrifugation
			22-2.4 Elutriation (Fig. 22-4A)
			22-2.5 Extracting Heterodera Cysts
		22-3 Extraction of Nematodes from Plant Material
			22-3.1 Direct Examination of Plant Material
			22-3.2 Baermann Funnel Extraction
			22-3.3 Root-Incubation Technique
			22-3.4 Mist Chamber Extraction (Seinhorst, 1950)
		22-4 Microscopic Observation and Identification of Nematodes
			22-4.1 Temporary Mounts
			22-4.2 Permanent Mounts
		22-5 Nematode Identification
			22-5.1 Nematodes with Stylets
			22-5.2 Nematodes without Stylets
		References
23
	Chapter 23 Protozoa
		23-1 First Considerations
			23-1.1 Considering Soil
			23-1.2 Environmental Selection
		23-2 Protozoan Ecology
			23-2.1 Ecological Roles
			23-2.2 Protozoan Communities
			23-2.3 Numbers and Food Resources
			23-2.4 Escape from Adverse Conditions
		23-3 Methods of Enumeration
			23-3.1 Direct Observation Methods
			23-3.2 Indirect Enumeration
		23-4 Identification
		23-5 Summary
		Acknowledgment
		References
24
	Chapter 24 Arthropods
		24-1 Principles
		24-2 Methods
			24-2.1 Evaluation of Biota in the Field
			24-2.2 Sampling Soil Cores
			24-2.3 Extraction of Soil Arthropods by Physical Methods
		24-3 Processing the Extracted Biota Sample
			24-3.1 Comments
		24-4 Biota Identification
		24-5 Preservation and Archiving
			24-5.1 Preservation
		24-6 Archiving
			24-6.1 Assigning Morphospecies to Functional Ecological Groupings
			24-6.2 Transforming Census Data
		24-7 Rearing
		24-8 Statistical Methods to Analyze Diversity
		References
25
	Chapter 25 Carbon Utilization and Fatty Acid Profiles for Characterization of Bacteria
		25-1 Characterization of Bacteria
		25-2 Carbon Source Utilization
			25-2.1 Principles
			25-2.2 Materials
			25-2.3 Procedure
		25-3 Fatty Acid Analysis
			25-3.1 Principles
			25-3.2 Materials
			25-3.3 Procedure
		References
26
	Chapter 26 Multilocus Enzyme Electrophoresis Methods for the Analysis of Bacterial Population Genetic Structure
		26-1 Principles
		26-2 Methods
			26-2.1 Preparation of Enzyme Extracts
			26-2.2 Electrophoresis
			26-2.3 Enzyme Staining
			26-2.4 Collecting and Analyzing Data
		References
27
	Chapter 27 Spontaneous and Intrinsic Antibiotic Resistance Markers
		27-1 Selection of Spontaneous Antibiotic Resistant Strains
			27-1.1 Principles
			27-1.2 Materials
			27-1.3 Procedures
		27-2 Selection of Intrinsic Antibiotic Resistant Strains
			27-2.1 Principles
			27.2.2 Materials
			27-2.3 Procedures
		27-3 Evaluation of SAR and IAR Strains
			27-3.1 Principles
			27-3.2 Materials
			27-3.3 Procedures
		References
28
	Chapter 28 Serology and Conjugation of Antibodies
		28-1 Antibodies
			28-1.1 Microbes as Antigens
			28-1.2 Antibodies
			28-1.3 Antibodies Against Microorganisms
			28-1.4 Procedures for Utilization of Antibodies
		28-2 Adsorption of Cross-Reactive Antibodies from Polyclonal Antiserum
			28-2.1 Principles
			28-2.2 Materials
			28-2.2 Procedure
		28-3 Purification of Antibodies from Antiserum
			28-3.1 Principles
			28-3.2 Materials
			28-3.3 Procedure
		28-4 Conjugation of Antibodies with Fluorescein Isothyocyanate
			28-4.1 Principles
			28-4.2 Materials
			28-4.3 Procedure
		28-5 Storage of Antibodies and Antibody Conjugates
		28-6 Immunoassays
			28-6.1 Principles
			28-6.2 Peroxidase-Linked Immunosorbent Assay
			28-6.3 Alkaline Phosphatase-Linked Immunosorbent Assay
			28-6.4 Checkerboard Titration
			28-6.5 Biotinylated Second Antibody for Enhanced ELISA
			28-6.6 Immunoblot to Identify Colonies on a Dilution Plate
		References
29
	Chapter 29 Whole-Cell Protein Profiles of Soil Bacteria by Gel Electrophoresis
		29-1 Principles
		29-2 Methods
			29-2.1 Growth and Collection of Cells
			29-2.2 Disruption of Cells
			29-2.3 Preparation of Protein Extracts for SDS-PAGE
			29-2.4 Electrophoresis
			29-2.5 Visualizing Protein Profiles
			29-2.6 Storing and Recording of Protein Profiles
		29-3 General Comments
		References
30
	Chapter 30 Plasmid Profiles
		30-1 Plasmid Chromosome Relationships
			30-1.1 Types of Plasmids
			30-1.2 Functions of Plasmids
		30-2 Plasmid Profile Analysis
			30-2.1 Introduction
			30-2.2 In Situ Lysis of Bacteria Containing Plasmids Greater than 20 kb
			30-2.3 Extraction of Plasmids from Cells, followed by CsCI Ultracentrifugation Purification and Gel Electrophoresis
			30-2.4 Alkaline Lysis Miniprep
			30-2.5 Storage of Plasmid DNA
		30-3 Applications of Plasmid Profile Analyses for Soil Bacteria
			30-3.1 Plasmid Functions
			30-3.2 Classification of Soil Bacteria
		References
31
	Chapter 31 DNA Fingerprinting and Restriction Fragment Length Polymorphism Analysis
		31-1 DNA Fingerprinting
			31-1.1 Introduction
			31-1.2 DNA Isolation and Purification
			31-1.3 Restriction Enzyme Selection and Use
			31-1.4 Gel Electrophoresis
			31-1.5 Principles
			31-1.6 Methods
		31-2 RFLP Analyses
			31-2.1 Introduction
			31-2.2 Principles
			31-2.3 Methods
		References
32
	Chapter 32 Nucleic Acid Probes
		32-1 Probe Selection
		32-2 Isolation and Purification of Fragments to be Used as Probes
			32-2.1 Principles
			32-2.2 Materials
			32-2.3 Procedure
		32-3 Labeling of Probes
			32-3.1 Introduction
			32-3.2 Nonradioactive Probes
			32-3.3 Nick Translation
			32-3.4 Random Primer Fill-in
			32-3.5 Single Stranded Probes
			32-3.6 End Labeling of Synthetic Oligonucleotides
		32-4 Hybridization
			32-4.1 Introduction
			32-4.2 Colony Hybridization and Dot/Slot Blots
		32-5 Detection Systems
			32-5.1 Principles
		References
33
	Chapter 33 Marking Soil Bacteria with lacZY
		33-1 Principles
		33-2 Materials
			33-2.1 Cells
			33-2.2 Media
			33-2.3 Chemicals and Reagents
		33-3 Procedure
			33-3.1 Mating and Selection
			33-3.2 Confirmation
			32-3.3 Identification
		33-4 Exconjugant-Type Isolate Vigor
		33-5 Marker Stability
		33-6 Recovery from Nonsterile Soil
		33-7 Attributes and Deficiencies
		33-8 Comments
		References
34
	Chapter 34 Detection of Specific DNA Sequences in Environmental Samples Via Polymerase Chain Reaction
		34-1 Theory
		34-2 Primer Design and Amplification Protocol
			34-2.1 Design of Primers
			34-2.2 Special Apparatus and Reagents
			34-2.3 Procedures
		34-3 Optimization of Amplification
			34-3.1 Amplification Cycles
			34-3.2 Primer Concentration
			34-3.3 Mg2+ Concentration
			34-3.4 Temperature Cycling Parameters
			34-3.5 Nucleotide Concentrations
			34-3.6 Taq Polymerase
		34-4 Identification of Amplified Products
		34-5 Quality Control
		34-6 Specificity of Amplification
		34-7 Sensitivity of Amplification
		34-8 Applications in Environmental Microbiology
			34-8.1 Detection of Specific DNA Target Sequences in Soil Samples
			34-8.2 Detection of Marker Enzyme Synthesis for Bioassay Studies
			34-8.3 Evolutionary and Biodiversity Studies
			34-8.4 Horizontal Gene Transfer and Genomic Rearrangements
		References
35
	Chapter 35 Isolation and Purification of Bacterial DNA from Soil
		35-1 General Considerations
			35-1.1 Biomass
			35-1.2 Organic/Humic Content of Soils
			35-1.3 Clay Content of Soils
		35-2 Bacterial Fractionation Approach for Recovery of Bacterial Community DNA
			35-2.1 Principles
		35-3 Direct Lysis Approach for the Recovery of Total Bacterial Community DNA
			35-3.1 Principles
		35-4 Bacterial Fractionation Protocol
			35-4.1 Materials
			35-4.2 Reagents
			35-4.3 Procedure
		35-5 Direct Lysis Protocol
			35-5.1 Materials
			35-5.2 Reagents
			35-5.3 Procedure
		35-6 Fractionation of DNA Gradients, Final Purification and Quantitation of Bacterial Community DNA
			35-6.1 Fractionation of DNA Bands from Cesium Chloride Gradients
			35-6.2 Isopropanol Extraction of Ethidium Bromide from DNA
			35-6.3 Desalting and Concentration of DNA
			35-6.4 Spectrophotometric Quantitation of DNA
			35-6.5 Quantitation of DNA by Ultraviolet Fluorescence in the Presence of Ethidium Bromide
		Acknowledgments
		References
36
	Chapter 36 Microbial Biomass
		36-1 Soil Sampling, Preparation, and Storage
		36-2 Physiological Methods
			36-2.1 Chloroform Fumigation Incubation Method
			36-2.2 Substrate-Induced Respiration Method
		36-3 Chemical Methods
			36-3.1 Chloroform Fumigation Extraction Method
			36-3.2 ATP Determinations
		36-4 Comparison of Methods
		References
37
	Chapter 37 Soil Enzymes
		37-1 Principles
			37-1.1 Mechanism of Enzyme Action
		37-2 Factors Affecting Rates of Enzyme Reactions
			37-2.1 Concentration of Enzyme and Substrate
			37-2.2 Temperature
			37-2.3 pH
			37-2.4 Cofactors, Inhibitors, and Ionic Environment
			37-2.5 Enzyme Inhibition
			37-2.6 Enzymes in Soils
		37-3 Assay of Enzymes in Soils
			37-3.1 Amidohydrolases (L-Asparaginase, L-Glutaminase, Amidase, and Urease)
			37-3.2 Phosphatases
			37-3.3 Arylsulfatase
			37-3.4 Rhodanese
			37-3.5 Dehydrogenases
			37-3.6 B-Glucosidase
			37-3.7 Other Enzymes
		References
38
	Chapter 38 Carbon Mineralization
		38-1 General Principles
			38-1.1 Field vs. Laboratory Experiments
		38-2 Experimental Principles
			38-2.1 Alkali Trapping and Detection of Carbon Dioxide
			38-2.2 Detection of Carbon Dioxide in Gaseous Samples
			38-2.3 Detection of Oxygen
			38-2.4 Aeration
			38-2.5 Chambers
			38-2.6 Comparison of Methods
		38-3 Field Methods
			38-3.1 Carbon Dioxide Detection by Soda Lime Absorption
			38-3.2 Soil Carbon Dioxide and Oxygen by Gas Chromatography
		38-4 Laboratory Methods
			38-4.1 Dynamic Method for Carbon Dioxide
			38-4.2 Static Methods for Carbon Dioxide and Oxygen
		References
39
	Chapter 39 Isotopic Methods for the Study of Soil Organic Matter Dynamics
		39-1 Decomposition of 14C-Labeled Organic Matter in Soils
			39-1.1 Introduction
			39-1.2 Obtaining 14C-Labeled Organic Materials
			39-1.3 Methods and Approach to Incubations of 14C-Labeled Organic Materials
		39-2 13C Natural Abundance Technique: Background and Principles
			39-2.1 Introduction
			39-2.2 13C Natural Abundance Technique: Methodology
		39-3 Decomposition of 15N-Labeled Organic Matter in Soils
			39-3.1 Introduction
			39-3.2 Labeling Organic Matter with 15N
			39-3.3 Determination of Mineralization Rates
			39-3.4 Preparation of Samples for 15N Analysis
			39-3.5 Calculations
			39-3.6 Comments
		39-4 Extraction of Labeled Organic Fractions in Studies of Soil Organic Matter Dynamics
			39-4.1 Introduction
			39-4.2 Extraction of Organic Matter Containing Labeled Carbon
			39-4.3 Extraction of Organic Matter Containing Labeled Nitrogen
		39-5 Conclusions
		References
40
	Chapter 40 Practical Considerations in the Use of Nitrogen Tracers in Agricultural and Environmental Research
		40-1 Preparing 15N-Labeled Materials
			40-1.1 Principles
			40-1.2 Determining Nitrogen-15 Concentration Needed
			40-1.3 Fertilizers
		40-2 Field Study Techniques
			40-2.1 Principles
			40-2.2 Managing Field Variability
			40-2.3 Application Techniques
			40-2.4 Plot Type and Size
			40-2.5 Sample Collection and Preparation
		40-3 Preparing for and Measuring Nitrogen-Isotope Ratio
			40-3.1 Principles
			40-3.2 Conversion of Labeled Nitrogen to Ammonium
			40-3.3 Direct Conversion of Labeled Nitrogen to Dinitrogen
			40-3.4 Measuring Nitrogen-Isotope Ratio
		40-4 Sources of Nitrogen-15 Supply and Analytical Service
		References
42
	Chapter 41 Nitrogen Availability Indices
		41-1 Current Status of Nitrogen Availability Indices
			41-1.1 Previous Summaries
			41-1.2 Selective Review of Recent Work
		41-2 Methods
			41-2.1 Field Methods
			41-2.2 Laboratory Methods
			41-2.3 Methods for Inorganic Nitrogen
		References
43
	Chapter 43 Dinitrogen Fixation
		43-1 Acetylene Reduction
			43-1.1 Principles
			43-1.2 Acetylene Reduction for Nodulated Root Systems
			43-1.3 Acetylene Reduction for Grass Systems
		43-2 Nitrogen Difference
			43-2.1 Introduction
			43-2.2 Nitrogen-Difference Method for Controlled Environment
			43-2.3 Nitrogen Difference Method-Field
		43-3 Nitrogen-15 Isotope Techniques
			43-3.1 Introduction
			43-3.2 Isotope Dilution
			43-3.3 Nitrogen-15 Natural Abundance Method
		43-4 Use of Dinitrogen-15 Gas
			43-4.1 For Measuring Dinitrogen Fixation
			43-4.2 Calibration of Acetylene-Reduction Method
		References
44
	Chapter 44 Measuring Denitrification in the Field
		44-1 Methods
			44-1.1 The Acetylene Inhibition Method
			44-1.2 The Nitrogen-15 Method
		44-2 Experimental Protocols
			44-2.1 Static Core Protocol
			44-2.2 Protocol for Closed Chamber Field Gas Flux Measurements Using Acetylene or Nitrogen-15
			44-2.3 Measuring Dinitrogen Emissions from Applied Nitrogen-15-Labeled Fertilizer
		44-3 Problems with Gas Sampling and Storage Containers
		44-4 Gas Diffusion Problems
		References
45
	Chapter 45 Sulfur Oxidation and Reduction in Soils
		45-1 Sulfur Oxidation
			45-1.1 Principles
			45-1.2 Methods
		45-2 Sulfate Reduction
			45-2.1 Principles
			45-2.2 Methods
		References
46
	Chapter 46 Iron and Manganese Oxidation and Reduction
		46-1 Iron-Depositing and Manganese-Oxidizing Heterotrophs
			46-1.1 Enrichment and Isolation
			46-1.2 Dilution Spread Plate Counts
		46-2 Iron-Oxidizing Autotrophs
			46-2.1 Acidophiles (Enrichment, Isolation, and Enumeration)
			46-2.2 Neutrophiles (Enrichment, Isolation, and Enumeration)
		46-3 lron- and Manganese-Reducing Heterotrophs
			46-3.1 Enumeration of Non-Enzymatic Iron- and Manganese-Reducers
			46-3.2 Enrichment and MPN Enumeration of Iron- and Manganese-Respiring Bacteria
		Acknowledgment
		References
47
	Subject Index