Table Of ContentSPRINGER
LAB MANUAL
Springer
Berlin
Heidelberg
New York
Barcelona
Hong Kong
London
Milan
Paris
Tokyo
Franz Schinner Richard Ohlinger
Ellen Kandeler Rosa Margesin (Eds.)
Methods
in Soil Biology
With 32 Figures
PROF. DR. FRANZ SCHtNNER Doz. DR. ELLEN KANDELER
University of Innsbruck Federal Agency and Research Centre
Institute of Microbiology for Agriculture
TechnikerstraBe 25 Institute for Soil Management
A-6020 Innsbruck SpargelfeldstraBe 191
Austria A-I220 Vienna
Austria
DR, RICHARD OHLINGER
Federal Office for Agrobiology DR. ROSA MARGESIN
Wieningerstra6e 8 University of lnnsbruck
A-4025 lim: Institute of Microbiology
Austria TechnikerstraBe 25
A-6020 Innsbruck
Austria
ISBN-13: 978-3-642-64633-1 Springer-Verlag Berlin Heidelberg New York
Library of Congr~$$ Coraloging-in-Puhlicarinn Oara
Biologis<h~ Aro.ilimorhoden, English.
Merhod! in .oil biology I Franz Schino,r ... ler aLl (ed,.I,
p. cm.
Includes bihliographical ref~r.nc •• and indu.
ISBN-13: 91&-3_642-64633_1 c-ISBN-13: 918-3-642-60966-4
DOl: 10.10 071978·3-642-60966-4
I. ~il bioloS)'-Merhodology. 1. Schinn~r Franz, 1947-
11. Tirl~.
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Springer
Preface
After many requests of colleagues from different European countries
we decided to produce an English version of the second German
edition of Bodenbiologische Arbeitsmethoden. The content of this
book was supplemented with some actual and proved methods.
This book introduces a large spectrum of relevant soil biological
methods that augment classical pedological investigations. They fur
ther facilitate a rapid evaluation of the effects of active substances
and the characterization of soil management, soil types and plant
communities. These methods can be applied to agricultural and for
est soils as well as to virgin soils. With slight variations, composts,
sludges and sediments may also be investigated.
Some methods apply to the determination of metabolic processes
ofliving soil organisms, other methods apply to the determination of
soil enzyme activity. The spectrum of methods described allows the
analysis of the carbon, nitrogen, phosphorus and sulfur cycles of soil
samples. A number of methods for the determination of bacterial
counts and biomass, fungal biomass, mycorrhizae, as well as soil
microbial community structure, were added. Further, methods to
determine various groups of the micro-, meso-, and macrofauna and
algae of soils, and methodological approaches to study microbial
faunal interactions are also included.
The selection of methods in this book was based on thorough
experimental testing of numerous procedures described in the litera
ture. Emphasis was placed on a comprehensive description of all
experi-mental steps and calculation and/or evaluation of results.
Many of the standardized methods were intercalibrated by laborato
ries in different countries.
This book touches on research and applied interests of agricul
ture, forest and soil science, biology, ecology, engineering, and con
servation. It is recommended as a source of standardized methods
for research in the soil sciences, as well as for laboratory courses for
VIII Preface
university, college and upper grade students. For federal and con
tract laboratories, this book may suggest further methods for soil
testing. We thank all authors for contributing, testing and optimizing
the described methods. We also thank the Production Editor and the
editorial staff of Springer-Verlag for publishing this book.
F. Schinner
R. Ohlinger
E. Kandeler
R. Margesin
Contents
I Methods in Soil Microbiology 1
1 Introduction 3
2 Soil Sampling and Sample Preparation 7
2.1 Soil Sampling 7
2.2 Sample Preparation 9
3 Bacterial Biomass 12
3.1 Counting by Fluorescence Microscopy 15
3.2 Plate Count Technique 20
3.3 Physiological Groups by Most Probable Number
Method 26
3.4 Denitrifiers by MPN Method 28
3.5 Nitrifiers by MPN Method 32
3.6 Free-living Aerobic and Microaerophilic Nitrogen
Fixers by MPN Method 37
3.7 Identification and Quantification with Monoclonal
Antibodies 41
4 Indirect Estimation of Microbial Biomass 47
4.1 Fungal Biomass by Ergosterol Content 49
4.2 Biomass-C by Fumigation-Incubation Technique 52
4.3 Biomass-C by Fumigation-Extraction Technique 56
4.4 Biomass-N by Fumigation-Extraction Technique 58
4.5 Ninhydrin-Reactive N by Fumigation-Extraction
Technique 60
4.6 Biomass-P by Fumigation-Extraction Technique 62
4.7 Substrate-Induced Respiration 64
4.8 Substrate-Induced Heat Output 68
4.9 Mathematical Analysis of Respiration Curves 73
X Contents
5 Community Structure of Soil Microorganisms 76
5.1 Fatty Acid Patterns of Microbial Phospholipids
and Lipopolysaccharides 80
6 Soil Respiration 93
6.1 Soil Respiration by Titration 95
6.2 Soil Respiration by Infrared Gas Analysis 98
6.3 Basal Respiration by a Syringe Micromethod 102
6.4 Soil Respiration by Oxygen Uptake 107
7 Dynamics of Soil Organic Matter III
7.1 In Situ Studies of Litter Decomposition ll4
7.2 Humification ll6
7.3 Soil Organic Matter Turnover by Carbon-Stable
Isotopes ll9
8 Non-Symbiontic Nitrogen Fixation 122
8.1 Nitrogenase Activity by Acetylene Reduction 125
8.2 Microbial Ethylene Production 129
8.3 Nitrogenase Activity by 15N -Fixation 130
2
9 Nitrogen Mineralization l35
9.1 In Situ Studies of N -Mineralization 136
9.2 N-Mineralization Under Aerobic Conditions l39
9.3 N-Mineralization Under Waterlogged Conditions 141
10 Nitrification and Denitrification 144
10.1 Potential Nitrification 146
10.2 Nitrification During Long-Term Incubation 149
10.3 Actual and Potential Denitrification Rates by
Acetylene-Inhibition Technique 151
10.4 In Situ Quantification of Total Denitrification
Losses by Acetylene-Inhibition Technique 155
11 Enzymes Involved in Nitrogen Metabolism 162
ILl Protease Activity 165
11.2 Arginine Deaminase Activity 168
11.3 Urease Activity by Colorimetric Technique 171
11.4 Urease Activity by Distillation Technique 174
ll.5 Nitrate Reductase Activity 176
11.6 Denitrification Enzyme Activity 179
Contents XI
12 Enzymes Involved in Carbon Metabolism 185
12.1 CM-Cellulase Activity 190
12.2 Xylanase Activity 193
12.3 Invertase Activity 196
12.4 ~-Glucosidase Activity 198
12.5 Chitinase Activity 201
12.6 Lipase Activity by Fluorometry 204
12.7 Lipase Activity by Titration 206
13 Enzymes Involved in Phosphorus Metabolism 208
13.1 Phosphomonoesterase Activity with the
Substrate Phenylphosphate 210
13.2 Phosphomonoesterase Activity with the
Substrate p-Nitrophenyl Phosphate 213
13.3 Phosphodiesterase Activity 217
13.4 Phosphotriesterase Activity 220
13.5 Inorganic Pyrophosphatase Activity 222
13.6 Phospholipase-C Activity 225
14 Enzymes Involved in Sulfur Metabolism 228
14.1 Arylsulfatase Activity 230
14.2 Dimethyl Sulfoxide Reduction 232
15 Enzymes Involved in Intracellular Metabolism 235
15.1 Catalase Activity 237
15.2 Dehydrogenase Activity with the Substrate TTC 241
15.3 Dehydrogenase Activity with the Substrate INT 243
16 Adenosine Phosphates 246
16.1 Adenylates and Adenylate Energy Charge (AEC) 249
16.2 ATP by TCA-Extraction Technique 252
16.3 ATP by Sulfuric Acid-Extraction Technique 260
17 Indolic and Phenolic Compounds 266
17.1 Auxins, L-Tryptophan and Related Indolic and
Phenolic Catabolites 268
18 Ectomycorrhizae 281
18.1 Quantification 282
18.2 Morphological and Anatomical Characterization 286
