Front Matter

LABOR ATORY PROTOCOLS in Applied Life Sciences

Downloaded by [Prakash Bisen] at 06:14 18 March 2014

LABOR ATORY PROTOCOLS Downloaded by [Prakash Bisen] at 06:14 18 March 2014

in Applied Life Sciences

Prakash S. Bisen

Emeritus Scientist Defense Research Development Establishment Defense Research Development Organization Ministry of Defense, Government of India Gwalior, India


CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2014 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S. Government works Version Date: 20140114 International Standard Book Number-13: 978-1-4665-5315-6 (eBook - PDF) This book contains information obtained from authentic and highly regarded sources. Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the validity of all materials or the consequences of their use. The authors and publishers have attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged please write and let us know so we may rectify in any future reprint. Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright.com (http:// www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com


I would like to dedicate this work to one of my mentors, late Professor G. L. Farkas, former director of the Institute of Plant Physiology, Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary. He was not only a great teacher, scientist, and author but also a wonderful human being who helped me develop my interest in biological research.


Contents

Preface lxxxiii About the Book

lxxxv


Acknowledgments lxxxvii Author lxxxix

1. Microscopy

1

1.1 Introduction

1

1.2

Light Microscopy

1

1.3

Atomic Force Microscopy

5

1.4

Bright Field Microscopy

5

1.5

Dark Ground Microscopy

7

1.6

Phase Contrast Microscopy

7

1.7

Confocal Scanning Laser Microscopy

8

1.8

Differential Interference Contrast Microscopy

9

1.9

Polarization Microscopy

10

1.10 Fluorescence Microscopy 1.10.1 Antibodies Can Be Used to Detect Specific Molecules by Fluorescence Microscopy

10 10

1.11 Experiments on Microscopy 1.11.1 Introduction 1.11.2 Microscope Operation 1.11.3 Smear Preparation 1.11.4 Dyes 1.11.5 Staining Procedure 1.11.6 Microscopic Examination of Microorganisms 1.11.7 Hanging Drop Technique 1.11.8 Hemocytometer 1.11.9 Ocular Meter and Stage Micrometer for Micrometry

14 14 15 16 18 18 19 19 20 20

1.12 Electron Microscopy 1.12.1 Transmission Electron Microscopy

21 21

vii

viii

Contents


1.12.2 Scanning Electron Microscopy 1.12.3 Scanning Tunneling Microscope

24 25

1.13 Sample Preparation for Electron Microscopy 1.13.1 Preparation of Specimen Supports 1.13.1.1 Glow Discharge 1.13.1.2 Polylysine Procedure 1.13.2 Sample Preparation and Contrast Enhancement 1.13.3 Embedding, Sectioning, and Staining 1.13.3.1 Replica Formation 1.13.3.2 Freeze-Etching and the Critical Point Technique 1.13.3.3 Freeze-Fracture 1.13.3.4 Shadow-Casting 1.13.3.5 Negative Contrast Technique 1.13.3.6 Positive Staining 1.13.3.7 Kleinschmidt Spreading with Positive Staining and Rotary Shadowing 1.13.3.8 Special Protocol Used with the Kleinschmidt Technique 1.13.4 Visualization of Nucleic Acid Protein Complexes on Polylysine Films 1.13.5 Improving the Quality of the Image and Image Reconstruction 1.13.6 Minimal Beam Exposure 1.13.7 Three-Dimensional Reconstruction of Molecular Structure from Unstained Sample 1.13.8 Image Rotation and Rotational Filtering 1.13.9 Special Mechanisms of Image Formation

39 40 40

1.14 Dark Field Electron Microscopy

40

1.15 Crewe Microscope

41

1.16 Backscatter Scanning Microscope

41

1.17 Experiment on Scanning Electron Microscopy of Cyanobacteria

41

1.18 Transmission Electron Microscopy of Phages (Bacteriophage and Cyanophage)

42

Suggested Readings

44

Important Links

46

2. Microtome

26 27 29 29 30 30 31 31 32 32 34 35 35 38 38 38 39

47

2.1 Introduction

47

2.2 Sectioning

47

2.3

48 48 49 50 50 52

Traditional Histology 2.3.1 Fixation, Dehydration, Embedding, and Staining 2.3.1.1 Fixation 2.3.1.2 Dehydration 2.3.1.3 Embedding 2.3.1.4 Staining

2.4 Ultramicrotome

52

2.5 Cryostat 2.5.1 Tissue Chilling 2.5.1.1 Apparatus and Materials 2.5.1.2 Protocol

53 54 54 54

Contents


2.5.2 Tissue Mounting 2.5.3 Preparation for Sectioning 2.5.4 Cryosectioning 2.5.5 Section Picking

ix 55 55 56 60

2.6 Incubation 2.6.1 Methods of Incubation 2.6.2 Microcell Method

60 60 60

2.7

Electron Microscopy Technique 2.7.1 Special Preparation for Electron Microscope 2.7.1.1 Metal Shadowing 2.7.1.2 Freeze-Fracture and Freeze-Etch Electron Microscopy 2.7.1.3 Spectroscopy (Especially FTIR or Infrared Spectroscopy)

63 63 64 65 66

2.8

Botanical Microtomy

66

2.9

Processing of Tissue 2.9.1 Methods of Tissue Processing 2.9.2 Fixatives 2.9.3 Staining Procedures 2.9.4 Fixation, Dehydration, Embedding, and Staining 2.9.4.1 Introduction 2.9.4.2 Material Preparation 2.9.4.3 Material Required 2.9.4.4 Protocol

67 67 68 69 73 73 74 74 75

Suggested Readings

80

Important Links

80

3. pH and Buffers

81

3.1 Introduction

81

3.2

Acids and Bases 3.2.1 Definitions 3.2.1.1 Acids and Bases 3.2.1.2 Alkali 3.2.1.3 Ampholytes 3.2.2 Strength 3.2.2.1 Strong Acids or Bases 3.2.2.2 Weak Acids or Bases

81 81 81 82 82 83 83 83

3.3

Hydrogen Ion Concentration and pH 83 3.3.1 Definition of pH 83 3.3.2 Water Dissociation 83 3.3.2.1 Temperature and Kw 84 3.3.3 Dissociation of Acids and Bases 84 3.3.3.1 Strong Acids 84 3.3.3.2 Weak Acids 85

3.4

Measurement of pH 3.4.1 pH Indicators 3.4.2 Measurement of pH 3.4.2.1 Glass Electrode 3.4.2.2 Calomel Electrode

86 86 87 87 87


x

Contents 3.5

Titration Curves 3.5.1 Strong Acid and a Strong Base 3.5.2 Weak Acid and a Strong Base

88 88 89

3.6

Buffer Solutions 3.6.1 Theory

89 89

3.7

What Is the pH of a Mixture of 5 mL of 0.1 mol/L Sodium Acetate and 4 mL of 0.1 mol/L Acetic Acid?

90

3.8

How the pH Changed on Adding 1 mL of 0.1 mol/L HCl in the Previous Mixture 90

3.9

Buffers Used in Biochemical Experiments 3.9.1 Common Laboratory Buffers 3.9.1.1 Bicarbonate 3.9.1.2 Phosphate 3.9.1.3 Tris 3.9.1.4 Zwitterionic Buffers

91 91 91 91 92 92

3.10 pH and Life 3.10.1 Animals 3.10.2 Plants 3.10.3 Bacteria

92 92 93 93

3.11 Care and Use of the pH Meter

93

3.12 Titration Curves 3.12.1 Practical Limits of Titration Curves 3.12.2 Solvent Correction 3.12.3 Determination of pKa Values Can Be Obtained from Titration Data by Three Methods

94 94 94

3.13 Determination of pH Using Indicators 3.13.1 Materials 3.13.2 Procedure

95 95 95

3.14 Titration of a Mixture of a Strong and a Weak Acid 3.14.1 Principle 3.14.2 Materials 3.14.3 Procedure

96 96 96 96

3.15 Titration of a Strong Acid with a Strong Base 3.15.1 Materials 3.15.2 Procedure

96 96 97

3.16 Titration of a Weak Acid with a Strong Base 3.16.1 Materials 3.16.2 Procedure

97 97 97

95

3.17 Determination of pKa 97 3.17.1 Materials 97 3.17.2 Procedure 98 3.18 pKa Values of a Dicarboxylic Acid 3.18.1 Materials 3.18.2 Procedure

98 98 98


Contents

xi

3.19 Acetate Buffers 3.19.1 Materials 3.19.2 Procedure

98 98 98

3.20 Titration Curves of Amino Acids 3.20.1 Principle 3.20.2 Materials 3.20.3 Protocol 3.20.4 Results and Calculations

99 99 99 99 99

3.21 Determination of pH of the Given Water/Soil Sample 3.21.1 Principle 3.21.2 Materials 3.21.3 Protocol 3.21.4 Precautions

100 100 101 101 102

3.22 Determination of pH of Fruit Juice 3.22.1 Principle 3.22.2 Protocol

102 102 102

3.23 Measurement of pH of Soil Sample 3.23.1 Principle 3.23.2 Materials 3.23.3 Protocol

103 103 103 103

3.A

Appendix A Indicators of Hydrogen Ion Concentration

104

3.B

Appendix B Buffer Solutions 3.B.1 Introduction 3.B.2 Tris Buffers 3.B.3 Ideal Buffers 3.B.4 Preparation of Imidazole, MOPS, and HEPES Buffers 3.B.5 Preparation of Phosphate Buffers (Gomori Buffers) 3.B.6 Acids and Bases 3.B.7 Buffers and Stock Solutions 3.B.7.1 Phosphate-Buffered Saline 3.B.7.2 10× Tris EDTA (TE) 3.B.7.3 Tris–HCl (1 M) 3.B.7.4 Tris Magnesium Buffer (TM) 3.B.7.5 Tris-Buffered Saline (TBS)

104 104 105 106 106 108 109 109 109 110 110 111 111

Suggested Readings

111

Important Links

112

4. Spectrophotometer

113

4.1 Introduction

113

4.2

Beer–Lambert Law 4.2.1 Molar Extinction Coefficient 4.2.2 Specific Extinction Coefficient

114 118 118

4.3

Limitations of the Beer–Lambert Law

118

4.4

Measurement of Extinction

119


xii

Contents 4.5 Colorimeter 4.5.1 Filters 4.5.2 Output 4.5.3 Directions for Use of the Spectronic 20 Spectrocolorimeter 4.5.3.1 Description of the Optical System 4.5.3.2 Description of the Electrical System 4.5.4 Choice of Instruments for Colorimetry 4.5.4.1 Absorbance Curves of Two Dyes 4.5.4.2 Demonstration of Beer’s Law 4.5.4.3 Colorimetric Estimation of Inorganic Phosphate 4.5.4.4 Validity of Beer’s Law for the Colorimetric Estimation of Creatinine

119 120 120 120 120 122 122 123 123 124

4.6 Spectrophotometry 4.6.1 Applications 4.6.2 Direction for Operations

125 125 129

124

4.7

Infrared Spectroscopy 130 4.7.1 Introduction 130 4.7.2 Principle 131 4.7.3 Instrumental Components 131 4.7.4 Sources 132 4.7.4.1 Globar Source 133 4.7.4.2 Incandescent Wire Source 133 4.7.5 Detectors 133 4.7.6 Sample Preparation 133 4.7.7 Applications 134 4.7.7.1 Absorption Spectrum of p-Nitrophenol 135 4.7.7.2 Absorption Spectra of Coenzyme Cytochrome c 135 4.7.7.3 Determination of the pKa Value of p-Nitrophenol 136 4.7.7.4 Estimation of Barbiturates with the UV–Vis Spectrophotometer 137 4.7.7.5 Estimation of Hemoglobin Spectra 139 4.7.7.6 Ultraviolet Absorption of Proteins and Amino Acids 140

4.8

Fluorescence Spectroscopy 4.8.1 Fluorescence 4.8.2 Quenching 4.8.3 Applications 4.8.4 Intensity of Fluorescence and Concentration 4.8.5 Description of the Instrument 4.8.5.1 Light Source 4.8.5.2 Filters 4.8.5.3 Photomultiplier

141 141 141 142 142 143 143 143 143

4.9

Sensitivity of Fluorescence Assays 4.9.1 Principle 4.9.2 Materials 4.9.3 Protocol

144 144 144 144

4.10 Fluorescence Quenching 4.10.1 Materials 4.10.2 Protocol

145 145 145

4.11 Measurement of α-Naphthyl Phosphatase Activity 4.11.1 Principle

145 145

Contents 4.11.2 Materials 4.11.3 Protocol 4.11.4 Calculations

145 145 147

Suggested Readings

148

Important Links

148

5. Centrifugation


xiii

149

5.1 Introduction

149

5.2 Centrifuges

150

5.3 Ultracentrifugation 5.3.1 Preparative Centrifugation 5.3.2 Analytical Centrifugation 5.3.3 Differential Centrifugation 5.3.4 Density Gradient Centrifugation 5.3.5 Isopycnic Centrifugation

150 150 150 150 153 153

5.4

155

Suspending Medium

5.5 Rotors

155

5.6

Fractionation of Rat Liver 5.6.1 Principle 5.6.2 Materials 5.6.3 Protocol

156 156 156 157

5.7

Estimation of DNA, RNA, and Protein in the Isolated Cell Fractions 5.7.1 Principle 5.7.2 Materials 5.7.3 Protocol

158 158 158 159

5.8

Enzyme Distribution in the Cell 5.8.1 Principle 5.8.2 Protocol 5.8.3 Results and Calculations

159 159 160 160

5.9 Glucose-6-Phosphatase 5.9.1 Principle 5.9.2 Materials 5.9.3 Protocol 5.9.4 Results and Calculations

160 160 161 162 162

5.10 Glutamate Dehydrogenase 5.10.1 Principle 5.10.2 Materials 5.10.3 Protocol 5.10.4 Result and Calculations

162 162 162 163 163

5.11 Acid Phosphatase 5.11.1 Principle 5.11.2 Materials 5.11.3 Protocol 5.11.4 Results and Calculation

163 163 163 164 164

5.12 Mitochondrial Swelling 5.12.1 Principle

164 164

xiv

Contents


5.12.2 Materials 5.12.3 Protocol

164 165

5.13 Determination of Lysosomal Integrity 5.13.1 Principle 5.13.2 Materials 5.13.3 Protocol 5.13.4 Calculations

165 165 166 167 167

5.14 Effect of Detergents on the Stability of the Lysosomal Membrane 5.14.1 Principle 5.14.2 Materials 5.14.3 Method 5.14.4 Calculations and Result

167 167 168 168 168

5.15 Density Gradient Centrifugation 5.15.1 Principle 5.15.2 Materials 5.15.3 Protocol 5.15.4 Result

169 169 170 171 171

5.16 Fractionation of Pig Brain by Density Gradient Centrifugation 5.16.1 Principle 5.16.2 Materials 5.16.3 Protocol 5.16.4 Precautions

171 171 172 172 173

5.17 Distribution of Enzymes in the Fractions Obtained from Pig Brain 5.17.1 Principle 5.17.2 Materials 5.17.3 Protocol 5.17.4 Results and Conclusions

174 174 174 175 175

5.18 Isolation of Chloroplasts 5.18.1 Protocol 5.18.2 Materials 5.18.3 Method 5.18.4 Result

175 175 176 176 178

5.19 Isolation of Chloroplast DNA 5.19.1 Principle 5.19.2 Precautions 5.19.3 Materials 5.19.4 Protocol 5.19.5 Result

178 178 178 178 179 179

5.20 Isolation of Mitochondria and Assay of a Marker Enzyme 5.20.1 Principle 5.20.2 Materials 5.20.3 Isolation of Mitochondria 5.20.3.1 Protocol I 5.20.3.2 Protocol II 5.20.3.3 Protocol III (by Clinical Centrifuge)

179 179 180 180 180 181 182

5.21 Isolation of Genomic DNA 5.21.1 Principle 5.21.2 Precautions

184 184 184

Contents 5.21.3 Materials 5.21.4 Method 5.21.5 DNA Digestion

184 184 185

5.22 Isolation of Chloroplasts from Spinach Leaves by Differential Centrifugation 5.22.1 Materials 5.22.2 Protocol

186 186 186

5.23 Isolation of Plasmid DNA by Centrifugation 5.23.1 Principle 5.23.2 Materials 5.23.3 Protocol

186 186 187 187

Suggested Readings

188

Important Links

189

6. Radiation Biology


xv

191

6.1 Introduction

191

6.2 Radioisotopes 6.2.1 Atomic Structure 6.2.1.1 Radioisotopes Are Used to Trace Molecules in Cells and Organisms 6.2.2 Units of Radioactivity 6.2.3 Measurement of Radioactivity 6.2.4 Proportional Counters 6.2.4.1 Scintillation Counters 6.2.4.2 Types of Scintillation Counting 6.2.4.3 Mechanism of the Liquid Scintillation Detection 6.2.5 Counting of Dual-Labeled Samples 6.2.5.1 Determination of Counting Efficiency

192 192 193 195 196 196 197 198 199 201 202

6.3

204

Sample Preparation

6.4 Autoradiography 6.4.1 Film Emulsion 6.4.2 Determination of Exposure Time 6.4.3 Tissue Preparation and Artifacts 6.4.4 Specific Autoradiographic Techniques

205 205 206 206 207

6.5

Distribution of 14CO2 in Different Plant Parts 207 6.5.1 Principle 207 6.5.2 Precautions 208 6.5.3 Disposal of Radioactive Waste 208 6.5.4 Where to Buy the Isotopes 208 6.5.5 Materials 209 6.5.6 Protocol 209 6.5.6.1 Feeding of Plants with 14CO2 209 6.5.6.2 Extraction and Measurement of Radioactivity 210 6.5.7 Calculations 211 6.5.8 Result 211

6.6

Photosynthetic Reduction of 14CO2 to Primary Metabolic Product 6.6.1 Principle 6.6.2 Precautions 6.6.3 Materials

212 212 212 212

xvi

Contents

6.7

6.6.4 Protocol 6.6.5 Conclusions 6.6.6 Result 6.6.7 Autoradiography

212 214 214 214

Incorporation of Labeled Acetate into Leucoplastic Fatty Acids of Developing Brassica Seeds 6.7.1 Principle 6.7.2 Precautions 6.7.3 Materials 6.7.4 Protocol 6.7.5 Result

214 214 215 215 215 216

Suggested Readings

216

Important Links

216


7. Chromatography

217

7.1 Chromatography

217

7.2

Paper Chromatography 7.2.1 Introduction 7.2.2 Sample Preparation 7.2.3 Paper 7.2.4 Solvent 7.2.5 Component Detection 7.2.6 Identification

217 217 218 219 219 219 220

7.3

Ascending Chromatography

220

7.4

Descending Chromatography 7.4.1 Multiple Developments 7.4.2 Continuous Development 7.4.3 2D (Bidimensional) Chromatography 7.4.4 Reversed-Phase Chromatography 7.4.4.1 Partition Chromatography

220 221 221 221 221 222

7.5

Separation and Identification of Amino Acids by Descending Paper Chromatography 7.5.1 Principle 7.5.2 Materials 7.5.3 Protocol 7.5.4 Observations 7.5.5 Conclusion

223 223 224 224 225 226

7.6

Separation and Identification of Amino Acids by Ascending Paper Chromatography 226 7.6.1 Materials 226 7.6.2 Protocol 226 7.6.3 Observations 227

7.7

Separation and Identification of Amino Acids in a Given Mixture by 2D Paper Chromatography 7.7.1 Principle 7.7.2 Materials 7.7.3 Procedure 7.7.4 Observations

227 227 227 228 229


Contents

xvii

7.8

Thin Layer Chromatography 7.8.1 Introduction 7.8.2 Principle 7.8.3 Production of Thin Layer 7.8.4 Development 7.8.5 Location

229 229 229 230 230 230

7.9

Thin-Layer Gel Chromatography

231

7.10 Separation and Identification of Sugars by Adsorption Thin Layer Chromatography 7.10.1 Principle 7.10.2 Materials 7.10.3 Protocol 7.10.4 Observations

232 232 232 232 233

7.11 Identification of Lipids in a Given Sample by Thin Layer Chromatography 7.11.1 Principle 7.11.2 Materials 7.11.3 Protocol 7.11.4 Observation and Calculations

233 233 234 234 234

7.12 Identification of Sugars in Fruit Juices Using Thin Layer Chromatography 7.12.1 Materials 7.12.2 Protocol

235 235 235

7.13 Column Chromatography

235

7.14 Gradient Preparation

237

7.15 Adsorption Chromatography

238

7.16 Separation of Pigments from Leaves or Flowers by Adsorption Column Chromatography 7.16.1 Principle 7.16.2 Materials 7.16.3 Protocol 7.16.3.1 Sample Preparation 7.16.3.2 Column Preparation 7.16.3.3 Sample Application 7.16.3.4 Column Development 7.16.3.5 Result

240 240 240 240 240 241 241 241 241

7.17 Ion-Exchange Chromatography 7.17.1 Introduction 7.17.2 Theory 7.17.2.1 Matrix 7.17.2.2 Ionizable Groups 7.17.2.3 Ion-Exchange Equilibria 7.17.2.4 Elution of Bound Ions 7.17.2.5 Preparation of Material

241 241 242 242 242 243 243 244

7.18 Separation of Amino Acids by Ion-Exchange Chromatography 7.18.1 Materials 7.18.2 Protocol 7.18.2.1 Preparation of the Column 7.18.2.2 Elution of Amino Acid 7.18.2.3 Detection of Amino Acids

245 245 246 246 246 246


xviii

Contents 7.19 Separation of Proteins from Human Serum by Ion-Exchange Chromatography 7.19.1 Principle 7.19.2 Materials 7.19.3 Protocol 7.19.3.1 Recovery of Protein 7.19.3.2 Characterization of Protein

247 247 248 248 248 248

7.20 Gel Filtration/Size-Exclusion Column Chromatography 7.20.1 Molecular Weight Determination

248 251

7.21 Separation of Blue Dextran and Cobalt Chloride on Sephadex G-25 7.21.1 Principle 7.21.2 Materials 7.21.3 Protocol

253 253 253 253

7.22 Desalting of Protein Sample by Gel Filtration 7.22.1 Introduction 7.22.2 Materials 7.22.3 Protocol 7.22.3.1 Column Preparation 7.22.3.2 Running of Column 7.22.3.3 Column Separation

253 253 254 254 254 254 254

7.23 Determination of Molecular Weight of a Given Protein by Gel Filtration 7.23.1 Principle 7.23.2 Materials 7.23.3 Protocol 7.23.4 Molecular Weight Determination

255 255 255 255 256

7.24 Concentration of Dilute Protein Solutions Using Sephadex G-25 7.24.1 Principle 7.24.2 Materials 7.24.3 Protocol 7.24.4 Result

256 256 256 256 256

7.25 Affinity Chromatography 7.25.1 Introduction 7.25.2 Principle 7.25.3 Affinity Material 7.25.4 Spacer Arm

257 257 257 258 259

7.26 Hydrophobic Interaction Chromatography or Affinity Elution

261

7.27 Purification by Affinity Chromatography

262

7.28 Preparation of an Affinity Column 7.28.1 Principle 7.28.2 Materials 7.28.3 Protocol 7.28.3.1 Initial Washing and Swelling 7.28.3.2 Addition of the Spacer Arm 7.28.3.3 Preparation of the Ligand 7.28.3.4 Coupling of Ligand 7.28.3.5 Suggested Timetable

263 263 263 263 263 263 264 265 265

7.29 Preparation of Rat Brain Acetylcholinesterase 7.29.1 Principle 7.29.1.1 Extraction

266 266 266

Contents



xix 266 266

7.30 Acetylcholinesterase Assay 7.30.1 Principle 7.30.1.1 Enzyme Hydrolysis 7.30.1.2 Production of Color 7.30.2 Materials 7.30.3 Protocol 7.30.4 Calculation of Enzyme Activity

267 267 267 267 267 267 268

7.31 Purification of Acetylcholinesterase by Affinity Chromatography 7.31.1 Principle 7.31.1.1 Binding 7.31.1.2 Elution 7.31.1.3 Reactivation of Acetylcholinesterase 7.31.1.4 Removal of Protein Bound Nonspecifically 7.31.1.5 Protein Assay 7.31.2 Materials 7.31.3 Protocol 7.31.3.1 Concentration of the Purified Protein

268 268 268 268 268 268 269 269 269 269

7.32 MALDI-TOF

274

7.33 Nuclear Magnetic Resonance Spectroscopy

276

7.34 Maintenance of Enzyme Activity

278

7.35 Addition of Chaotropes

278

7.36 Role of Polyols/Hydrophilic Polymers in Stabilizing Structures

278

7.37 Stabilization by Side-Chain Modification

279

7.38 Criteria of Purity

279

7.39 Biuret

280

7.40 Folin (Lowry’s Method)

281

7.41 Bradford

281

Suggested Readings

282

Important Links

283

8. Separation Technology

285

8.1 Electrophoresis

285

8.2 Principle

285

8.3

Types of Electrophoresis 8.3.1 Paper Electrophoresis 8.3.2 Low-Voltage Paper Electrophoresis 8.3.3 High-Voltage Paper Electrophoresis

286 286 287 287

8.4

Separation of Amino Acids by Paper Electrophoresis 8.4.1 Principle 8.4.2 Materials 8.4.3 Protocol

288 288 288 289

8.5

Cellulose Acetate Strip Electrophoresis

289

xx

Contents 8.5.1

8.5.2


8.6

Separation of Serum Proteins by Electrophoresis on Cellulose Acetate 8.5.1.1 Principle 8.5.1.2 Materials 8.5.1.3 Protocol Identification of Proteins by Cellulose Acetate Electrophoresis Following the Fractionation of Human Blood Plasma 8.5.2.1 Principle 8.5.2.2 Materials 8.5.2.3 Protocol

290 290 290 290 290 290 291 291

Gel Electrophoresis 8.6.1 Starch Gel Electrophoresis 8.6.1.1 Isolation of Lactate Dehydrogenase Isoenzymes by Starch Block Electrophoresis 8.6.1.2 Separation of Isoenzymes of Malate Dehydrogenase Starch Gel Electrophoresis

294

8.7

Polyacrylamide Gel Electrophoresis 8.7.1 Apparatus 8.7.1.1 Rod Gel Electrophoresis

296 298 298

8.8

Polyacrylamide Disc Gel Electrophoresis 8.8.1 Principle 8.8.2 Materials 8.8.2.1 Stock Solutions 8.8.2.2 Working Solutions 8.8.2.3 Samples 8.8.2.4 Staining Solutions 8.8.2.5 Equipments 8.8.3 Protocol 8.8.3.1 Separation Gel 8.8.3.2 Spacer Gel 8.8.3.3 Sample Application 8.8.3.4 Electrophoresis 8.8.4 Staining

300 300 300 300 301 301 301 301 301 301 301 302 302 302

8.9

SDS Gel Electrophoresis 8.9.1 Principle 8.9.2 Materials 8.9.3 Method 8.9.3.1 Preparation of Gel 8.9.3.2 Preparation of Samples 8.9.3.3 Electrophoresis 8.9.3.4 Fixing and Staining 8.9.4 Mobility Determination

302 302 303 304 304 304 304 304 305

8.10 Slab Gel Electrophoresis 8.10.1 Buffer Systems 8.10.1.1 Native Continuous Polyacrylamide Gel Electrophoresis 8.10.1.2 Native Discontinuous Polyacrylamide Gel Electrophoresis (Disc Gel Electrophoresis) 8.10.1.3 Native Discontinuous Polyacrylamide Gel Electrophoresis (Nonreducing) (SDS-PAGE) 8.10.1.4 Gel Electrophoresis SDS-Polyacrylamide (Reducing) 8.10.2 Staining of Proteins in Polyacrylamide Gels 8.10.3 Detection, Estimation, and Recovery of Protein Gel

292 292 293

305 305 305 307 309 310 311 311


Contents

xxi

8.11 Native Disc Gel Electrophoresis of Proteins 8.11.1 Principle 8.11.2 Materials 8.11.3 Sample Preparation 8.11.4 Preparation of the Gel Rods

311 311 311 312 313

8.12 SDS-Polyacrylamide Slab Gel Electrophoresis of Proteins under Reducing Conditions (SDS-PAGE) 8.12.1 Principle 8.12.2 Materials 8.12.2.1 Reagents 8.12.3 Protocol 8.12.3.1 Sample Preparation 8.12.3.2 Preparation of Slab Gels 8.12.3.3 Electrophoresis of Sample

314 314 314 315 317 317 317 317

8.13 Slab Gel Gradient Electrophoresis of Protein and Multiple Molecular Forms of Acetylcholinesterase 8.13.1 Principle 8.13.2 Materials 8.13.3 Protocol 8.13.3.1 Electrophoresis 8.13.3.2 Staining

318 318 319 319 319 319

8.14 Protein (Western) Blotting 8.14.1 Electroblotting (Western Blotting of Proteins from SDS-PAGE) 8.14.1.1 Introduction 8.14.1.2 Materials 8.14.1.3 Protocol

320 320 320 321 321

8.15 Isoelectric Focusing 8.15.1 Separation of Proteins by Isoelectric Focusing 8.15.1.1 Introduction 8.15.1.2 Materials 8.15.1.3 Protocol

322 324 324 324 325

8.16 Agarose Gel Electrophoresis 8.16.1 Apparatus 8.16.2 Buffers 8.16.3 Nucleic Acids 8.16.4 Detection of Nucleic Acids in Gels 8.16.5 Agarose Gel Electrophoresis of Double-Stranded DNA 8.16.6 Separation of DNA by Molecular Weight

326 327 327 327 328 328 329

8.17 Determination of Molecular Weight of Plasmid DNA by Agarose Gel Electrophoresis 8.17.1 Introduction 8.17.2 Materials 8.17.2.1 Reagents 8.17.3 Protocol

330 330 330 330 331

8.18 Electrophoretic Evaluation of Quality of High Molecular Weight DNA Isolated from Plant Material 8.18.1 Introduction 8.18.2 Materials 8.18.2.1 Reagents

332 332 332 332

xxii

Contents 8.18.3 Protocol 8.18.4 Result 8.19 Large-Scale Preparative Electrophoresis 8.19.1 Block Electrophoresis 8.19.2 Column Electrophoresis with Polyacrylamide 8.19.3 Immunoelectrophoresis 8.19.4 Capillary Electrophoresis 8.19.5 Two-Dimensional Electrophoresis

333 333 333 333 334 335

Suggested Readings

336

Important Links

339

9. Histochemical Techniques


333 333

341

9.1 Introduction 9.1.1 Histochemistry

341 341

9.2 Protein 9.2.1 Reactions for Protein 9.2.1.1 Dinitrofluorobenzene Method 9.2.1.2 Tetrazotized Dianisidine Method 9.2.1.3 Baker’s Method for Tyrosine 9.2.1.4 Fast Green Method for Basic Protein 9.2.1.5 Sakaguchi Reaction for Arginine 9.2.1.6 Bennett’s Mercurial Method for Sulfhydryl Groups

342 345 345 346 347 348 348 349

9.3

350 350 350 353 354 354 354 355 356

Acidic Compounds 9.3.1 Introduction 9.3.1.1 Acidic Compounds (Nucleic Acids) 9.3.1.2 Use of Deoxyribonuclease 9.3.1.3 Use of Ribonuclease 9.3.1.4 Hydrochloric Acid Method for RNA 9.3.1.5 Toluidine Blue Method for Basophilia 9.3.1.6 Metachromatic Staining with Toluidine Blue 9.3.1.7 Acridine Orange Method for DNA and RNA

9.4 Carbohydrates–Polysaccharides 9.4.1 Introduction 9.4.1.1 Reactions for Polysaccharides 9.4.1.2 Alcian Blue Method 9.4.1.3 Lugol’s Iodine Method

356 356 357 358 358

9.5 Lipids 9.5.1 Introduction 9.5.2 Reactions for Lipids 9.5.2.1 Sudan Black Method 9.5.2.2 Oil Red O Method for Staining Fats 9.5.2.3 Benzopyrene Method for Lipids 9.5.2.4 Rhodamine B Method for Lipids

359 359 359 360 361 361 362

9.6

362 362 362

Minor Compounds 9.6.1 Reaction with Minor Components 9.6.1.1 Iron

9.7 Autoradiography 9.7.1 Introduction

363 363

Contents

9.8


10.

xxiii

9.7.2 Autoradiography Methods and Uses 9.7.3 Developer D 19 B 9.7.4 Protocol 9.7.5 Results

363 364 364 366

Enzyme Localization in Embedded Material 9.8.1 Introduction 9.8.1.1 ATPase Localization 9.8.2 Peroxidase Localization 9.8.2.1 Protocol 9.8.3 Dehydrogenases 9.8.3.1 Introduction

367 367 367 368 368 368 368

Suggested Readings

372

Important Links

372

Cytogenetics

373

10.1 Introduction

373

10.2 Study Different Stages in Root Tip Cells of Plant

373

10.3 Preparation of Root Tips for Mitotic Studies

375

10.4 Preparation and Study of Slides for Mitosis Using Acetocarmine Squash Techniques from Onion Root Tips 10.4.1 Materials 10.4.2 Protocol 10.4.3 Results and Observations

376 376 377 377

10.5 Perform Carmine or Orcein Staining Techniques 378 10.5.1 Materials 378 10.5.2 Protocol I 378 10.5.3 Protocol II 378 10.5.4 Protocol III 379 10.5.5 Protocol IV (for Aloe vera) 379 10.6 Feulgen Staining Techniques 10.6.1 Materials 10.6.2 Protocol I 10.6.3 Protocol II 10.6.4 Results and Observations 10.6.4.1 Interphase 10.6.4.2 Prophase 10.6.4.3 Metaphase 10.6.4.4 Anaphase 10.6.4.5 Telophase 10.6.4.6 Cytokinesis

379 379 379 380 380 380 380 380 381 381 381

10.7 Preparation of Onion Flower Buds for Meiosis Studies 10.7.1 Preparation and Study of Slides for Meiotic Stages in Onion Flower Buds by the Squash Technique 10.7.1.1 Carmine Staining Technique 10.7.2 Feulgen Staining Techniques 10.7.2.1 Materials 10.7.2.2 Protocol 10.7.2.3 Results and Observations

381 383 383 383 383 383 384

xxiv

Contents


10.7.3 Homotypic or Second Meiotic Division 10.7.3.1 Prophase II 10.7.3.2 Metaphase II 10.7.3.3 Anaphase II 10.7.3.4 Telophase II

387 387 387 389 389

10.8 Fixation 389 10.8.1 Prefixing 389 10.8.1.1 Colchicines 389 10.8.1.2 Acenaphthene 390 10.8.1.3 Chloral Hydrate 390 10.8.1.4 8-Hydroxyquinoline 390 10.8.1.5 α-Bromonaphthalene 391 10.8.2 Preparation 391 10.8.3 Fixation 391 10.8.3.1 Ethanol or Ethyl Alcohol (C2H5OH) 392 10.8.3.2 Acetic Acid (CH3COOH) 392 10.8.3.3 Formaldehyde (HCHO) 393 10.8.3.4 Propionic Acid (C2H5COOH) 393 10.8.3.5 Chloroform (CHCl3) 393 10.8.4 Fixing Mixtures 393 10.8.4.1 Navashin’s Fluid 393 10.8.4.2 Carnoy’s Fixative 393 10.8.4.3 Carnoy’s Fluid I 394 10.8.4.4 Carnoy’s Fluid II 394 10.8.4.5 Carnoy and Lebrun’s Fluid 394 10.8.4.6 G. S. Sansom’s Mixture 394 10.8.4.7 Cutter’s Mixture 394 10.8.4.8 Mark’s Mixture 394 10.8.4.9 Newcomer’s Mixture 395 10.8.4.10 Iron Acetate–Acetic Alcohol Mixture 395 10.8.4.11 Newcomer’s Fluid 395 10.8.4.12 Original Navashin’s Fluid 395 10.8.4.13 Karpechenko’s Fluid 395 10.8.4.14 Belling’s Fluid 395 10.8.4.15 Levitsky’s Fixative 395 10.8.4.16 La Cour’s Fluid 396 10.8.5 Staining 396 10.8.5.1 Basic Fuchsin 396 10.8.5.2 Feulgen Reagent (Fuchsin Sulfurous Acid or Leuco-Basic Fuchsin) 397 10.8.5.3 Chemical Basis of the Reaction 397 10.8.5.4 Carmine 397 10.8.5.5 Orcein 400 10.8.5.6 Giemsa 400 10.9 Mounting and Making Temporary Slides Permanent 10.9.1 Mounting Medium 10.9.1.1 Balsam 10.9.2 Procedure for Making Slides Permanent 10.9.3 Preparation of Albuminized Cover Slip

401 401 401 401 402

Contents

xxv


10.10 Study the Effect of Colchicines or Acenaphthene or Chloral Hydrate on Chromosomes of Allium cepa 402 10.10.1 Principle 402 10.10.2 Materials 403 10.10.3 Protocol 403 10.11 Estimate the Chiasma Frequency at Diplotene–Diakinesis 10.11.1 Introduction 10.11.2 Chiasma Frequency 10.11.3 Single Crossing Over 10.11.4 Multiple Crossing Over 10.11.5 Materials 10.11.6 Protocol 10.11.7 Observations

404 404 404 405 405 406 406 406

10.12 Study the Effect of γ Radiation on Chromosomes 10.12.1 Principle 10.12.2 Effect of Radiation 10.12.3 Physiological Effects 10.12.4 Mutational Effects 10.12.5 Chromosomal Effect 10.12.6 Types of Breaks 10.12.7 Radiation Hazards 10.12.8 Mechanism of Changes due to Radiation 10.12.8.1 Materials 10.12.8.2 Source of Gamma Rays 10.12.8.3 Observations

407 407 407 407 408 408 408 408 408 409 409 409

10.13 To Prepare Slides Showing Diplotene Stages in Spermatocytes of Desert Locust (Schistocerca gregaria) or in Oocytes of the Newt (Triturus viridescens) Showing Lampbrush Chromosomes 410 10.13.1 Principle 410 10.13.2 Materials 411 10.13.3 Protocol 411 10.13.3.1 Protocol 1 (for Triturus, Newt, or Any Other Amphibian) 411 10.13.3.2 Protocol 2 (for S. gregaria) 411 10.14 Study Karyotypes of Allium cepa and Vicia faba 411 10.14.1 Principle 411 10.14.2 Karyotype Concept 412 10.14.3 Idiogram 412 10.15 Micrometry 10.15.1 Materials 10.15.2 Protocol 10.15.3 Idiogram

413 414 414 414

10.16 To Demonstrate Reciprocal Translocation in Pollen Mother Cells of Rhoeo discolor (Tradescantia) 415 10.16.1 Introduction 415 10.16.1.1 Homozygous Translocation 415 10.16.1.2 Heterozygous Translocation 415 10.16.2 Materials 415 10.16.3 Protocol 416


xxvi

Contents 10.17 To Study the Spiral Nature of Chromosomes Using Any Plant Material 10.17.1 Principle 10.17.2 Materials (Shock Treatment: For Procedure I and II) 10.17.2.1 Protocol I (for Mitotic Chromosome) 10.17.2.2 Protocol II (for Meiotic Chromosome) 10.17.2.3 Protocol III (by Dissolving the Outer Nucleic Envelope) 10.17.2.4 Protocol IV (Precipitation of DNA as a Metallic Salt and then Partially Digest Proteins) 10.17.2.5 Protocol V (Pretreatment)

416 416 417 417 418 418

10.18 To Show the Different Banding Patterns in Eukaryotic Chromosomes 10.18.1 Principle 10.18.2 Q-Band Technique 10.18.2.1 Materials 10.18.2.2 Protocol 10.18.3 G-Band Technique 10.18.3.1 Materials 10.18.3.2 Protocol 10.18.4 Modified Technique for Giemsa Staining 10.18.5 Giemsa Staining Technique

418 418 419 419 419 419 419 420 420 420

10.19 Fungal Cytogenetics 10.19.1 Principle

421 421

10.20 Harvesting of Neurospora 10.20.1 Flamed-Loop Method 10.20.1.1 Materials 10.20.1.2 Protocol 10.20.2 Spore Suspension Method 10.20.2.1 Materials 10.20.2.2 Protocol

422 422 422 422 422 422 423

10.21 To Study the Ordered (Tetrad) and Random Ascospore Analysis in Neurospora sp. 10.21.1 Principle

424 424

10.22 To Demonstrate the Tetrad and Random Analysis 10.22.1 Crossing of Two Mating Types 10.22.1.1 Materials 10.22.1.2 Protocol

426 426 426 426

10.23 Tetrad Analysis 10.23.1 Materials 10.23.2 Protocol

426 427 427

10.24 Pigmentation and Nutritional Requirement Analysis 10.24.1 Materials 10.24.2 Protocol

427 427 428

10.25 Random Spore Analysis 10.25.1 Materials 10.25.2 Protocol 10.25.3 Scoring Tubes for Pigmentation

429 429 430 430

10.26 Test for Nutritional Requirement 10.26.1 Materials 10.26.2 Protocol

431 431 431

418 418

Contents


10.26.3 Scoring for Nutritional Requirement 10.26.4 Analysis

xxvii 431 431

10.27 Suggested Media for Neurospora, Sordaria, and Aspergillus Experiments 10.27.1 Vogel’s Medium 10.27.2 Biotin Solution 10.27.3 Medium N Stock Solution (50X) 10.27.4 Medium N Final Solution 10.27.5 Wastegaard–Mitchell Crossing Medium 10.27.6 Biotin Solution 10.27.7 Wastegaard–Mitchell Crossing Medium Stock Solution (20X) 10.27.8 Crossing Medium Final Solution

433 433 433 433 433 434 434 434 434

10.28 Study of Heterokaryosis in Neurospora from the Nutritionally Deficient Mutants 10.28.1 Principle 10.28.2 Complementation between Genes or Nonallelic Mutants 10.28.3 Materials 10.28.4 Protocol

434 434 436 436 436

10.29 General Information and Methods for Drosophila Experiments 437 10.29.1 Introduction 437 10.29.2 Life Cycle 437 10.29.3 Sex Differences 438 10.29.4 Hereditary Traits 438 10.29.4.1 Body Color 438 10.29.4.2 Wings 438 10.29.4.3 Bristles 439 10.29.4.4 Eyes 439 10.29.5 Etherization 439 10.29.6 How to Get Virgin Female Flies 440 10.29.7 Study of Random Mating in Population of Drosophila, Heterozygous for a Single Gene Pair 440 10.29.7.1 Introduction 440 10.29.7.2 Materials 444 10.29.7.3 Observation and Destruction of Parents (P1) 444 10.29.7.4 Observation and Crossing of First Filial Generation (F1) 444 10.29.7.5 Tabulation of Results 445 10.30 Study of Salivary Gland Chromosomes of Drosophila melanogaster for Nature of Polyteny and Regions of Chromosome Puffs 10.30.1 Introduction 10.30.2 Materials 10.30.3 Protocol

446 446 447 447

10.31 Human Genetics 10.31.1 Introduction

448 448

10.32 Human Sex Chromosomes and Barr Bodies 10.32.1 Study of Barr Body in the Nuclei by Means of Buccal Smear 10.32.1.1 Introduction 10.32.1.2 Drumsticks 10.32.1.3 Lyon’s Hypothesis 10.32.1.4 Amniotic Fluid Examination 10.32.2 Study of Blood Groups 10.32.2.1 To Study ABO and Rh Blood Types in Man

449 449 449 450 450 451 451 451

xxviii

Contents


10.32.2.2 Rh Factor 10.32.2.3 The MN Blood Groups 10.32.2.4 Sickle Cell Anemia 10.32.3 PTC Test, Color Blindness, and the Inheritance of These Traits in a Human Population: To Work Out an Inheritance Pattern of a Dominant Gene (PTC) and a Sex-Linked Gene (Color Blindness) in a Human Population 10.32.3.1 Introduction 10.32.3.2 PTC Testing 10.32.3.3 Color Blindness

11.

453 453 453

454 454 456 456

10.33 Autoradiography

456

10.34 Photomicrography

458

Suggested Readings

459

Important Links

459

Biomolecules

461

11.1 Introduction

461

11.2 Carbohydrates 11.2.1 Function of Carbohydrates in the Biosphere 11.2.1.1 Photosynthesis 11.2.1.2 Respiration 11.2.2 Structure of Cells and Molecules 11.2.3 Structure of Carbohydrates 11.2.3.1 Introduction 11.2.3.2 Stereochemistry 11.2.4 Macromolecules 11.2.4.1 Homopolysaccharides 11.2.4.2 Heteropolysaccharides 11.2.5 Experiment: Benedict’s Test for Reducing Sugars 11.2.5.1 Principle 11.2.5.2 Materials 11.2.5.3 Protocol 11.2.6 Experiment: Iodine Test for Polysaccharides 11.2.6.1 Principle 11.2.6.2 Materials 11.2.6.3 Protocol 11.2.7 Optical Activity 11.2.7.1 Polarimeter 11.2.7.2 Specific Rotation 11.2.8 Experiment: Mutarotation of Glucose 11.2.8.1 Principle 11.2.8.2 Materials 11.2.8.3 Protocol 11.2.8.4 Results 11.2.9 Experiment: Estimation of Carbohydrates by the Anthrone Method 11.2.9.1 Principle 11.2.9.2 Materials 11.2.9.3 Protocol 11.2.9.4 Results

461 461 462 462 463 464 464 464 470 470 471 473 473 474 474 474 474 474 474 474 475 475 476 476 476 476 476 477 477 477 477 477


Contents

xxix

11.2.10 Experiment: Determination of Reducing Sugars Using 3,5-Dinitrosalicylic Acid 477 11.2.10.1 Principle 477 11.2.10.2 Materials 478 11.2.10.3 Protocol 478 11.2.10.4 Result 478 11.2.11 Experiment: Determination of Glucose by Means of the Enzyme Glucose Oxidase (β-d-Glucose: Oxygen Oxidoreductase) 478 11.2.11.1 Principle 478 11.2.11.2 Materials 479 11.2.11.3 Protocol 479 11.2.12 Experiment: Determination of Starch in Plant Tissues 479 11.2.12.1 Principle 479 11.2.12.2 Materials 480 11.2.12.3 Protocol 480 11.2.12.4 Results and Calculations 480 11.2.13 Experiment: Isolation and Assay of Glycogen from the Liver and Skeletal Muscle of Rats 480 11.2.13.1 Principle 480 11.2.13.2 Materials 481 11.2.13.3 Protocol 481 11.2.13.4 Results 482 11.2.14 Experiment: Acid Hydrolysis of Polysaccharides 482 11.2.14.1 Principle 482 11.2.14.2 Materials 482 11.2.14.3 Protocol 482 11.2.15 Experiment: Enzymatic Hydrolysis of Glycogen by α- and β-Amylase 483 11.2.15.1 Principle 483 11.2.15.2 Materials 483 11.2.15.3 Protocol 483 11.2.15.4 Result 483 11.2.16 Experiment: Breakdown of Glycogen and the Production of Glucose-l-Phosphate by Muscle Phosphorylase 484 11.2.16.1 Principle 484 11.2.16.2 Materials 484 11.2.16.3 Protocol 484 11.2.16.4 Results 485 11.3 Amino Acids and Proteins 485 11.3.1 Amino Acids 485 11.3.2 Protein Structure 486 11.3.2.1 Metalloproteins 487 11.3.3 Amino Acid Chemistry 487 11.3.3.1 Acid–Base Properties 487 11.3.3.2 Isoelectric Point 488 11.3.3.3 Stereochemistry 488 11.3.4 Amino Acid Composition of Proteins 490 11.3.4.1 Formulae of Amino Acids 490 11.3.4.2 Peptide Bond 490 11.3.5 Protein Structure 490 11.3.5.1 Primary Structure 490 11.3.5.2 Secondary Structure 492


xxx

Contents 11.3.5.3 Tertiary Structure 493 11.3.5.4 Quaternary Structure 493 11.3.6 Function in the Living Organism 494 11.3.6.1 Amino Acids 494 11.3.6.2 Peptides 494 11.3.6.3 Proteins 495 11.3.6.4 Membranes 496 11.3.6.5 Plasma Proteins 496 11.3.7 Experiment: Determination of Crude Protein by Micro-Kjeldahl Method 497 11.3.7.1 Principle 497 11.3.7.2 Precautions 497 11.3.7.3 Materials 497 11.3.7.4 Protocol 498 11.3.7.5 Results and Calculations 499 11.3.8 Experiment: Quantitative Estimation of Amino Acids Using the Ninhydrin Reaction 499 11.3.8.1 Principle 499 11.3.8.2 Materials 499 11.3.8.3 Protocol 500 11.3.9 Experiment: Biuret Assay 500 11.3.9.1 Principle 500 11.3.9.2 Materials 500 11.3.9.3 Protocol 500 11.3.10 Experiment: Folin–Lowry Method of Protein Assay 501 11.3.10.1 Principle 501 11.3.10.2 Materials 501 11.3.10.3 Protocol 501 11.3.11 Isolation of Proteins 501 11.3.11.1 Experiment: Isolation of Casein from Milk 502 11.3.12 Experiment: Preparation and Properties of Cytochrome c 502 11.3.12.1 Principle 502 11.3.12.2 Materials 502 11.3.12.3 Protocol 502 11.3.12.4 Results and Conclusion 504 11.3.13 Protein Structure 504 11.3.13.1 Experiment: Identification of the C-Terminal End of Amino Acid of a Protein 504 11.3.14 Experiment: Determination of the Free Amino End Group of Some Proteins 505 11.3.14.1 Principle 505 11.3.14.2 Materials 505 11.3.14.3 Protocol 506 11.3.14.4 Results and Conclusion 506 11.3.15 Experiment: Detection of Changes in the Conformation of Bovine Serum Albumin by Viscosity Measurements 506 11.3.15.1 Principle 506 11.3.15.2 Materials 507 11.3.15.3 Protocol 507 11.3.15.4 Results and Conclusion 508 11.3.16 Experiment: Effect of pH on the Conformation of Bovine Serum Albumin 508 11.3.16.1 Materials 508

Contents


11.3.16.2 Method 11.3.16.3 Results and Conclusions 11.4 Lipids 11.4.1 Introduction 11.4.2 Classification and Biological Role of Lipids 11.4.2.1 Simple Lipids 11.4.2.2 Compound Lipids 11.4.2.3 Derived Lipids 11.4.3 Qualitative Test of Lipids 11.4.3.1 Solubility Test 11.4.3.2 Emulsification Test 11.4.3.3 Paper Spot Test 11.4.3.4 Saponification Test 11.4.4 Unsaturated Lipids 11.4.4.1 Liebermann–Burchard Test 11.4.4.2 Odor Test 11.4.4.3 Sudan Black B Test 11.4.4.4 Sudan III Test 11.4.5 Quantitative Analysis of Lipids 11.4.5.1 Introduction 11.4.6 Experiment: Determination of the Acid Value of a Fat 11.4.6.1 Principle 11.4.6.2 Materials 11.4.6.3 Protocol 11.4.6.4 Results and Conclusion 11.4.7 Experiment: Saponification Value of Fat 11.4.7.1 Principle 11.4.7.2 Materials 11.4.7.3 Protocol 11.4.7.4 Results and Conclusion 11.4.8 Experiment: Iodine Number of a Fat 11.4.8.1 Principle 11.4.8.2 Materials 11.4.8.3 Protocol 11.4.8.4 Results and Conclusion 11.4.9 Experiment: Estimation of Blood Cholesterol 11.4.9.1 Principle 11.4.9.2 Materials 11.4.9.3 Protocol 11.4.9.4 Results and Conclusion 11.4.10 Separation and Isolation of Lipids 11.4.10.1 Experiment: Lipid Composition of Wheat Grain 11.4.11 Experiment: Preparation of Cholesterol from Brain 11.4.11.1 Principle 11.4.11.2 Materials 11.4.11.3 Protocol 11.4.12 Fat-Soluble Vitamins 11.4.12.1 Experiment: Effect of Ultraviolet Light on Vitamin A 11.4.13 Experiment: Preparation of the D Vitamins by Irradiation of Their Precursors with Ultraviolet Light 11.4.13.1 Principle 11.4.13.2 Materials

xxxi 508 508 508 508 509 509 510 512 514 514 514 514 514 516 516 516 516 517 517 517 517 517 517 517 517 517 517 518 518 519 519 519 519 519 520 520 520 520 520 521 521 521 522 522 522 522 522 522 523 523 524

xxxii

Contents


11.4.13.3 Protocol 11.4.13.4 Results and Conclusion

524 524

11.5 Nucleic Acids 524 11.5.1 Introduction 524 11.5.2 Chemical Composition of the Nucleic Acids 525 11.5.3 Biological Significance of Nucleic Acids 530 11.5.4 DNA 531 11.5.4.1 Occurrence 531 11.5.4.2 Function 531 11.5.5 RNA 531 11.5.5.1 Occurrence 531 11.5.5.2 Protein Synthesis 532 11.5.6 Nucleic Acids of Viruses 532 11.5.7 Experiment: Isolation of RNA from Yeast 532 11.5.7.1 Principle 532 11.5.7.2 Materials 532 11.5.7.3 Protocol 532 11.5.8 Experiment: Isolation of Total RNA from Plant Tissue by SDS–Phenol Method 533 11.5.8.1 Principle 533 11.5.8.2 Precaution 533 11.5.8.3 Materials 533 11.5.8.4 Protocol 533 11.5.9 Experiment: Electrophoresis of RNA Nucleotides 534 11.5.9.1 Principle 534 11.5.9.2 Materials 534 11.5.9.3 Protocol 534 11.5.9.4 Results and Conclusion 535 11.5.10 Experiment: Separation of RNA Nucleotides by Ion-Exchange Chromatography 535 11.5.10.1 Principle 535 11.5.10.2 Materials 536 11.5.10.3 Protocol 536 11.5.10.4 Results and Conclusion 536 11.5.11 Experiment: Base Composition of RNA 536 11.5.11.1 Principle 536 11.5.11.2 Materials 537 11.5.11.3 Protocol 537 11.5.12 Experiment: Isolation of DNA from Coconut 537 11.5.12.1 Principle 537 11.5.12.2 Precautions 537 11.5.12.3 Material 538 11.5.12.4 Protocol 538 11.5.13 Experiment: Isolation of DNA from Pig Spleen 539 11.5.13.1 Principle 539 11.5.13.2 Materials 539 11.5.13.3 Protocol 539 11.5.14 Experiment: Ultraviolet Absorption of the Nucleic Acids 540 11.5.14.1 Principle 540 11.5.14.2 Materials 540 11.5.14.3 Protocol 541


Contents 11.5.15 Experiment: Viscosity of DNA Solutions 11.5.15.1 Principle 11.5.15.2 Materials 11.5.15.3 Protocol 11.5.15.4 Result and Conclusion 11.5.16 Experiment: Estimation of DNA by the Diphenylamine Reaction 11.5.16.1 Principle 11.5.16.2 Materials 11.5.16.3 Protocol 11.5.17 Experiment: Estimation of RNA by Means of the Orcinol Reaction 11.5.17.1 Principle 11.5.17.2 Materials 11.5.17.3 Protocol 11.5.18 Experiment: Determination of the Phosphorus Content of a Nucleic Acid 11.5.18.1 Principle 11.5.18.2 Materials 11.5.18.3 Protocol

12.

xxxiii 541 541 542 542 542 543 543 543 543 543 543 543 543 544 544 544 544

Suggested Readings

544

Important Links

545

Enzymology

547

12.1 Introduction

547

12.2 Enzymes as Catalysts 12.2.1 Catalysis

547 547

12.3 Classification

548

12.4 Measurement of Enzyme Activity 550 12.4.1 Enzyme Assay 550 12.4.1.1 Controls 550 12.4.1.2 Progress Curve 550 12.4.1.3 Enzyme Units 550 12.4.2 Enzyme Activity and Substrate Concentration 551 12.4.2.1 Michaelis–Menten Enzymes 551 12.4.2.2 Michaelis Equation 553 12.4.2.3 Kinetic Constants Km and V 553 12.4.2.4 Validity of the Michaelis Equation 555 12.4.3 Allosteric Enzymes 555 12.4.3.1 Effect of Substrate 555 12.4.3.2 Allosteric Kinetics 556 12.4.3.3 Kinetic Constants 556 12.4.3.4 Metabolic Control 557 12.4.4 Factors Affecting Enzyme Activity 557 12.4.4.1 Cofactors 557 12.4.4.2 Coenzymes 557 12.4.4.3 Activators 558 12.4.4.4 Inhibitors 558 12.4.5 Temperature 559 12.4.5.1 Effect of the Enzyme Reaction 559

xxxiv

Contents


12.4.5.2 12.4.5.3 12.4.6 pH 12.4.6.1 12.4.6.2

Energy of Activation of an Enzyme Effect of Temperature on Denaturation pH Optimum Enzyme Stability

560 561 561 561 562

12.5 Time Course Studies of the Reaction Catalyzed by Alkaline Phosphatase (EC 3.1.3.1) 12.5.1 Principle 12.5.2 Materials 12.5.3 Protocol 12.5.4 Result

562 562 563 563 564

12.6 Effect of Enzyme Concentration on the Rate of Enzyme-Catalyzed Reaction 12.6.1 Principle 12.6.2 Materials 12.6.3 Protocol 12.6.4 Result and Observations

564 564 565 565 565

12.7 Determination of Temperature Optima for Alkaline Phosphatase 12.7.1 Principle 12.7.2 Materials 12.7.3 Protocol 12.7.4 Result and Observations

565 565 566 566 567

12.8 Determination of Effect of pH on the Activity of Alkaline Phosphatase 12.8.1 Principle 12.8.2 Materials 12.8.3 Protocol

567 567 567 567

12.9 Effect of Substrate Concentration on Activity of Alkaline Phosphate and Determination of the Km and Vmax of the Reaction 12.9.1 Principle 12.9.2 Materials 12.9.3 Protocol 12.9.4 Results and Calculations

568 568 568 568 569

12.10 Progress Curve of Serum Alkaline Phosphatase (Orthophosphoric Acid Monoester Phosphohydrolase, 3.1.3.1) 12.10.1 Principle 12.10.2 Materials 12.10.3 Protocol 12.10.4 Results and Conclusion

569 569 569 569 570

12.11 Effect of Variation of Serum Alkaline Phosphatase Activity with Enzyme Concentration 570 12.11.1 Principle 570 12.11.2 Materials 570 12.11.3 Protocol 570 12.11.4 Results and Conclusion 570 12.12 Effect of Substrate Concentration and Inhibitors on Oxheart Lactate Dehydrogenase 571 12.12.1 Principle 571 12.12.2 Materials 571 12.12.3 Protocol 571

Contents 12.12.4 Results and Conclusion 12.12.4.1 Determination of the Michaelis Constant 12.12.4.2 Effect of Inhibitors 12.12.4.3 High Substrate Concentration

xxxv 572 572 572 573


12.13 Effect of Temperature on the Activity of α-Amylase 573 12.13.1 Principle 573 12.13.2 Materials 573 12.13.3 Protocol 573 12.13.3.1 Determination of the Energy of Activation 573 12.13.3.2 Temperature and Enzyme Stability 574 12.14 Determination of the Molecular Weight and Purity of Chymotrypsin from the Enzyme Kinetics 12.14.1 Principle 12.14.2 Materials 12.14.3 Protocol 12.14.4 Results and Conclusion

574 574 575 575 575

12.15 Yeast Isocitrate Dehydrogenase: Allosteric Enzyme 12.15.1 Principle 12.15.2 Materials 12.15.3 Protocol 12.15.3.1 Preparation of the Enzyme 12.15.3.2 Enzyme Assay

576 576 576 576 576 577

12.16 Purification and Characterization of Enzymes 12.16.1 Introduction 12.16.2 Method of Enzyme Isolation 12.16.2.1 Cell Lysis Method 12.16.2.2 Ultrasonic Cell Disruption 12.16.2.3 Grinding 12.16.2.4 Cell Lysis by High-Pressure Homogenizers 12.16.2.5 French Pressure Cell 12.16.2.6 Waring Blender and Virtis Homogenizer 12.16.2.7 Bead Mills 12.16.2.8 Enzymatic Lytic Method 12.16.2.9 Detergent 12.16.3 Clarification of the Extract 12.16.3.1 Aqueous Biphasic Separation 12.16.4 Enzyme Concentration 12.16.4.1 Ammonium Sulfate Precipitation 12.16.4.2 Dialysis of Protein 12.16.4.3 Enzyme Purification and Characterization 12.16.4.4 Purification Testing 12.16.4.5 Ultracentrifugation 12.16.4.6 Differential Centrifugation 12.16.4.7 Isopycnic Centrifugation

577 577 578 579 580 580 580 580 580 581 583 583 583 583 583 583 584 585 585 585 586 587

12.17 Isolation of Muramidase (Mucopeptide N-Acetylmuramyl Hydrolase, 3.21.17) from Egg White 12.17.1 Principle 12.17.2 Materials 12.17.3 Protocol 12.17.3.1 Enzymes and Protein Assay 12.17.3.2 Purification

587 587 588 589 589 589

xxxvi

Contents 12.18 Separation of the Isoenzymes of Lactate Dehydrogenase by Electrophoresis on Polyacrylamide Gel 12.18.1 Principle 12.18.2 Materials 12.18.3 Protocol

590 590 591 591


12.19 Enzyme Engineering 592 12.19.1 Introduction 592 12.19.2 Enzyme Production by Gene Manipulation 592 12.19.2.1 Future Uses 594 12.19.3 Advantages of Genetic Engineering 595 12.19.4 Manipulation of Enzyme Design 595 12.19.4.1 Rational Protein Design 596 12.19.4.2 Site-Directed Mutagenesis 596 12.19.4.3 Random Mutagenesis 601 12.19.4.4 Combinatorial Mutagenesis 602 12.19.5 Directed Evolution 603 12.19.5.1 Gene Shuffling 604 12.19.5.2 Family Shuffling 606 12.19.5.3 Error-Prone PCR 609 12.19.6 Selection Parameters 609 12.19.6.1 In Vivo Display in Bacteria 609 12.19.6.2 Phage Display or Phagemid Display Can Be Used to Facilitate the Selection of Mutant Peptides 609 12.19.6.3 Ribosome Display (In Vitro) 610 12.19.6.4 High-Throughput Product Analysis 612 12.19.6.5 Agar Plate-Based Assay 613 12.19.7 Application of Enzyme Engineering 613 12.19.7.1 Subtilisin 613 12.19.7.2 Protein Engineering of Laundry Detergent 614

13.

12.20 Artificial Enzymes 12.20.1 Synzymes 12.20.2 Abzymes 12.20.3 Promises of Enzyme Technology

614 614 615 615

Suggested Readings

616

Important Links

616

Membranes

619

13.1 Introduction

619

13.2 Composition of Membranes 13.2.1 Phospholipids 13.2.2 Cholesterol and Other Lipids 13.2.3 Proteins

619 620 622 622

13.3 Membrane Transport 13.3.1 Diffusion 13.3.1.1 Simple Passive Diffusion 13.3.1.2 Facilitated Diffusion 13.3.2 Active Transport

622 622 622 624 624

13.4 Effect of Lipid Composition on the Permeability of a Lipid Monolayer 13.4.1 Principle

625 625

Contents

xxxvii



625 625

13.5 Effect of Detergents and Other Membrane-Active Reagents on the Erythrocyte Membrane 13.5.1 Principle 13.5.2 Materials 13.5.3 Protocol 13.5.4 Results and Conclusion

626 626 626 626 626

13.6 Permeability of Model Membrane (Liposome) 13.6.1 Principle 13.6.1.1 Liposomes 13.6.1.2 Membrane Permeability 13.6.1.3 Ionophores 13.6.2 Materials 13.6.3 Protocol 13.6.3.1 Preparation of Liposomes 13.6.4 Results and Conclusions

627 627 627 627 627 628 628 628 628

13.7 Effect of Cholesterol on the Anion Permeability of a Phospholipid Membrane 13.7.1 Principle 13.7.2 Materials 13.7.3 Protocol 13.7.4 Results and Conclusion

629 629 630 630 630

13.8 Effect of Insulin on the Transport into Isolated Fat Cell 13.8.1 Principle 13.8.1.1 Glucose Uptake 13.8.1.2 Isolated Fat Cells 13.8.2 Materials 13.8.3 Protocol 13.8.3.1 Preparation of Isolated Fats Cells 13.8.3.2 Glucose Uptake 13.8.4 Results and Conclusion

630 630 630 630 631 631 631 632 632

13.9 Transport of Amino Acids across the Small Intestine 13.9.1 Principle 13.9.1.1 Transport of Metabolites 13.9.1.2 Everted Sac 13.9.1.3 Histidine Estimation 13.9.2 Materials 13.9.2.1 Incubation Medium (KRP Containing 18 mmol/L Glucose) 13.9.2.2 Inhibitor Solutions 13.9.3 Protocol 13.9.3.1 Preparation of the Everted Sac 13.9.3.2 Amino Acid Transport 13.9.3.3 Determination of Histidine 13.9.4 Results and Calculation

632 632 632 632 633 633 633 633 634 634 635 635 635

13.10 Absorption of Xylose from the Gut in Man 13.10.1 Principle 13.10.1.1 Absorption of Xylose 13.10.1.2 Xylose Estimation 13.10.2 Materials 13.10.2.1 p-Bromoaniline Reagent

635 635 635 636 636 636

xxxviii

Contents


13.10.3 Protocol 636 13.10.3.1 Test 636 13.10.3.2 Estimation of d-Xylose 636 13.10.4 Results and Calculation 636 Suggested Readings

637

Important Links

637

14. Photosynthesis and Respiration

639

14.1 Introduction 14.1.1 Bioenergetics

639 639

14.2 Measurement 14.2.1 Principle 14.2.2 Practice 14.2.3 Calibration of the Electrode 14.2.4 Applications

639 639 641 642 643

14.3 Photosynthesis 14.3.1 Light Reaction 14.3.2 Dark Reaction

644 644 644

14.4 Isolation of Chloroplasts from Spinach Leaves 14.4.1 Principle 14.4.2 Materials 14.4.3 Protocol 14.4.4 Results and Observations

646 646 647 647 649

14.5 Determination of Chlorophyll Concentration in the Chloroplast Suspension 14.5.1 Principle 14.5.2 Protocol

649 649 649

14.6 Evolution of Oxygen by Isolated Chloroplast Using Hill Oxidants 14.6.1 Principle 14.6.2 Materials 14.6.3 Protocol 14.6.3.1 Oxygen Electrode Assays 14.6.3.2 Hill Oxidants 14.6.4 Results and Conclusion

650 650 651 651 651 651 651

14.7 Methyl Viologen as a Terminal Electron Acceptor 14.7.1 Principle 14.7.2 Materials 14.7.3 Protocol 14.7.3.1 Oxygen Uptake with Methyl Viologen as Electron Acceptor 14.7.3.2 Activity of PSI Measured with Methyl Viologen 14.7.4 Results and Conclusion

651 651 652 652 652 652 652

14.8 Spectrophotometric Assay of the Hill Reaction and the Estimation of Chlorophyll 653 14.8.1 Principle 653 14.8.2 Materials 653 14.8.3 Protocol 653 14.8.3.1 Assay of Hill Reaction 653 14.8.3.2 Assay of Chlorophyll Content 653

Contents


14.8.3.3 Oxidation and Reduction in Living Cells 14.8.3.4 Electron Transport Chain 14.8.4 Results and Interpretation

15.

xxxix 653 655 656

14.9 Respiration of Mitochondria and Oxidative Phosphorylation 14.9.1 Principle 14.9.2 Materials 14.9.3 Protocol

656 656 657 657

14.10 Effect of Inhibitors on the Respiratory Chain 14.10.1 Principle 14.10.2 Materials 14.10.3 Protocol 14.10.4 Results and Conclusion

657 657 659 659 660

14.11 Compounds That Affect the High-Energy State of Mitochondria 14.11.1 Principle 14.11.2 Materials 14.11.3 Protocol 14.11.4 Results and Conclusion

660 660 661 661 661

Suggested Reading

661

Important Links

662

Microbiology

663

15.1 Introduction

663

15.2 Good Microbiological Practice 15.2.1 Laboratory Safety 15.2.1.1 Preparation of Temporary Cotton Plugs 15.2.1.2 Preparation of Permanent Cotton Plugs

663 663 666 667

15.3 Culture Media

668

15.4 Types of Media 15.4.1 Synthetic Media 15.4.2 Complex Media 15.4.3 Natural Media

669 669 669 669

15.5 Preparation of Culture Media 15.5.1 Procedure

670 670

15.6 Maintenance and Preservation of Cultures 15.6.1 Transfer on Fresh Media 15.6.2 Overlaying with Mineral Oil 15.6.3 Freeze-Drying (Lyophilization) 15.6.4 Storage at Low Temperature

670 671 671 671 671

15.7 Preparation of Basic Liquid Media (Broth) for the Cultivation of Bacteria 15.7.1 Principle 15.7.2 Materials 15.7.2.1 Nutrient Broth 15.7.2.2 Glucose Broth 15.7.3 Protocol

671 671 672 672 672 672

15.8 Solid Media for Growth of Microorganisms 15.8.1 Introduction

672 672

xl

Contents


15.8.2 Common Culture Media Used in Laboratories 674 15.8.2.1 Natural Media 674 15.8.2.2 Semisynthetic Media 675 15.8.2.3 Synthetic Media 675 15.8.2.4 Some Special Media for Cultivation of Members of Actinomycetes 676 15.8.2.5 Media for Soil Bacteria 676 15.8.2.6 Media for Soil Fungi 677 15.8.2.7 Selective Media for the Isolation of Plant Pathogens 677 15.9 Isolation and Maintenance of Organisms 15.9.1 Isolation and Maintenance of Organisms by Streak Plate Method 15.9.1.1 Principle 15.9.1.2 Materials 15.9.1.3 Protocol 15.9.1.4 Results and Observations

677 677 677 678 678 678

15.10 Isolation of Organisms by Pour Plate Method and Serial Dilation Method 15.10.1 Introduction 15.10.2 Materials 15.10.3 Protocol 15.10.4 Results and Observations

679 679 681 682 683

15.11 Spread Plate Technique 15.11.1 Introduction 15.11.2 Materials 15.11.3 Protocol 15.11.4 Result

683 683 683 683 685

15.12 Isolation of Vibrio parahaemolyticus from Seafood Sample 15.12.1 Introduction 15.12.2 Materials 15.12.2.1 TCBS Media Composition (g/L) 15.12.3 Protocol 15.12.4 Results and Observation

685 685 686 686 686 686

15.13 Isolation of Lactic Acid Bacteria from Milk and Buttermilk Sample 15.13.1 Introduction 15.13.2 Materials 15.13.3 Protocol 15.13.4 Results and Observation

686 686 687 688 688

15.14 Isolation of Staphylococcus aureus from Food Sample 15.14.1 Introduction 15.14.2 Materials 15.14.3 Protocol 15.14.4 Results and Observations

689 689 689 689 689

15.15 Sauerkraut Fermentation 15.15.1 Introduction 15.15.2 Materials 15.15.3 Protocol 15.15.4 Results and Observations

690 690 690 691 691

15.16 Isolation of Microorganisms from Air 15.16.1 Materials

692 692

Contents


15.16.2 Protocol 15.16.3 Observation

xli 692 692

15.17 Phyllosphere/Phylloplane Microbiology 15.17.1 Introduction 15.17.2 Isolation of Microbial Flora from Phyllosphere and Phylloplane 15.17.2.1 Principle 15.17.2.2 Materials 15.17.2.3 Protocol 15.17.2.4 Results and Observation

692 692 692 692 693 693 693

15.18 Measurement of Microbial Growth 15.18.1 Principle 15.18.2 Bacterial Growth

693 693 696

15.19 Methods to Measure Bacterial Growth 15.19.1 Turbidimetry Determinations

696 697

15.20 Growth Curve Measurement of Bacterial Population by Serial Dilution Method 15.20.1 Principle 15.20.2 Materials 15.20.3 Protocol 15.20.4 Results and Observations

698 698 701 701 701

15.21 Serial Dilutions 15.21.1 Principle 15.21.2 Materials 15.21.3 Protocol 15.21.4 Results and Observations

702 702 702 702 703

15.22 Measurement of Fungal Growth by Colony Diameter Method 15.22.1 Principle 15.22.2 Materials 15.22.3 Protocol 15.22.4 Results and Observations

703 703 704 704 704

15.23 Biomass Measurement of Fungal Growth (Dry Weight Method) 15.23.1 Principle 15.23.2 Materials 15.23.3 Protocol 15.23.4 Results and Observations

705 705 705 705 706

15.24 Effect of Incubation Temperature on Growth 15.24.1 Materials 15.24.2 Protocol 15.24.3 Results and Observations

706 706 706 706

15.25 Effect of pH on Microbial Growth 15.25.1 Introduction 15.25.2 Materials 15.25.3 Protocol 15.25.4 Result and Observations

707 707 707 707 708

15.26 Microscopic Examination of Bacteria 15.26.1 Hanging Drop Preparation or Motility Test 15.26.1.1 Introduction 15.26.1.2 Materials

708 708 708 708

xlii

Contents


15.26.1.3 Protocol 15.26.1.4 Results and Observations

708 709

15.27 Microscopic Examination of Pathogens by One Tube One Plate Method 15.27.1 Introduction 15.27.2 Materials 15.27.3 Protocol 15.27.4 Results and Observation

709 709 710 710 710

15.28 Smear Preparation 15.28.1 Introduction 15.28.2 Protocol 15.28.2.1 From Liquid Media 15.28.2.2 From Solid Medium

710 710 711 711 711

15.29 Staining 15.29.1 Type of Stains 15.29.1.1 Simple Stains 15.29.1.2 Negative Stain 15.29.1.3 Differential Stains 15.29.1.4 Structural Stains

712 712 712 713 715 719

15.30 Biochemical Tests 15.30.1 Introduction 15.30.1.1 Amylase Activity 15.30.1.2 Caseinase 15.30.1.3 Carbohydrate Digestion 15.30.1.4 Catalase Activity 15.30.1.5 Urease 15.30.1.6 H2S Production 15.30.1.7 IMVIC Reactions 15.30.1.8 Litmus-Milk Reaction 15.30.1.9 Nitrate Reduction 15.30.1.10 Oxidase Test 15.30.1.11 Pectinase 15.30.1.12 Gelatinase 15.30.1.13 Lipase 15.30.1.14 Gluconate Test

725 725 725 726 727 731 732 734 734 739 740 741 744 744 746 746

15.31 Microbial Genetics 15.31.1 Introduction 15.31.1.1 Mutant Isolation by Gradient Plate Method 15.31.1.2 Demonstration of Streaking Plates Using Loop/Toothpick 15.31.1.3 Isolation of Streptomycin-Resistant Mutant by Replica Plating Method 15.31.1.4 Bioassay for Evaluating the Mutagen for Carcinogen by Ames Test

747 747 748 749 751 754

15.32 Bacterial Recombination 15.32.1 Introduction 15.32.1.1 To Demonstrate Transformation in E. coli or in Any Other Bacterium

757 757

Suggested Readings

768

Important Links

769

759

Contents


16.

xliii

Soil Biology

771

16.1 Introduction

771

16.2 Estimation of Humus in Soil/Manure 16.2.1 Principle 16.2.2 Materials 16.2.3 Protocol

772 772 773 773

16.3 Determination of Microbial Carbon Biomass 16.3.1 Principle 16.3.2 Materials 16.3.3 Protocol 16.3.3.1 Sample Collection 16.3.3.2 Analysis 16.3.4 Results and Observations

773 773 774 774 774 774 776

16.4 Estimation of Algal (Photosynthetic) Biomass in Soil Cores 16.4.1 Introduction 16.4.2 Materials 16.4.3 Protocol 16.4.4 Result and Observations

776 776 776 776 776

16.5 Soil Decomposition and Microbial Community Structure (Winogradsky Technique) 16.5.1 Principle 16.5.2 Materials 16.5.3 Protocol 16.5.4 Observation and Results

777 777 777 777 778

16.6 Soil Algae Enumeration 16.6.1 Algae Test 16.6.2 Introduction 16.6.3 Materials 16.6.4 Protocol 16.6.4.1 Soil Metabolic Assessment

778 778 778 779 779 781

16.7 Phosphate Solubilizing Bacteria 16.7.1 Isolation Phosphate Solubilizing Bacteria from Soil 16.7.1.1 Principle 16.7.1.2 Materials 16.7.1.3 Protocol 16.7.1.4 Result and Observation

785 785 785 786 786 786

16.8 Isolation of Sulfur Oxidizing Bacteria from Soil 16.8.1 Principle 16.8.2 Materials 16.8.3 Protocol 16.8.4 Results and Observation

788 788 788 788 789

16.9 Nitrogen Cycle

789

16.10 Demonstrate the Process of Ammonification in Soil 16.10.1 Principle 16.10.2 Material 16.10.3 Protocol 16.10.4 Result and Observation

790 790 791 791 791

xliv

Contents 16.11 Demonstrate the Process of Nitrification in Soil 792 16.11.1 Principle 792 16.11.2 Materials 793 16.11.2.1 Reagents 793 16.11.3 Protocol 794 16.11.3.1 Test for Nitrosomonas 794 16.11.3.2 Test for Nitrobacter 794 16.11.4 Results 794


16.12 MPN Method for Nitrosomonas and Nitrobacter 795 16.12.1 Principle 795 16.12.2 Materials 795 16.12.3 Protocol 796 16.12.4 Results and Observations 796 16.13 Isolation of Azospirillum sp. from Soil and Plant Root 16.13.1 Introduction 16.13.2 Materials 16.13.3 Protocol 16.13.4 Result and Observations

796 796 797 797 797

16.14 Enumeration of Azospirillum by MPN Method 16.14.1 Introduction 16.14.2 Materials 16.14.3 Protocol 16.14.4 Result and Observations

798 798 798 798 798

16.15 Demonstration of Microbial Denitrification in Soil 16.15.1 Principle 16.15.2 Materials 16.15.3 Protocol 16.15.4 Results and Observation

799 799 800 800 800

16.16 Isolation of Plant Growth Promoting Rhizobacteria 16.16.1 Principle 16.16.2 Protocol 16.16.2.1 Characterization of PGPR 16.16.2.2 Production of Ammonia 16.16.2.3 IAA 16.16.2.4 HCN Production 16.16.2.5 Antibiotic Production 16.16.2.6 Antifungal Activity

800 800 801 801 802 802 803 803 804

16.17 Isolation of Photosynthetic Bacteria 804 16.17.1 Principle 804 16.17.2 Materials 804 16.17.3 Protocol 805 16.17.4 Results and Observations 805 16.18 Cyanobacteria 16.18.1 Introduction 16.18.2 Isolation and Purification of Cyanobacteria 16.18.2.1 Isolation 16.18.2.2 Inoculation Procedure 16.18.2.3 Incubation Conditions 16.18.2.4 Culture Room

805 805 806 806 809 809 809

Contents


16.18.2.5 Isolation Procedures 16.18.2.6 Purification Methods 16.18.2.7 Test for Purity 16.18.2.8 Culture Media 16.18.3 Isolation of Cyanobacteria from Soil/Water 16.18.3.1 Introduction 16.18.3.2 Materials 16.18.3.3 Protocol 16.18.3.4 Results and Observations 16.18.4 Measurement of Specific Growth Rate and Generation Time in a Given Cyanobacterium 16.18.4.1 Introduction 16.18.4.2 Materials 16.18.4.3 Protocol 16.18.4.4 Results and Observations

xlv 809 811 812 812 813 813 813 813 813 814 814 814 814 815

16.19 Nitrogen Fixation 16.19.1 Free Living 16.19.1.1 Introduction 16.19.1.2 Isolation of Free-Living Nitrogen-Fixing Bacteria from Soil 16.19.2 Symbiotic Nitrogen-Fixing Bacteria 16.19.2.1 Introduction 16.19.2.2 Sampling 16.19.2.3 Laboratory Work

816 816 817 817 820 820 822 822

16.20 Mycorrhizal Symbiotic Association 16.20.1 Introduction 16.20.2 Sampling 16.20.3 Manipulation and Staining of Spores and Roots 16.20.3.1 Determination of Percent Mycorrhizal Colonization in Root 16.20.3.2 Spore and Root Extraction from Pot Cultures/Field Samples 16.20.3.3 Preparation of Permanent Slide Mount for Reference 16.20.4 Histochemical Visualization of Total AMF Mycelium in Roots 16.20.4.1 Principle 16.20.4.2 Materials 16.20.4.3 Protocol 16.20.4.4 Results and Observations 16.20.5 Histochemical Localization of Lipids in Mycorrhizal Roots 16.20.5.1 Principle 16.20.5.2 Materials 16.20.5.3 Protocol 16.20.5.4 Results and Observations 16.20.6 Histochemical Localization of SDH 16.20.6.1 Principle 16.20.6.2 Materials 16.20.6.3 Protocol 16.20.6.4 Results and Observations 16.20.7 Histochemical Localization of ALP and Acid Phosphatase 16.20.7.1 Principle 16.20.7.2 Materials 16.20.7.3 Protocol 16.20.7.4 Results and Observation 16.20.8 Histochemical Localization of Peroxidase 16.20.8.1 Principle

825 825 826 827 827 827 829 830 830 831 831 832 832 832 832 832 833 833 833 833 833 834 834 834 834 834 834 834 834

xlvi

Contents


16.20.8.2 Materials 834 16.20.8.3 Protocol 834 16.20.8.4 Results and Observation 835 16.20.9 Estimation of Mycorrhizal Colonization 835 16.20.9.1 Protocol 835 16.20.9.2 Results and Observations 836 16.20.10 Extraction and Measurement of AM Fungal Hyphae in Soil 836 16.20.10.1 Protocol 836 16.20.11 Estimation of SDH and ALP Active Hyphae in Soil 836 16.20.11.1 Materials 836 16.20.11.2 Protocol 838 16.20.11.3 Results and Observations 838 16.21 Actinomycetes 16.21.1 Introduction 16.21.2 Isolation of Actinomycetes from Garden Soil on Agar Plates 16.21.2.1 Materials 16.21.2.2 Protocol 16.21.2.3 Results and Observation 16.21.3 Qualitative Determination of Antagonistic Properties of the Isolated Streptomyces from Soil 16.21.3.1 Introduction 16.21.3.2 Material 16.21.3.3 Protocol

838 838 839 839 839 839

16.22 Aquatic Molds 16.22.1 Introduction 16.22.2 Isolation and Purification of Aquatic Molds 16.22.2.1 Materials 16.22.2.2 Protocol 16.22.2.3 Results and Observations 16.22.3 Isolation of Cellular Slime Molds 16.22.3.1 Introduction 16.22.3.2 Material 16.22.3.3 Protocol 16.22.3.4 Results and Observations

843 843 843 843 844 844 844 844 845 845 846

16.23 Fungi 16.23.1 Introduction 16.23.2 Isolation and Microscopic Examination of Fungi 16.23.2.1 Materials 16.23.2.2 Protocol 16.23.2.3 Results and Observations 16.23.3 Serial Dilution Plate Technique 16.23.3.1 Materials 16.23.3.2 Protocol 16.23.3.3 Results and Observations 16.23.4 Slide Culture Mount (Riddell Mount) 16.23.4.1 Introduction 16.23.4.2 Materials 16.23.4.3 Protocol 16.23.4.4 Results and Observation 16.23.5 Isolation of Pure Culture 16.23.5.1 Principle 16.23.5.2 Protocol

846 846 847 847 847 847 847 847 848 848 849 849 849 849 849 850 850 850

839 839 839 841

Contents 16.23.6 Isolation of Microorganisms from Soil and Water 16.23.6.1 Introduction 16.23.6.2 Methods for the Isolation of Pure Cultures from Soil/Water 16.23.6.3 Experiment Isolation of Pure Cultures from Water/Soil 16.23.6.4 Isolation of Yeast from Soil Ripened Grape


16.24 Rhizosphere 16.24.1 Introduction 16.24.2 Isolation of Microorganisms from Rhizosphere 16.24.2.1 Materials 16.24.2.2 Protocol 16.24.2.3 Observations

xlvii 851 851 851 853 854 856 856 856 856 856 858

16.25 Microbial Production of Citric Acid by Aspergillus niger 858 16.25.1 Introduction 858 16.25.2 Materials 858 16.25.3 Protocol 858 16.25.4 Result 858 16.26 Isolation of Industrially Important Antibiotic Producing Microorganisms 16.26.1 Introduction 16.26.2 Materials 16.26.3 Methods 16.26.4 Results and Observations

861 861 861 861 861

16.27 Assay of Antibiotics and Demonstration of Antibiotic Resistance 16.27.1 Introduction 16.27.2 Materials 16.27.3 Antibiotic Sensitivity Test 16.27.3.1 Protocol (Kirby–Bauer Method) 16.27.3.2 Results and Observation 16.27.4 Determination of MIC of Antibiotic 16.27.4.1 Protocol 16.27.4.2 Result and Observations

861 861 862 862 862 862 862 862 863

16.28 Characterize and Identify Aflatoxin by Thin-Layer Chromatography 16.28.1 Introduction 16.28.2 Materials 16.28.3 Procedure 16.28.3.1 Extraction of Toxins 16.28.3.2 Preparation of TLC Plates

863 863 864 865 865 866

16.29 Protozoa 16.29.1 Introduction 16.29.2 Isolation of Protozoa from Soil 16.29.2.1 Materials 16.29.2.2 Protocol 16.29.2.3 Results and Observations 16.29.3 Staining of Free-Living (Nonpathogenic) Protozoa 16.29.3.1 Introduction 16.29.3.2 Materials 16.29.3.3 Protocol 16.29.3.4 Results and Observations

867 867 867 867 867 868 868 868 868 868 868

16.30 Observation of Microscopic Invertebrates from Pond Water 16.30.1 Introduction 16.30.2 Materials

868 868 871

xlviii

Contents


16.30.3 Protocol 16.30.4 Results and Observations

871 871

16.31 Observation of Algae from Pond Water 16.31.1 Introduction 16.31.2 Protocol

872 872 872

16.32 Microscopic Examination of Soil Microbes 16.32.1 Observe Soil Bacteria 16.32.2 Preparation of a Smear Mount 16.32.2.1 Materials 16.32.2.2 Protocol 16.32.3 Preparation of Wet Mounts 16.32.3.1 Protocol 16.32.3.2 Gram Stain for Bacteria 16.32.3.3 Protocol

872 872 873 873 873 873 873 874 874

16.33 Bacteria Frequently Isolated from Soil 16.33.1 Gram-Negative Chemolithotrophs 16.33.1.1 Nitrobacteraceae 16.33.1.2 Sulfur Metabolizing 16.33.2 Gram-Negative Aerobic Rods and Cocci 16.33.2.1 Pseudomonadaceae 16.33.2.2 Azotobacteraceae 16.33.2.3 Rhizobiaceae 16.33.2.4 Uncertain Affiliation 16.33.3 Gram-Negative Facultative Anaerobic Rods 16.33.4 Gram-Negative Cocci and Coccobacilli 16.33.5 Gram-Positive Cocci 16.33.6 Endospore-Forming Rods 16.33.7 Budding and/or Appendaged 16.33.8 Coryneform Group 16.33.9 Mycobacteriaceae

874 874 874 874 875 875 875 875 875 875 875 875 876 876 876 876

16.34 Fungi Frequently Isolated from Soil 16.34.1 Kingdom Protista (Protozoans) 16.34.2 Kingdom Stramenophila 16.34.3 Phylum Heterokonta 16.34.4 Class Hyphochytridiomycetes 16.34.5 Class Oomycetes 16.34.6 Kingdom Eumycota (Fungi) 16.34.7 Phylum Chytridiomycota 16.34.8 Phylum Zygomycota 16.34.9 Class Zygomycetes 16.34.10 Phylum Ascomycota 16.34.11 Subphylum Euascomycotina 16.34.12 Subphylum Laboulbeniomycotina 16.34.13 Subphylum Saccharomycotina 16.34.14 Phylum Basidiomycotina 16.34.15 Classes Urediniomycetes and Ustomycetes 16.34.16 Class Gelimycetes 16.34.17 Class Homobasidiomycetes 16.34.18 Form Phylum Deuteromycotina 16.34.19 Form Class Blastomycetes 16.34.20 Form Class Coelomycetes

877 877 877 877 877 878 878 878 879 879 879 879 879 879 880 880 880 880 880 880 881


17.

Contents

xlix

16.34.21 Form Class Hyphomycetes 16.34.22 Series Aleuriospore 16.34.23 Series Annellosporae 16.34.24 Series Arthrosporae 16.34.25 Series Blastosporae 16.34.26 Series Botryoblastosporae 16.34.27 Series Meristem Blastosporae 16.34.28 Series Phialosporae

881 881 881 881 881 881 881 882

16.A Appendix 16.A.1 Common Culture Media for Soil Microorganisms 16.A.2 Some Common Media for Cyanobacteria

882 882 884

Suggested Reading

885

Important Links

888

Virology

889

17.1 Introduction

889

17.2 Isolation of Bacteriophage from Sewage 17.2.1 Principle 17.2.2 Materials 17.2.2.1 Media 17.2.3 Protocol 17.2.3.1 First Step: Filtration and Amplification of Bacterial Viruses 17.2.3.2 Second Step: Bacteriophage Isolation and Plating 17.2.4 Results and Observations 17.2.4.1 Phage Typing

890 890 890 890 891 891 892 893 893

17.3 Preparation of High Titer of Bacteriophage of Isolated Bacterial Virus 17.3.1 Principle 17.3.2 Materials 17.3.3 Protocol 17.3.4 Results and Observations

893 893 894 894 895

17.4 One-Step Growth Curve of Coliphage and Determining the Number of Phage Particles Released by the Lysis of the Single Infected Bacterial Cell (Burst Size) 17.4.1 Introduction 17.4.2 Materials 17.4.3 Protocol 17.4.3.1 Bacteriophage Production Burst Size Determination (Single-Step Growth Curve) 17.4.4 Result 17.4.4.1 Calculation for Example

895 895 896 896 896 898 898

17.5 Demonstration of Lysogeny and Isolation of Rhizobium Phage 17.5.1 Introduction 17.5.2 Materials 17.5.3 Protocol 17.5.4 Results and Observations

899 899 899 899 900

17.6 Isolation of Cyanophage LPP from Pond Water 17.6.1 Introduction 17.6.2 Materials

900 900 903

l

Contents


17.6.2.1 Modified Chu 10 Medium 17.6.2.2 Magnesium Saline Composition g/L in Distilled Water 17.6.3 Protocol 17.6.3.1 PFU Assay 17.6.4 Results and Observations

18.

903 903 903 903 904

17.7 Demonstration for the Presence of Actinophages in Soil 17.7.1 Introduction 17.7.2 Materials 17.7.3 Protocol 17.7.4 Result and Observations

906 906 906 906 906

17.8 Isolation of Phage from Flies 17.8.1 Introduction 17.8.2 Materials 17.8.3 Protocol 17.8.3.1 Extraction 17.8.3.2 PFU Assay 17.8.4 Results and Observations

907 907 907 907 907 909 909

17.9 Virus Culture in Embryonating Chicken Eggs 17.9.1 Introduction 17.9.2 Materials 17.9.3 Protocol

909 909 909 910

17.10 Extraction and Isolation of Tobacco Mosaic Virus 17.10.1 Introduction 17.10.2 Materials 17.10.3 Protocol 17.10.3.1 Extraction and Pathogenicity Test 17.10.3.2 Purification of TMV Virus 17.10.4 Result and Observations

911 911 912 912 912 912 915

17.11 Cultivation of Viruses in Tissue Culture 17.11.1 Introduction 17.11.2 Experiment Study of Morphology of Chicken Fibroblast Monolayers 17.11.2.1 Protocol

915 915 915 915

17.12 Viral Hemagglutination Test for the Presence of Antigens 17.12.1 Introduction 17.12.2 Materials 17.12.3 Protocol 17.12.4 Results

917 917 917 918 919

Suggested Readings

919

Important Links

920

Immunochemical Methods

921

18.1 Introduction

921

18.2 Immunity 18.2.1 Innate Immunity/Native Immunity/Nonspecific Immunity 18.2.1.1 Species Immunity 18.2.1.2 Racial Immunity 18.2.1.3 Individual Immunity

921 922 922 922 922

Contents 18.2.2


18.2.3

Acquired Immunity 18.2.2.1 Active Natural Immunity 18.2.2.2 Active Artificial Immunity Passively Acquired Immunity 18.2.3.1 Passive Natural Immunity 18.2.3.2 Passive Artificial lmmunity

li 923 923 923 923 924 924

18.3 Ags

924

18.4 Antigenic Determinant or Epitope

925

18.5 Haptens

925

18.6 Specificity

926

18.7 T-Cell Dependent (TD) and T-Cell Independent (TI) Ags

926

18.8 Immunoglobulins 18.8.1 Classification 18.8.2 Structure

926 927 927

18.9 IG Determinants 18.9.1 Isotypes 18.9.2 Allotypes 18.9.3 Idiotypes

931 931 931 932

18.10 Ig Classes 18.10.1 IgG 18.10.2 IgM 18.10.3 IgA 18.10.4 IgD 18.10.5 IgE 18.10.6 Abnormal Igs

932 932 932 933 934 935 935

18.11 Monoclonal Antibodies 18.11.1 Production 18.11.2 Uses

936 936 938

18.12 Polyclonal Antibodies

938

18.13 Ag–Ab Reaction 18.13.1 Affinity and Avidity 18.13.1.1 Affinity (Intrinsic Affinity) 18.13.1.2 Avidity (Functional Affinity) 18.13.2 Sensitivity and Specificity 18.13.3 Titer 18.13.4 Zeta Phenomenon 18.13.5 Mechanism of Ag–Ab Reactions (Marrack’s Hypothesis) 18.13.6 Zone Phenomenon

938 939 939 939 939 939 939 940 941

18.14 Determination of Ag–Ab Reactions 941 18.14.1 Precipitation 941 18.14.1.1 Definition 941 18.14.1.2 Applications 941 18.14.1.3 Ring Test 942 18.14.1.4 Slide Test 942 18.14.1.5 Tube Test 942 18.14.1.6 Immunodiffusion (or) Precipitation in Gel (or) Gel Precipitation 942


lii

Contents 18.14.1.7 Immunoelectrophoresis 18.14.1.8 Electroimmunodiffusion (Electroimmunoassay) 18.14.2 Agglutination Reactions 18.14.2.1 Definition 18.14.2.2 Slide Agglutination Test 18.14.2.3 Tube (Direct) Agglutination Test 18.14.2.4 Passive (Indirect) Agglutination Test 18.14.2.5 Reverse Passive Agglutination Test 18.14.2.6 Coagglutination 18.14.3 Complement Fixation Test 18.14.3.1 Principle 18.14.3.2 Procedure 18.14.3.3 Results and Observations 18.14.4 Immunolabeled Tests 18.14.4.1 Immunofluorescence 18.14.4.2 ELISA 18.14.4.3 Radioimmunoassay 18.14.4.4 Immunoelectroblotting 18.14.4.5 Immunochromatographic Technique 18.14.4.6 Immunoelectron Microscopy 18.14.5 Neutralization 18.14.5.1 Toxin Neutralization Tests 18.14.6 Opsonization 18.14.7 Chemiluminescence Assay

945 946 947 948 948 949 949 951 951 955 955 956 957 958 958 960 962 965 965 965 966 966 966 969

18.15 Collection of Blood Sample

969

18.16 Experiment: To Determine the ABO Blood Group by Using Slide Agglutination Test 18.16.1 Introduction 18.16.2 Materials 18.16.3 Protocol 18.16.4 Interpretation

969 969 970 970 970

18.17 Experiment: Determination of Rh Factor in Human Being 18.17.1 Introduction 18.17.2 Materials 18.17.3 Protocol 18.17.4 Observation and Result

970 970 972 973 973

18.18 Experiment: Demonstration of Agglutination Reaction of Unknown Bacterial Culture by Slide Agglutination Technique 18.18.1 Introduction 18.18.2 Materials 18.18.3 Protocol 18.18.4 Results and Observations

973 973 974 974 975

18.19 Experiment: Estimate the Total Leukocyte Count by Hemocytometer 18.19.1 Introduction 18.19.1.1 Causes of Leukocytosis 18.19.1.2 Causes of Leukopenia 18.19.1.3 Normal Values 18.19.2 Materials 18.19.2.1 Specimens 18.19.3 Protocol

975 975 975 976 976 976 976 977

Contents


18.19.4 Calculations 18.19.5 Error of the Total White Cell Count

liii 977 978

18.20 Experiment: Determine the Differential Count of WBCs (Leucocyte) by Staining Method 18.20.1 Introduction 18.20.1.1 Hypersegmented Neutrophil 18.20.1.2 Eosinophil 18.20.1.3 Basophil 18.20.1.4 Lymphocyte 18.20.1.5 Monocyte 18.20.2 Protocol 18.20.2.1 Preparation of Blood Smears 18.20.2.2 Staining the Cells 18.20.3 Observation

978 978 979 979 979 979 979 979 979 980 981

18.21 Experiment: Determine the Total Erythrocyte Count by Hemocytometer 18.21.1 Introduction 18.21.1.1 Specimens 18.21.2 Materials 18.21.3 Protocol 18.21.4 Calculations 18.21.5 Observations 18.21.5.1 Additional Information 18.21.5.2 Sources of Error 18.21.6 Clinical Significance 18.21.7 Results and Observations

981 981 982 982 982 982 983 983 983 983 984

18.22 Experiment: Lymphoid Cells Identification in Blood Smears 18.22.1 Introduction 18.22.2 Materials 18.22.3 Protocol 18.22.4 Results

984 984 985 985 985

18.23 Experiment: Spleen Cell Preparation from Mouse 18.23.1 Introduction 18.23.2 Materials 18.23.2.1 Reagents 18.23.3 Protocol 18.23.4 Result

985 985 986 986 986 987

18.24 Experiment: Identification of Lymphocyte Populations 18.24.1 General View 18.24.2 Experiment: Identification of FC Receptor-Bearing Cells 18.24.2.1 Introduction 18.24.2.2 Materials 18.24.2.3 Protocol 18.24.2.4 Calculations 18.24.2.5 Result 18.24.3 Experiment: Identification of B Lymphocytes by Detection of Cell Surface Ig 18.24.3.1 Introduction 18.24.3.2 Precautions 18.24.3.3 Materials 18.24.3.4 Protocol 18.24.3.5 Result

987 987 988 988 989 990 991 991 991 991 992 992 994 994

liv

Contents 18.25 Experiment: Isolation of Monocytes by Adherence 18.25.1 Introduction 18.25.2 Materials 18.25.3 Protocol 18.25.4 Result

995 995 995 995 996


18.26 Experiment: Isolation of Neutrophils 996 18.26.1 Introduction 996 18.26.2 Materials 996 18.26.3 Protocol 997 18.26.3.1 Enrichment for Neutrophils by Dextran Sedimentation 997 18.26.3.2 Remove Mononuclear Cells by Ficoll-Hypaque Centrifugation 997 18.26.3.3 Remove RBC and Count Neutrophils 997 18.27 Experiment: Isolation of Human Basophils 18.27.1 Introduction 18.27.2 Materials 18.27.3 Protocol 18.27.4 Result

998 998 998 998 999

18.28 Experiment: Isolation of Tissue Mast Cells 18.28.1 Introduction 18.28.2 Materials 18.28.3 Protocol 18.28.3.1 Prepare Tissue Fragments 18.28.3.2 Recover Mast Cells 18.28.3.3 Acid Toluidine Blue Staining of Mast Cells

999 999 999 999 999 1000 1000

18.29 Experiment: Measurement of Percent β-Glucuronidase Secretion 18.29.1 Introduction 18.29.2 Materials 18.29.2.1 Reagents and Solutions 18.29.3 Protocol 18.29.4 Calculations

1001 1001 1001 1001 1002 1002

18.30 Experiment: Generation of Polyclonal Antibodies in Rabbits 18.30.1 Introduction 18.30.2 Protocol 18.30.2.1 Adjuvants 18.30.2.2 Protein Ags 18.30.2.3 Expressed or Purified Proteins 18.30.2.4 Protein from Bands in a Gel Slice 18.30.2.5 Housing of Rabbits 18.30.2.6 Rabbit Injection Protocol 18.30.2.7 Immunization/Boost 18.30.2.8 Handling of Blood from Rabbits 18.30.2.9 Determination of Ab Titer in Rabbit Antiserum 18.30.2.10 Storage of Antiserum

1002 1002 1003 1003 1004 1004 1004 1004 1004 1005 1005 1005 1005

18.31 Experiment: Gel Diffusion/Ouchterlony Immunodiffusion 18.31.1 Introduction 18.31.2 Materials 18.31.2.1 Staining and Destaining Solution 18.31.2.2 Preparation of Solutions

1005 1005 1008 1008 1008

Contents


18.31.3 Protocol 18.31.3.1 Preparation of Slides with Sample Wells 18.31.3.2 Loading the Samples 18.31.3.3 (Optional) Staining Slides 18.31.4 Results and Conclusions

lv 1008 1008 1009 1010 1010

18.32 Experiment: Demonstration of Radial Immunodiffusion 18.32.1 Introduction 18.32.2 Materials 18.32.3 Protocol 18.32.4 Result

1012 1012 1012 1012 1013

18.33 Experiment: Counter Current Immunoelectrophoresis 18.33.1 Principle 18.33.2 Materials 18.33.3 Protocol 18.33.4 Result and Conclusion

1013 1013 1013 1014 1014

18.34 Experiment: Serum Electrophoresis 18.34.1 Principle 18.34.2 Materials 18.34.3 Protocol 18.34.4 Results and Conclusion

1014 1014 1015 1016 1016

18.35 Experiment: Demonstration of Immunoelectrophoresis 18.35.1 Introduction 18.35.2 Materials 18.35.3 Protocol 18.35.4 Result

1016 1016 1017 1017 1017

18.36 Experiment: Determination of Albumin by Laurell Rocket Immunoelectrophoresis 1018 18.36.1 Principle 1018 18.36.2 Materials 1018 18.36.3 Protocol 1019 18.36.4 Results and Conclusion 1019 18.37 Experiment: Purification of Igs by Ion Exchange Chromatography 18.37.1 Principle 18.37.2 Materials 18.37.3 Protocol 18.37.4 Result

1019 1019 1020 1020 1021

18.38 Experiment: The Detection and Assay of a Myeloma Protein 18.38.1 Principle 18.38.2 Materials 18.38.3 Protocol 18.38.4 Result and Conclusion

1021 1021 1021 1021 1022

18.39 Experiment: ELISA 18.39.1 Introduction 18.39.1.1 Direct ELISA 18.39.1.2 Indirect ELISA 18.39.1.3 Sandwich ELISA 18.39.1.4 Competitive ELISA 18.39.1.5 Inhibition ELISA

1022 1022 1023 1023 1023 1024 1024

lvi

Contents


18.39.2 Precautions 18.39.3 Materials 18.39.4 Protocol 18.39.4.1 Ag Application to Plate 18.39.4.2 Ab Dilution in Tubes 18.39.4.3 Ab Addition to Plate 18.39.4.4 Substrate Addition to Plate 18.39.5 Result

1026 1027 1028 1028 1029 1029 1030 1030

18.40 Experiment: Pregnancy Testing by Using the Immunological Methods 18.40.1 Introduction 18.40.1.1 Latex Agglutination Inhibition Test 18.40.1.2 Hemagglutination Inhibition Test 18.40.1.3 Pregnancy Testing through Commercially Available Kits

1031 1031 1031 1032 1032

18.41 Experiment: HIV DOT Test of AIDS Patients 18.41.1 Introduction 18.41.2 Materials 18.41.3 Protocol 18.41.4 Results

1035 1035 1035 1035 1036

18.42 Experiment: Demonstration of Immunofluorescence Assay 18.42.1 Introduction 18.42.2 Materials 18.42.3 Protocol 18.42.3.1 Anti-Human IgG Ab–FITC Conjugate 18.42.3.2 Immunofluorescence Assay 18.42.4 Results and Observations

1036 1036 1036 1037 1037 1037 1037

18.43 Experiment: Identification of Bacteria by Using Fluorescent Ab Technique (FAT) 18.43.1 Introduction 18.43.2 Materials 18.43.3 Protocol 18.43.4 Result

1037 1037 1038 1038 1038

18.44 Experiment: Immunofluorescence Labeling of Cultured Cells 18.44.1 Introduction 18.44.2 Materials 18.44.2.1 Reagents and Solutions 18.44.3 Protocol 18.44.4 Results and Observations

1038 1038 1039 1040 1040 1042

18.45 Experiment: Isolation of IgG from Serum 1042 18.45.1 Introduction 1042 18.45.1.1 Isolation of Ig-Rich Fraction from Serum by Salt Precipitation 1042 18.45.1.2 Removal of Salt from Protein Fraction 1043 18.45.1.3 Ultra-Membrane Filtration 1044 18.45.1.4 Isolation of IgG from Ig-Rich Fraction 1045 18.46 Experiment: Purification of IgM 18.46.1 Introduction 18.46.2 Materials 18.46.3 Protocol

1045 1045 1046 1046

Contents


19.

lvii

Suggested Reading

1046

Important Links

1047

Genetic Engineering

1049

19.1 Introduction

1049

19.2 Tools of Genetic Engineering 19.2.1 Enzymes 19.2.1.1 Restriction Endonucleases 19.2.1.2 DNA Ligase 19.2.1.3 Exonucleases 19.2.1.4 DNA Polymerases 19.2.1.5 Alkaline Phosphatase 19.2.1.6 Polynucleotide Kinase 19.2.1.7 Terminal Deoxynucleotidyl Transferase 19.2.1.8 Reverse Transcriptase 19.2.2 Cloning Vectors 19.2.2.1 Plasmid Cloning Vectors 19.2.2.2 Bacteriophages 19.2.2.3 M13 Phage Vectors 19.2.3 Transformation 19.2.3.1 Selection of Recombinants 19.2.4 Electrophoretic Techniques 19.2.4.1 Decontamination of EtBr Containing Solutions

1051 1051 1051 1051 1052 1053 1053 1053 1053 1053 1053 1054 1058 1061 1065 1066 1068 1070

19.3 DNA Sequencing 19.3.1 Chemical Cleavage and Modification Method 19.3.1.1 G Cleavage 19.3.1.2 A + G Cleavage 19.3.1.3 C + T Cleavage 19.3.1.4 C Cleavage 19.3.2 Dideoxy Chain Termination Method 19.3.2.1 PCR

1070 1070 1070 1071 1071 1071 1071 1072

19.4 To Isolate Genomic DNA from E. coli 1077 19.4.1 Principle 1077 19.4.2 Precautions 1077 19.4.3 Materials 1077 19.4.4 Protocol 1078 19.4.5 Result 1078 19.5 Rapid Method for Isolating Plasmid DNA from Yeast 19.5.1 Principle 19.5.2 Precautions 19.5.3 Materials 19.5.4 Protocol 19.5.5 Results and Observations

1078 1078 1079 1079 1080 1080

19.6 Isolation of Plasmid DNA from Yeast (Protocol 1) 19.6.1 Introduction 19.6.2 Precautions 19.6.3 Materials 19.6.4 Protocol 19.6.5 Results and Observations

1080 1080 1080 1081 1081 1081


lviii

Contents 19.7 Isolation of Yeast Plasmid DNA (Protocol 2) 19.7.1 Introduction 19.7.2 Precautions 19.7.3 Material 19.7.4 Protocol 19.7.5 Results and Observations

1081 1081 1081 1082 1082 1082

19.8 Isolation of Drosophila Genomic DNA 19.8.1 Introduction 19.8.2 Precautions 19.8.3 Materials 19.8.4 Protocol 19.8.4.1 Extraction 19.8.4.2 Crude Purification and Precipitation/Resuspension 19.8.5 Results and Observation

1083 1083 1083 1083 1083 1083 1083 1084

19.9 To Determine λmax Value of the Isolated DNA Sample 19.9.1 Principle 19.9.2 Materials 19.9.3 Protocol 19.9.4 Results

1084 1084 1084 1085 1085

19.10 To Determine the Purity of the Isolated DNA Sample 19.10.1 Principle 19.10.2 Materials 19.10.3 Protocol 19.10.4 Result

1085 1085 1085 1086 1086

19.11 Determine the Quantity of DNA in the Sample 19.11.1 Principle 19.11.2 Materials 19.11.3 Protocol 19.11.4 Result

1086 1086 1086 1086 1086

19.12 To Perform Agarose Gel Electrophoresis for the Given DNA Sample 19.12.1 Principle 19.12.1.1 Electrophoresis 19.12.1.2 Agarose Gel 19.12.1.3 Electrophoresis Buffer 19.12.1.4 Gel Size 19.12.1.5 Sample Concentration 19.12.1.6 Sample Load Solution 19.12.1.7 Gel Staining 19.12.2 Materials 19.12.3 Protocol 19.12.4 Results

1086 1086 1086 1087 1087 1087 1087 1087 1088 1088 1088 1088

19.13 To Isolate Plasmid DNA from Given Bacterial Culture 1089 19.13.1 Principle 1089 19.13.1.1 Alkaline Lysis 1089 19.13.1.2 Rapid Boiling Method: For Small Plasmids in E. coli 1091 19.13.1.3 Plasmid Isolation Using Concert High Purity Plasmid Purification Systems 1091 19.14 Electrophoresis of DNA: Linear, Circular, and Super Coiled 19.14.1 Principle

1092 1092

Contents


19.14.2 Materials 19.14.2.1 Reagents 19.14.3 Protocol 19.14.4 Result

lix 1093 1093 1093 1094

19.15 To Perform Restriction Digestion of the Given DNA Sample 19.15.1 Principle 19.15.2 Materials 19.15.3 Protocol 19.15.3.1 Preparation of 1% Agarose Gel and Electrophoresis 19.15.4 Result

1094 1094 1094 1095 1095 1095

19.16 To Demonstrate Ligation 19.16.1 Principle 19.16.2 Materials 19.16.3 Protocol 19.16.4 Result

1095 1095 1096 1096 1096

19.17 Elution of DNA from Agarose Gel 19.17.1 Principle 19.17.2 Materials 19.17.3 Protocol

1096 1096 1097 1097

19.18 Purification of Phage λ DNA 19.18.1 Principle 19.18.2 Materials 19.18.3 Protocol 19.18.3.1 Preparation of E. coli Cells 19.18.3.2 Isolation/Preparation of λ Phage Stock 19.18.3.3 Phage Cultivation 19.18.3.4 Preparation of Phage Titer from Single Plaque 19.18.3.5 Preparation of λ DNA 19.18.3.6 DNA Precipitation 19.18.3.7 DNA Purification

1098 1098 1098 1099 1099 1099 1099 1100 1100 1101 1101

19.19 To Perform Restriction Digestion of Plasmid DNA with Different Restriction Endonucleases and to Determine the Position of Restriction Site Using Restriction Mapping 19.19.1 Principle 19.19.2 Materials 19.19.3 Protocol 19.19.3.1 RE Digest 19.19.3.2 Restriction Mapping 19.19.3.3 Result

1102 1102 1102 1103 1103 1103 1104

19.20 To Perform Transformation 19.20.1 Principle 19.20.2 Materials 19.20.2.1 Preparation of Competent Cells 19.20.2.2 Preparation of Plates 19.20.3 Protocol 19.20.3.1 Transformation 19.20.4 Result

1104 1104 1104 1104 1105 1105 1105 1105

19.21 Directional Cloning into Plasmid Vectors 19.21.1 Principle

1105 1105

lx

Contents



1106 1106

19.22 To Assay Reporter Chloramphenicol Acetyltransferase Gene 19.22.1 Principle 19.22.2 Materials 19.22.3 Protocol 19.22.3.1 Preparation of Transfected Cell Pellets 19.22.3.2 Preparation of Cell Extracts 19.22.3.3 Detection of CAT Activity Using TLC

1107 1107 1108 1108 1108 1109 1109

19.23 To Assay β-Gal in Extracts of Mammalian Cells 19.23.1 Principle 19.23.2 Materials 19.23.3 Protocol 19.23.4 Result

1111 1111 1113 1113 1114

19.24 Determination of Nucleotide Sequence of DNA by Dideoxy Chain Termination Method 19.24.1 Principle 19.24.2 Materials 19.24.2.1 Reagents 19.24.2.2 DNA Sequencing Apparatus 19.24.3 Protocol 19.24.3.1 Sequencing Reaction 19.24.3.2 Electrophoresis 19.24.3.3 Autoradiography

1114 1114 1116 1116 1116 1116 1116 1117 1117

19.25 Amplification of 16S rDNA Genes by the Polymerase Chain 19.25.1 Principle 19.25.2 Materials 19.25.3 Protocol 19.25.3.1 Preparation of Template DNA 19.25.3.2 Preparation of the PCR Amplifications 19.25.4 Result

1118 1118 1119 1119 1119 1120 1121

19.26 PCR from Fungal Spores/AMF (Arbuscular Mycorrhizal Fungi) 19.26.1 Introduction 19.26.2 Materials 19.26.3 Protocol 19.26.3.1 Preparation of Template DNA 19.26.3.2 Preparation of the PCR Amplifications 19.26.4 Result

1121 1121 1121 1121 1121 1122 1123

19.27 To Demonstrate the Nested PCR Reaction 19.27.1 Introduction 19.27.2 Materials 19.27.2.1 Solutions and Reagents 19.27.3 Protocol 19.27.3.1 Extraction of DNA from Fungal Spores 19.27.3.2 Extraction of DNA from Plant 19.27.3.3 DNA Extraction from AMF Spores 19.27.4 Result

1123 1123 1123 1123 1123 1123 1124 1124 1125

Suggested Readings

1125

Important Links

1126

Contents


20.

lxi

Molecular Biology

1127

20.1 Introduction

1127

20.2 Isolation of Nucleic Acids from Biological Samples 20.2.1 Homogenization or Disruption of Cells 20.2.2 Dissociation of Nucleoprotein Complexes 20.2.3 Removal of Contaminating Materials and Precipitation of Nucleic Acids

1128 1128 1129 1129

20.3 Demonstration of Southern Blotting 1129 20.3.1 Introduction 1129 20.3.2 Materials 1130 20.3.3 Protocol 1130 20.3.3.1 Digestion of DNA 1130 20.3.3.2 Agarose Gel Construction and Preparation 1130 20.3.3.3 Loading, Running, and Staining of the Gel 1131 20.3.3.4 Preparation of the Gel for the Pictures and Transfer 1131 20.3.3.5 Membrane Preparation 1131 20.3.3.6 Neutralization Solution 1131 20.3.3.7 Clean Up the Probe 1131 20.3.3.8 Preparation of the Prehybridization/Hybridization of the Membrane 1132 20.3.3.9 Preparation of the Labeled Probe 1132 20.3.3.10 Washing of Blot 1133 20.3.3.11 Wrapping and Preparing of the Blot for Developing Film 1133 20.3.3.12 Develop Film 1133 20.3.4 Result and Observation 1133 20.4 Demonstration of Experiment on RFLP Analysis 20.4.1 Introduction 20.4.2 Types of Markers 20.4.2.1 Morphological Markers 20.4.2.2 Biochemical Markers 20.4.2.3 Molecular Markers

1134 1134 1134 1134 1134 1134

20.5 Restriction Fragment Length Polymorphism 20.5.1 Experimental Outline 20.5.2 Materials 20.5.3 Protocol 20.5.3.1 DNA Fragment Separation on Agarose Gel 20.5.3.2 Hybridization 20.5.3.3 Southern Blotting (Described Earlier) 20.5.3.4 Probe Labeling 20.5.3.5 Autoradiography 20.5.4 Result

1135 1135 1135 1135 1135 1136 1136 1137 1137 1138

20.6 Isolation of RNA from Biological Sample 20.6.1 Introduction 20.6.2 Materials 20.6.3 Protocol 20.6.3.1 Optional 20.6.4 Results and Observations

1138 1138 1138 1139 1139 1139

20.7 Extraction of Polysomal RNA of Nuclear Sequences 20.7.1 Introduction

1141 1141

lxii

Contents 20.7.2 Materials 20.7.3 Protocol 20.7.3.1 Polysome Extraction 20.7.3.2 RNA Purification 20.7.3.3 RNA Wash and Final Concentration Determination 20.7.3.4 Electrophoresis on Polyacrylamide Gel 20.7.3.5 Electrophoresis on Agarose Gel 20.7.4 Result and Observations


20.8 Separation of RNA Species by Ion-Exchange Column Chromatography on Methylated Albumin Kiesselguher Columns 20.8.1 Introduction 20.8.2 Materials 20.8.2.1 Column Preparation 20.8.3 Protocol 20.8.4 Result and Observations

1141 1141 1141 1143 1144 1144 1144 1145 1145 1145 1146 1146 1146 1146

20.9 Northern Hybridization of RNA Fractionated by Agarose Formaldehyde Gel Electrophoresis 1147 20.9.1 Introduction 1147 20.9.2 Material 1147 20.9.3 Precautions and Preparation 1148 20.9.4 Protocol 1148 20.9.5 Results and Observation 1151 20.10 Preparation of Radiolabeled Probe by Random Primer Method 20.10.1 Introduction 20.10.2 Precautions 20.10.3 Preparation 20.10.4 Materials 20.10.5 Protocol 20.10.6 Result and Observation

1151 1151 1151 1151 1152 1152 1152

20.11 Detection of Specific Fragment of Plant DNA from Restriction Digest by Hybridization with a Labeled Probe 20.11.1 Introduction 20.11.2 Materials 20.11.3 Precautions 20.11.4 Preparation 20.11.5 Protocol 20.11.6 Result and Observation

1152 1152 1153 1153 1153 1153 1154

20.12 In Vitro Transcription 20.12.1 Introduction 20.12.2 Safety Guidelines 20.12.3 Troubleshooting: No RNA Is Synthesized 20.12.4 Experimental Outline 20.12.5 Materials 20.12.6 Protocol

1154 1154 1156 1156 1157 1158 1158

20.13 In Vitro Translation of RNA 20.13.1 Introduction

1160 1160

20.14 Reticulocytes and Their Use in the Study of Protein Synthesis 20.14.1 Introduction 20.14.2 Materials

1162 1162 1162

Contents


20.14.3 Protocol 20.14.4 Results

lxiii 1162 1163

20.15 Protein Synthesis in Intact Rabbit Reticulocytes 20.15.1 Experimental Outline 20.15.2 Precautions 20.15.3 Materials and Prelab Preparations 20.15.4 Protocol 20.15.5 Results and Observations

1163 1163 1163 1163 1164 1164

20.16 Metabolic Labeling of Proteins and Immunoprecipitation 20.16.1 Introduction 20.16.2 Safety Guidelines 20.16.3 Precautions 20.16.4 Materials 20.16.5 Protocol 20.16.6 Western Blots

1164 1164 1165 1165 1165 1165 1166

20.17 Induction of β-Galactosidase in Strains of E. coli 1166 20.17.1 Introduction 1166 20.17.2 Experimental Design 1168 20.17.3 Materials 1169 20.17.4 Protocol 1169 20.17.5 Results and Observations 1170 20.18 Effect of Different Inducers on the Induction of β-Galactosidase 1170 20.18.1 Introduction 1170 20.18.2 Materials and Protocol 1171 20.19 Effect of Protein Synthesis Inhibitors on the Induction of β-Galactosidase 1171 20.19.1 Introduction 1171 20.19.2 Materials and Protocol 1171 20.20 Turnover of β-Galactosidase 1171 20.20.1 Protocol 1171

21.

Suggested Readings

1171

Important Links

1172

Biosensors

1173

21.1 Introduction

1173

21.2 Biosensor 21.2.1 Membrane Entrapment 21.2.2 Physical Adsorption 21.2.3 Covalent Bonding 21.2.4 Cross-Linking

1174 1174 1174 1175 1175

21.3 Classification of Biosensors

1176

21.4 Characteristics of Biosensors

1177

21.5 Components of Biosensors 21.5.1 Bioactive Components 21.5.1.1 Enzymes 21.5.1.2 Antibodies 21.5.1.3 Cells

1178 1178 1178 1178 1179

lxiv

Contents


21.5.1.4 Nucleic Acids 21.5.1.5 Lipids 21.5.2 Sensing Device 21.5.2.1 Optical Biosensor 21.5.2.2 Piezoelectric Biosensor 21.5.2.3 Resonant Biosensor 21.5.2.4 Thermal Detection Biosensor 21.5.2.5 Ion-Sensitive Biosensor 21.5.3 Electrochemical Biosensor 21.5.3.1 Conductometric Biosensor 21.5.3.2 Amperometric Biosensor (Measurement of the Current Resulting from a Redox Reaction) 21.5.3.3 Potentiometric Biosensor

22.

1179 1179 1179 1179 1180 1180 1180 1180 1181 1182 1182 1183

21.6 Biochip 21.6.1 DNA Chip 21.6.2 Protein Chip

1184 1186 1187

21.7 Immunobiosensor

1187

21.8 Aptamers

1188

21.9 Surface Plasmon Resonance (SPR) Technology

1190

21.10 Working Principle of Biosensors

1190

21.11 Immunosensors

1191

21.12 Calorimetric Biosensors

1194

21.13 Potentiometric Biosensors

1197

21.14 Amperometric Biosensors 21.14.1 Generation of Amperometric Biosensor

1199 1202

21.15 Optical Biosensors

1203

21.16 Piezoelectric Biosensors

1204

21.17 Second-Generation Biosensors

1204

21.18 Third-Generation Biosensors

1205

21.19 Biosensor for Healthcare 21.19.1 Glucose Biosensor 21.19.2 Lactate Biosensors 21.19.3 Urea and Creatinine Biosensors 21.19.4 Cholesterol Biosensor 21.19.5 Uric Acid Biosensor

1207 1207 1208 1209 1209 1210

21.20 Conclusion and Future Challenges

1210

Suggested Readings

1210

Important Links

1211

Enzyme Immobilization

1213

22.1 Introduction

1213

22.2 Selection of Carrier and Support before Immobilization

1214


Contents

lxv

22.2.1 Physical Properties 22.2.2 Chemical Properties 22.2.3 Stability 22.2.4 Resistance 22.2.5 Safety 22.2.6 Economic 22.2.7 Kinetic Properties

1214 1214 1214 1215 1215 1215 1215

22.3 Carriers for Enzyme Immobilization 22.3.1 Alginates 22.3.2 Chitin and Chitosan 22.3.3 Carrageenans 22.3.4 Cellulose

1216 1216 1216 1217 1217

22.4 Limitations and Advantages of Enzyme Immobilization 22.4.1 Limitations 22.4.2 Advantages

1217 1217 1218

22.5 Methods of Immobilizations 22.5.1 Carrier-Binding Method 22.5.2 Adsorption of Enzymes 22.5.3 Covalent Binding/Coupling 22.5.4 Modification of Some Other Chemical Groups

1218 1219 1219 1222 1224

22.6 Cross-Linking 22.6.1 Glutaraldehyde 22.6.2 Cyanogen Bromide 22.6.3 Carbodiimides 22.6.4 Cross-Linked Enzyme Crystals 22.6.5 Cross-Linked Enzyme Aggregates 22.6.6 Molecular Imprinting Technique

1225 1226 1228 1229 1231 1232 1233

22.7 Entrapment Method 22.7.1 Membrane Confinement 22.7.2 Occlusion 22.7.3 Entrapment and Encapsulation

1233 1235 1235 1236

22.8 Whole-Cell Immobilization

1237

22.9 Industrial Application of Enzymes

1237

22.10 Cell Immobilization by Using Alginate 22.10.1 Principle 22.10.2 Materials 22.10.3 Protocol 25.10.4 Result

1241 1241 1242 1242 1242

22.11 Immobilization of Enzyme Horseradish Peroxidase (Donor: H2O2 Oxidoreductase; EC 1.11.1.7) by Aminoantipyrine–Phenol Assay 22.11.1 Principle 22.11.2 Materials 22.11.3 Protocol 22.11.4 Results and Observations

1242 1242 1243 1243 1245

Suggested Readings

1246

Important Links

1246

lxvi

Contents


23. Plant Tissue Culture

1247

23.1 Introduction

1247

23.2 Facilities 23.2.1 Sterile Transfer Facilities 23.2.2 Laboratory Layout 23.2.3 Instruments 23.2.4 Tools 23.2.5 Glassware for Media 23.2.6 Culture Vessels

1247 1248 1248 1249 1249 1250 1250

23.3 Glassware Preparation 23.3.1 Sterilization 23.3.2 Wet Heat Sterilization 23.3.3 Dry Sterilization 23.3.4 Filter Sterilization

1250 1251 1251 1251 1251

23.4 Media Preparation 23.4.1 Preparation of Stock Solution 23.4.2 Protocol

1252 1253 1253

23.5 Sterilization of Media

1257

23.6 Aseptic Manipulation

1258

23.7 Sterile Transfer Facilities

1258

23.8 Temperature

1258

23.9 Light

1259

23.10 Aeration

1259

23.11 Temperature

1261

23.12 Plant Cell Culture System

1262

23.13 Explants

1262

23.14 Calluses

1263

23.15 Cell Suspension

1265

23.16 Protoplast

1266

23.17 Surface Sterilization

1267

23.18 Common Culture Media

1267

23.19 Sterilization of Plant Materials 23.19.1 Introduction 23.19.2 Materials 23.19.3 Protocol 23.19.4 Observation and Result

1267 1267 1268 1268 1269

23.20 Preparation of Tissue Culture Media 23.20.1 Introduction 23.20.2 Materials 23.20.3 Protocol 23.20.4 Observation and Result

1269 1269 1270 1270 1272


Contents

lxvii

23.21 Organ Culture 23.21.1 Introduction 23.21.2 Material 23.21.3 Protocol 23.21.4 Observations and Results

1272 1272 1272 1272 1272

23.22 Callus Propagation 23.22.1 Introduction 23.22.2 Materials 23.22.3 Protocol 23.22.4 Observation and Result

1273 1273 1273 1274 1274

23.23 Plant Regeneration from Callus or Plant Tissue 23.23.1 Introduction 23.23.2 Materials 23.23.3 Protocol 23.23.4 Observations and Results

1275 1275 1275 1275 1276

23.24 Single Cell Culture 1276 23.24.1 Introduction 1276 23.24.1.1 Filter Paper Raft Nurse Tissue Technique 1276 23.24.1.2 Microchamber Technique 1278 23.24.1.3 Microdrop Method 1278 23.24.1.4 Bergmann’s Plating Technique 1278 23.24.1.5 Thin Layer Liquid Medium Culture 1278 23.24.2 Culture Requirements 1280 23.24.3 Cell Viability Test 1280 23.25 Preparation and Fusion of Protoplasts 23.25.1 Introduction 23.25.2 Isolation 23.25.2.1 Isolation of Single Cell from Intact Plant Organ 23.25.3 Protoplast Fusion and Somatic Hybridization 23.25.4 Mechanism of Protoplast Fusion

1280 1280 1281 1281 1283 1283

23.26 Protocols on Protoplast Fusion 23.26.1 Spontaneous Fusion 23.26.2 Induced Fusion 23.26.2.1 Mechanical Fusion 23.26.2.2 Chemofusion 23.26.2.3 Electrofusion

1285 1285 1285 1285 1285 1287

23.27 Fusion Products: Hybrids and Cybrids

1291

23.28 Methods of Somatic Hybridization

1291

23.29 Protoplast Fusion (Somatic Cell Hybridization) 23.29.1 Materials 23.29.2 Protocol 23.29.3 Results and Observations

1293 1293 1294 1294

23.30 Electrofusion Isolation of Oat and Corn Protoplast 23.30.1 Introduction 23.30.2 Materials 23.30.3 Protocol 23.30.4 Observations and Results

1295 1295 1295 1295 1295


lxviii

Contents 23.31 Culture of Protoplasts 23.31.1 Introduction 23.31.2 Materials 23.31.3 Protocol

1296 1296 1297 1297

23.32 Other Protocols 23.32.1 Droplet Culture 23.31.2 Coculture 23.32.3 Feeder Layer Techniques 23.32.4 Hanging Droplet Method 23.32.5 Bead Culture

1298 1298 1298 1298 1298 1300

23.33 Somatic Embryogenesis 23.33.1 Introduction 23.33.2 Material 23.33.3 Protocol 23.33.4 Results

1300 1300 1302 1302 1303

23.34 Production of Haploid Plants 23.34.1 Introduction 23.34.2 Anther Culture 23.34.2.1 Materials 23.34.2.2 Protocol 23.34.2.3 Results

1303 1303 1304 1304 1304 1304

23.35 Embryo Encapsulation: Production of Artificial Seeds 23.35.1 Introduction 23.35.2 Materials 23.35.3 Protocol 23.35.4 Results

1307 1307 1307 1308 1308

23.36 Cytological Examination of Regenerated Plants 23.36.1 Microtomy 23.36.2 Dehydration 23.36.3 Infiltration, Embedding, and Sectioning 23.36.4 Result

1308 1308 1309 1309 1310

23.37 Demonstration of Root Hair Culture of Different Explants Source of Cereal Using Agrobacterium rhizogenes 1310 23.37.1 Introduction 1310 23.37.2 Materials and Protocol 1311 23.37.2.1 Seed Sterilization and Germination 1311 23.37.2.2 Preparation of A. rhizogenes 1311 23.37.2.3 Callus Formation 1312 23.37.2.4 Establishment of Hairy Root Cultures 1312 23.37.3 Results and Observations 1312 23.38 Experiment Demonstration of Root Hair Culture of Different Explants Using Arbuscular Mycorrhizal Fungi 23.38.1 Introduction 23.38.2 Materials and Protocols 23.38.2.1 Seed Sterilization and Germination 23.38.2.2 Callus Formation 23.38.2.3 Root Regeneration 23.38.2.4 Culture Media

1313 1313 1313 1313 1315 1315 1315

Contents


23.38.2.5 Mycorrhizal Inoculum 23.38.2.6 Minimal Agarose Medium 23.38.3 Observation and Result

lxix 1315 1315 1316

23.39 Useful Secondary-Metabolite Production from Plant Tissue Culture 23.39.1 Introduction 23.39.2 Tropane Alkaloid Production from Plant Tissue Culture 23.39.3 Materials 23.39.4 Protocol 23.39.5 Thin Layer Chromatography 23.39.6 HPLC

1319 1319 1319 1320 1324 1324 1324

23.40 Genetic Transformation of Plants through Agrobacterium tumefaciens as Cloning Vector 23.40.1 Introduction 23.40.2 Materials 23.40.3 Protocol

1325 1325 1326 1326

23.41 Assay of β-Glucuronidase in Transformed Plant Tissue 23.41.1 Introduction 23.41.2 Taming Agrobacterium for Use in Biotechnology 23.41.3 Materials and Protocols 23.41.4 Observation

1326 1326 1327 1327 1328

23.42 Random Amplified Polymorphic DNA Analysis 23.42.1 Introduction 23.42.2 Materials 23.42.3 Protocol 23.42.4 RAPD Analysis

1328 1328 1328 1328 1329

23.43 Efficiency of Rhizobia for Nodulation in Leguminous Crops 23.43.1 Introduction 23.43.2 Materials 23.43.3 Protocol 23.43.4 Results and Observations

1330 1330 1332 1332 1334

23.44 Seed Inoculation with Rhizobia 23.44.1 Introduction 23.44.2 Materials 23.44.3 Protocol 23.44.4 Observations and Results

1335 1335 1335 1335 1335

Suggested Readings

1337

Important Links

1337

24. Animal Cell Science and Technology

1339

24.1 Introduction

1339

24.2 Chronology of Cell Culture Development 24.2.1 Primary Cell Culture 24.2.2 Continuous Cell Lines 24.2.2.1 Established Cell Lines 24.2.3 Monolayer Cultures 24.2.4 Suspension Cultures

1340 1340 1341 1342 1342 1342


lxx

Contents 24.3 Cell Culture Laboratory 24.3.1 Laminar Flow/Cell Culture Hoods 24.3.1.1 Class I Hoods 24.3.1.2 Class II Hoods 24.3.1.3 Class III Hoods 24.3.1.4 Safety Guidelines 24.3.2 CO2 Incubators 24.3.2.1 Safety Guidelines 24.3.3 Microscopes 24.3.4 Centrifuges 24.3.5 Filters 24.3.6 Other Common Equipment 24.3.6.1 Laboratory Accessories 24.3.7 Washing of Glassware 24.3.7.1 Washing of New Glassware 24.3.7.2 Washing of Used Glassware 24.3.7.3 Washing of Rubber Bungs 24.3.7.4 Sintered Glass Filters 24.3.7.5 Glass Pipettes 24.3.8 Disposable Syringes 24.3.9 Silicone Glassware

1342 1344 1344 1345 1345 1345 1346 1346 1347 1347 1347 1348 1348 1348 1348 1350 1350 1350 1350 1352 1352

24.4 Culture Medium 24.4.1 Cell Culture Media and Growth Requirements for Animal Cells 24.4.2 Preparation of an Animal Cell Culture Medium

1352 1352 1353

24.5 Cell Subculturing 24.5.1 Subculture of Adherent Cells 24.5.2 Harvesting of Cells Mechanically 24.5.3 Harvesting of Cells Using Proteolytic Enzymes 24.5.4 Subculture of Cells in Suspension

1354 1354 1354 1355 1356

24.6 Cell Quantification 24.6.1 Calculating Cell Number 24.6.2 Alternative Methods for Determination of Cell Number 24.6.3 Calculation of Cell Number

1356 1357 1358 1358

24.7 Seeding Cells onto Culture Plates

1359

24.8 Maintenance of Cells in Culture

1360

24.9 Growth Kinetics of Animal Cells in Culture

1360

24.10 Cryopreservation of Cells

1361

24.11 Resuscitation of Frozen Cells

1362

24.12 Determination of Cell Viability

1362

24.13 Safety Considerations in Cell Culture

1363

24.14 Aseptic Techniques and Good Cell Culture Practice 1364 24.14.1 Good Practice 1364 24.14.2 Identification and Eradication of Bacterial and Fungal Infections 1364 24.14.3 Identification of Mycoplasma Infections 1365 24.14.3.1 Eradication of Mycoplasma 1366 24.15 Preparation of Tissue Culture Medium 24.15.1 Introduction

1367 1367


Contents

lxxi

24.15.2 Functions of the Main Ingredients in Culture Media 24.15.2.1 Balanced Salt Solution 24.15.2.2 Amino Acids and Vitamins 24.15.2.3 Other Ingredients 24.15.2.4 Buffers 24.15.2.5 Antibiotics

1367 1367 1367 1367 1367 1368

24.16 Preparation of Single Cell Suspension of Spleen and Thymus 24.16.1 Introduction 24.16.2 Materials 24.16.3 Protocol

1368 1368 1369 1369

24.17 Cell Counting 24.17.1 Introduction 24.17.2 Hemocytometer 24.17.3 Materials 24.17.4 Protocol 24.17.4.1 Cell Sampling 24.17.4.2 Slide Preparation 24.17.5 Results

1369 1369 1370 1370 1370 1370 1371 1371

24.18 Cell Viability Assay 24.18.1 Introduction 24.18.2 Materials 24.18.3 Protocol 24.18.4 Result

1372 1372 1372 1373 1373

24.19 Estimation of Viability by Dye Uptake 24.19.1 Introduction 24.19.2 Materials 24.19.3 Protocol 24.19.4 Results

1373 1373 1373 1373 1374

24.20 Macrophage Monolayer from PEE and Measurement of Phagocytic Activity 24.20.1 Introduction 24.20.2 Materials 24.20.3 Protocol 24.20.4 Applications

1374 1374 1374 1374 1375

24.21 Trypsinization of Monolayer and Subculturing 24.21.1 Introduction 24.21.2 Materials 24.21.3 Protocol

1375 1375 1375 1375

24.22 Cryopreservation 24.22.1 Introduction 24.22.2 Importance 24.22.3 Preservation 24.22.3.1 Cell Line Selection 24.22.4 Materials 24.22.5 Protocol

1377 1377 1377 1377 1377 1378 1378

24.23 Thawing 24.23.1 Introduction 24.23.2 Precautions 24.23.3 Materials 24.23.4 Protocol

1379 1379 1379 1379 1379


lxxii

25.

Contents 24.24 Role of Serum in Cell Culture 24.24.1 Introduction 24.24.2 Precautions 24.24.3 Materials 24.24.4 Protocol

1380 1380 1380 1380 1380

24.25 Metaphase Chromosome Preparation from Cultured Cells 24.25.1 Introduction 24.25.2 Materials 24.25.3 Protocol 24.25.4 Results

1381 1381 1381 1382 1382

24.26 Isolation of DNA and Demonstration of Apoptosis of DNA Laddering 24.26.1 Introduction 24.26.2 Materials 24.26.3 Protocol

1382 1382 1383 1383

24.27 Demonstration of Apoptosis of DNA Laddering 24.27.1 Introduction 24.27.2 Materials 24.27.3 Protocol

1383 1383 1384 1384

24.28 MTT Assay for Cell Viability and Growth 24.28.1 Introduction 24.28.2 Materials 24.28.3 Protocol 24.28.4 Analysis

1384 1384 1385 1385 1386

24.29 Cell Fusion by PEG 24.29.1 Introduction 24.29.2 Materials 24.29.3 Methods 24.29.3.1 Monolayer Fusion 24.29.3.2 Suspension Fusion

1386 1386 1387 1387 1387 1387

Suggested Readings

1387

Important Links

1388

Bioprocess Engineering

1389

25.1 Introduction

1389

25.2 Isolation of Industrially Important Microorganisms 1389 25.2.1 Introduction 1389 25.2.2 Materials 1391 25.2.3 Protocol 1391 25.2.4 Isolation Methods Utilizing Selection of the Desired Characteristics as Selective Factors 1391 25.2.5 Enrichment Liquid Culture 1391 25.2.6 Disadvantages 1392 25.2.7 Solutions to the Problem of Early Washout 1393 25.2.7.1 Turbidostat 1393 25.2.7.2 Chemostat 1393 25.2.8 Enrichment Cultures Using Solidified Media 1394 25.2.9 Isolation Methods Not Utilizing Selection of the Desired Characteristic 1394

Contents


25.2.10 Screening Methods 25.2.11 Screening Mechanism

lxxiii 1395 1395

25.3 Determination of Thermal Death Point and Thermal Death Time of Microorganisms for Design of a Sterilizer 25.3.1 Effect of Incubation Temperatures on Growth 25.3.1.1 Precautions 25.3.1.2 Materials 25.3.1.3 Protocol 25.3.1.4 Observations and Results 25.3.2 Lethal Effects of Temperature on Microorganism: Thermal Death Point 25.3.2.1 Introduction 25.3.2.2 Precautions 25.3.2.3 Materials 25.3.2.4 Protocol 25.3.2.5 Observations and Results 25.3.3 Determination of Thermal Death Time of an Organism 25.3.3.1 Introduction 25.3.3.2 Materials 25.3.3.3 Protocol 25.3.3.4 Observations and Results

1397 1397 1397 1397 1398 1398 1399 1399 1400 1400 1400

25.4 Determination of Ethanol Production Using Different Substrates 25.4.1 Introduction 25.4.2 Materials 25.4.3 Protocol 25.4.4 Observations and Results

1401 1401 1401 1401 1402

25.5 Sugar Fermentation by Yeast (S. cerevisiae) for Production of Ethanol and Cell Biomass Using Laboratory Fermenter of 2 L Capacity 25.5.1 Introduction 25.5.2 Material 25.5.3 Protocol 25.5.4 Data Analysis Procedure

1403 1403 1403 1403 1406

25.6 Measurement of Yeast Biomass Using Hemocytometer Technique 25.6.1 Materials 25.6.2 Protocol 25.6.2.1 Calculations 25.6.2.2 Experimental Procedures

1406 1406 1406 1406 1406

25.7 Study Fermentation Process for Lactic Acid Production by Streptococcus thermophilus and Cell Biomass 25.7.1 Introduction 25.7.2 Material 25.7.3 Protocol 25.7.3.1 Fermentation Process

1407 1407 1408 1409 1409

1395 1396 1396 1396 1396 1396

25.8 Microbial Production of Citric Acid Using Aspergillus niger 1410 25.8.1 Introduction 1410 25.8.2 Material 1411 25.8.2.1 Organism and Culture Maintenance 1411 25.8.2.2 Preparation of Fermentation Medium for Citric Acid Production 1411

lxxiv

Contents


25.8.2.3 25.8.2.4 25.8.3 Method 25.8.3.1 25.8.3.2 25.8.4 Result

Pretreatment of Molasses Inoculum Preparation Fermentation Technique Product Recovery

1411 1411 1412 1412 1412 1413

25.9 Microbial Production of Antibiotics (Penicillin) 25.9.1 Introduction 25.9.1.1 Biotransformation of Antibiotics 25.9.2 Material 25.9.2.1 Strain Development 25.9.2.2 Storage of Strains 25.9.2.3 Inoculum Preparation 25.9.2.4 Fermentation Medium 25.9.3 Methods 25.9.3.1 Inoculation Methods 25.9.3.2 Fermentation Process 25.9.3.3 Downstream Processing 25.9.4 Production of Steroid Hormones through Biotransformation

1413 1413 1413 1414 1414 1416 1416 1416 1416 1416 1417 1417 1418

25.10 Production of Vinegar (Acetic Acid) 25.10.1 Introduction 25.10.2 Material 25.10.2.1 Substrate 25.10.2.2 Microorganisms 25.10.3 Process 25.10.4 Protocol 25.10.5 Result

1419 1419 1420 1420 1421 1421 1422 1424

25.11 Demonstration of Wine Production Using Grape Juice 25.11.1 Introduction 25.11.2 Materials 25.11.2.1 Inoculation of Must 25.11.3 Protocol 25.11.3.1 Fermentation 25.11.3.2 Aging 25.11.4 Results 25.11.4.1 Types of Wines

1424 1424 1424 1424 1425 1425 1426 1427 1427

25.12 Production and Manufacture of Beer 25.12.1 Introduction 25.12.2 Protocol 25.12.2.1 Malting 25.12.2.2 Mashing Process 25.12.2.3 Fermentation 25.12.2.4 Lagering or Aging 25.12.3 Kinds of Beers

1428 1428 1428 1428 1429 1429 1429 1429

25.13 Production and Estimation of Alkaline Protease 25.13.1 Introduction 25.13.2 Materials 25.13.2.1 Medium 25.13.2.2 Solutions 25.13.3 Protocol 25.13.4 Result

1430 1430 1430 1430 1430 1431 1431


Contents

lxxv

25.14 Mushroom Production Technology 25.14.1 Introduction 25.14.1.1 Morphology 25.14.1.2 Food Value of Edible Mushrooms 25.14.1.3 Cultivated Edible Mushrooms 25.14.1.4 Nutraceutical Properties of Mushrooms 25.14.1.5 Important Medicinal Mushrooms 25.14.1.6 Poisonous Mushrooms 25.14.1.7 Cultivation of Mushrooms

1431 1431 1432 1434 1435 1435 1435 1436 1436

25.15 Identification of Mushrooms by Spore Print Method 25.15.1 Introduction 25.15.2 Materials 25.15.3 Method 25.15.4 Results

1436 1436 1437 1437 1437

25.16 Isolation and Maintenance of Mushroom Culture 25.16.1 Materials 25.16.2 Method 25.16.3 Observation and Result

1438 1438 1438 1439

25.17 Production of Spawn for White Button Mushroom (A. brunnescens syn. A. bisporus) 1439 25.17.1 Introduction 1439 25.17.2 Materials 1440 25.17.3 Method 1441 25.17.4 Result 1443 25.17.5 Precautions 1443 25.18 Cultivation of White Button Mushroom (A. brunnescens = A. bisporus) 1444 25.18.1 Introduction 1444 25.18.2 Materials 1445 25.18.3 Protocol 1445 25.18.4 Precautions 1449 25.19 Cultivation of G. lucidum (Ling Zhi) 1449 25.19.1 Introduction 1449 25.19.2 Materials 1452 25.19.3 Protocol 1453 25.19.4 Observations and Results 1453 25.19.5 Submerged Cultivation of G. lucidum 1453 25.20 Cultivation of L. edodes 1456 25.20.1 Introduction 1456 25.20.2 Materials 1456 25.20.3 Protocol 1458 25.20.4 Observations and Results 1460 25.21 Cultivation of Paddy Straw Mushroom (V. volvacea) 1460 25.21.1 Introduction 1460 25.21.2 Materials 1462 25.21.3 Protocol 1462 25.21.4 Observations 1463 25.22 Cultivation Method of Oyster Mushroom 25.22.1 Materials

1463 1463

lxxvi

Contents 25.22.2 Protocol 25.22.3 Observations and Results


26.

1464 1464

Suggested Readings

1465

Important Links

1466

Environmental Biology

1467

26.1 Wastewater Treatment Process 26.1.1 Artificial Wetlands 26.1.2 Municipal Treatment Processes 26.1.3 Activated Sludge Process 26.1.4 Oxidation Ponds 26.1.5 Tertiary Treatment 26.1.6 Final Treatment 26.1.7 Solid Processing 26.1.8 Anaerobic Sludge Digestion 26.1.9 Composting 26.1.10 Landfills

1468 1470 1470 1472 1472 1473 1473 1474 1474 1474 1475

26.2 Measurement of pH 26.2.1 Principle 26.2.2 Materials 26.2.3 Protocol

1475 1475 1475 1476

26.3 Measurement of Alkalinity of Water 26.3.1 Principle 26.3.2 Materials 26.3.3 Protocol 26.3.4 Calculation 26.3.5 Result

1476 1476 1476 1476 1477 1477

26.4 Measurement of DO of Water 26.4.1 Principle 26.4.2 Materials 26.4.3 Protocol 26.4.4 Result

1477 1477 1478 1479 1479

26.5 Measurement of Chloride in Water 26.5.1 Principle 26.5.2 Materials 26.5.3 Protocol 26.5.4 Result

1480 1480 1480 1480 1481

26.6 Measurement of Nitrate in Water 26.6.1 Principle 26.6.2 Materials 26.6.3 Protocol 26.6.4 Result

1481 1481 1481 1481 1482

26.7 Measurement of Nitrite in Water 26.7.1 Principle 26.7.2 Materials 26.7.3 Protocol 26.7.4 Result

1482 1482 1482 1482 1483


Contents

lxxvii

26.8 Measurement of Ammonia in Water 26.8.1 Principle 26.8.2 Materials 26.8.3 Protocol 26.8.4 Result

1483 1483 1483 1483 1483

26.9 Measurement of Total Phosphorus in Water 26.9.1 Principle 26.9.2 Materials 26.9.3 Protocol 26.9.4 Result

1484 1484 1484 1484 1485

26.10 Measurement of Inorganic Phosphate in Water 26.10.1 Principle 26.10.2 Materials 26.10.3 Protocol 26.10.4 Result

1485 1485 1485 1485 1485

26.11 Measurement of Sulfate in Water 26.11.1 Principle 26.11.2 Materials 26.11.3 Protocol 26.11.4 Result

1486 1486 1486 1486 1486

26.12 Measurement of Sulfide in Water 1486 26.12.1 Principle 1486 26.12.2 Materials 1487 26.12.3 Protocol 1487 26.12.4 Result 1487 26.13 Measurement of Calcium and Magnesium in Water 1487 26.13.1 Principle 1487 26.13.2 Materials 1487 26.13.3 Protocol 1488 26.13.4 Result 1488 26.14 Detection of Coliforms for the Determination of the Purity of Potable Water 26.14.1 Principle 26.14.2 Experimental Outline 26.14.3 Materials 26.14.4 Protocol 26.14.5 Results

1488 1488 1489 1491 1491 1491

26.15 Determination of Total Dissolved Solids of Water 26.15.1 Principle 26.15.2 Materials 26.15.3 Protocol 26.15.4 Result

1492 1492 1492 1493 1493

26.16 Determination of DO Concentration of Water by Winkler’s Iodometric Method 1493 26.16.1 Principle 1493 26.16.2 Experimental Outline 1493 26.16.3 Materials 1494 26.16.4 Protocol 1494 26.16.5 Result 1495


lxxviii

Contents 26.17 Determination of Biochemical Oxygen Demand of Sewage Water 26.17.1 Principle 26.17.2 Experimental Outline 26.17.3 Materials 26.17.4 Protocol 26.17.5 Result

1495 1495 1495 1495 1496 1496

26.18 Determination of Chemical Oxygen Demand of Sewage Sample 26.18.1 Principle 26.18.2 Experimental Outline 26.18.3 Materials 26.18.4 Protocol 26.18.5 Result

1496 1496 1496 1497 1497 1497

26.19 Determination of the Efficiency of Removal of Air Pollutant, Using Fibrous Air Filter 26.19.1 Principle 26.19.2 Materials 26.19.3 Description 26.19.4 Protocol

1498 1499 1499 1499 1500

26.20 Isolation of Xenobiotic-Degrading Bacteria 26.20.1 Bioremediation of Xenobiotic Compounds 26.20.2 Principle 26.20.3 Materials 26.20.4 Protocol 26.20.4.1 Regulation of Toluene Concentration 26.20.5 Result and Observation

1501 1501 1503 1504 1504 1504 1505

26.21 Degradation of Aromatic Hydrocarbons by Bacteria 26.21.1 Principle 26.21.2 Experimental Outline 26.21.3 Materials 26.21.4 Protocol 26.21.4.1 Preculture Preparation 26.21.4.2 Gravimetric Analysis 26.21.4.3 Spectrofluorometric Analysis 26.21.5 Results

1505 1505 1505 1506 1506 1506 1506 1507 1507

26.22 Survey of Degradative Plasmids in Microbes Growing in Polluted Environment 1507 26.22.1 Introduction 1507 26.22.2 Catabolic Plasmids of Alkene Degradation 1508 26.23 Effect of SO2 on Crop Plants 26.23.1 Principle 26.23.2 Experimental Outline 26.23.3 Materials 26.23.4 Protocol 26.23.5 Results and Observations

1508 1508 1509 1510 1510 1510

26.24 Estimation of Heavy Metals in Water/Soil by Atomic Absorption Spectrophotometry 1510 26.24.1 Principle 1510 26.24.2 Sampling and Preservation 1511 26.24.2.1 Sample Containers 1511 26.24.2.2 Precaution 1511 26.24.2.3 Sampling Error 1512


Contents

lxxix

26.25 Preliminary Digestion for Metals 26.25.1 Open Digestion 26.25.1.1 Nitric Acid Digestion Apparatus 26.25.1.2 Nitric Acid–Hydrochloric Acid Digestion Apparatus 26.25.1.3 Nitric Acid–Sulfuric Acid Digestion Apparatus 26.25.1.4 Nitric Acid–Perchloric Acid Digestion 26.25.1.5 Nitric Acid–Perchloric Acid–Hydrofluoric Acid Digestion 26.25.1.6 Dry Ashing 26.25.2 Microwave Digestion System Scope and Application 26.25.2.1 Microwave-Assisted Digestion

1513 1514 1514 1514 1515 1515 1516 1516 1516 1517

26.26 Estimation of Metals 26.26.1 ICP Method 26.26.2 Analysis of Samples 26.26.3 Data Analysis and Calculations

1518 1518 1521 1522

26.27 Flame AAS 26.27.1 Scope and Application 26.27.2 Interferences 26.27.2.1 Chemical Interference 26.27.2.2 Background Correction 26.27.3 Apparatus and Equipment 26.27.4 Reagents and Standards 26.27.5 Protocol for Sample Preparation 26.27.5.1 Operating Conditions 26.27.5.2 Standardization 26.27.5.3 Data Analysis and Calculations

1522 1522 1522 1522 1522 1523 1524 1525 1525 1526 1526

26.28 Measurement of Aluminum in Soil/Water Sample 1526 26.28.1 Scope and Application 1526 26.28.2 Methods for Analysis 1526 26.28.2.1 ICP Method 1526 26.28.2.2 Eriochrome Cyanine R Method 1526 26.28.3 Protocol 1528 26.28.3.1 Preparation of Calibration Curve 1528 26.28.3.2 Sample Treatment in Absence of Fluorides and Complex Phosphate 1528 26.28.3.3 Removal of Phosphate Interference 1528 26.28.3.4 Correction for Samples Containing Fluoride 1529 26.28.4 Data Analysis and Calculation 1529 26.29 Measurement of Arsenic in Soil/Water Sample 26.29.1 Scope and Application 26.29.2 Methods for Analysis 26.29.2.1 Kit Method 26.29.2.2 Silver Diethyldithiocarbamate Method 26.29.2.3 ICP Method

1529 1529 1529 1529 1531 1534

26.30 Measurement of Cadmium in Soil/Water Sample 26.30.1 Scope and Application 26.30.2 Methods for Analysis 26.30.2.1 ICP Method 26.30.2.2 Dithizone Method

1534 1534 1534 1534 1534

26.31 Measurement of Mercury in Soil/Water Sample 26.31.1 Introduction 26.31.2 Scope and Application

1537 1537 1537


lxxx

27.

Contents 26.31.3 Methods for Analysis 26.31.3.1 Sampling 26.31.3.2 Preservation 26.31.3.3 Storage 26.31.3.4 Analytical Methods and Analytical Achievability 26.31.3.5 Reagents and Standards 26.31.3.6 Protocol 26.31.4 Data Analysis and Calculations 26.31.4.1 Calculations for the Bubbler System 26.31.4.2 Calculations for the Flow Injection System

1538 1538 1538 1538 1539 1540 1541 1543 1543 1543

26.32 Study on Biogenic Methane Production in Different Habitats (Biogas) 26.32.1 Principle 26.32.2 Experimental Outline 26.32.3 Materials 26.32.4 Protocol 26.32.5 Process 26.32.5.1 Methanogens 26.32.5.2 Mechanism of CH4 Formation 26.32.5.3 Production from Different Feedstocks

1544 1546 1546 1547 1547 1547 1549 1549 1549

Suggested Readings

1550

Important Links

1550

Bioinformatics

1551

27.1 Introduction

1551

27.2 Development 27.2.1 Organization of Biological Knowledge in Databases 27.2.2 Sequence Data Analysis 27.2.3 Structural Bioinformatics

1551 1552 1554 1555

27.3 Nucleotide Sequence Databases 27.3.1 Principle 27.3.2 Materials 27.3.3 Protocol

1556 1556 1556 1556

27.4 Protein Sequence Databases 27.4.1 Principle 27.4.2 Materials 27.4.3 Protocol

1556 1556 1556 1557

27.5 Biomolecular Structure Databases 27.5.1 Principle 27.5.2 Materials 27.5.3 Protocol

1557 1557 1557 1557

27.6 Pair-Wise Sequence Alignment 27.6.1 Principle 27.6.2 Materials 27.6.3 Protocol

1557 1557 1558 1558

27.7 Detection of CpG Island for Nucleotide Sequence 27.7.1 Principle 27.7.2 Materials 27.7.3 Protocol

1558 1558 1558 1558


Contents

lxxxi

27.8 Detection of Gene Structure of DNA Sequence 27.8.1 Principle 27.8.2 Materials 27.8.3 Protocol

1559 1559 1559 1559

27.9 Translation of Nucleotide Sequence to Protein Sequence 27.9.1 Principle 27.9.2 Materials 27.9.3 Protocol

1559 1559 1559 1560

27.10 Reports of Various Chemical and Physical Properties of Protein Sequence 27.10.1 Principle 27.10.2 Materials 27.10.3 Protocol

1560 1560 1560 1560

27.11 Database Search “Blast” 27.11.1 Principle 27.11.2 Materials 27.11.3 Protocol

1560 1560 1561 1561

27.12 Phylogenetic Analysis 27.12.1 Principle 27.12.2 Materials 27.12.3 Protocol

1561 1561 1561 1561

27.13 Molecular Pathways Database 27.13.1 Principle 27.13.2 Materials 27.13.3 Protocol

1562 1562 1562 1562

27.14 Proteomics 1562 27.14.1 Predicting Subcellular Location of a Protein 1562 27.14.1.1 Principle 1562 27.14.1.2 Materials 1563 27.14.1.3 Protocol 1563 27.14.1.4 Results and Observations 1563 27.14.2 Exploring Protein–Protein Interactions 1563 27.14.2.1 Principle 1563 27.14.2.2 Materials 1563 27.14.2.3 Protocol 1564 27.14.2.4 Results and Observations 1564 27.15 Metabolomics 27.15.1 Exploring Human Metabolites 27.15.1.1 Principle 27.15.1.2 Materials 27.15.1.3 Protocol 27.15.1.4 Results and Observations 27.15.2 Metabolite Profiling 27.15.2.1 Principle 27.15.2.2 Materials 27.15.2.3 Protocol 27.15.2.4 Result and Observation

1564 1564 1564 1564 1564 1565 1565 1565 1565 1565 1565

27.16 Genomics 27.16.1 Meta-Analysis of Gene Expression 27.16.1.1 Principle

1565 1565 1565

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Contents


27.16.1.2 Materials 27.16.1.3 Protocol 27.16.1.4 Results and Observations 27.16.2 Annotating Gene List Obtained through Expression Studies 27.16.2.1 Principle 27.16.2.2 Materials 27.16.2.3 Protocol 27.16.2.4 Results and Observations

1566 1566 1566 1566 1566 1566 1566 1567

27.17 Bioinformatics Approaches for DNA-Based Signatures of Species/ Populations/Breeds/Races/Variety/Strains 1567 27.17.1 Introduction 1567 27.17.2 DNA Bar Coding of Species 1568 27.17.3 Origin of Species Bar Code 1568 27.17.4 Examples of Species Bar Codes 1568 27.17.4.1 Identifying Birds by Species Bar Code 1568 27.17.4.2 Delimiting Cryptic Species by DNA Bar Code 1569 27.17.4.3 Identifying Flowering Plants by Species DNA Bar Code 1569 27.17.4.4 Genus DNA Bar Codes of Microbes 1569 27.17.5 DNA-Based Signature of Domestic Species 1572 27.17.6 DNA-Based Signature of Domestic Animal Breeds 1572 27.17.6.1 STR-Based Signatures of Breeds 1573 27.17.6.2 SNP Chip-Based DNA Signature of Breeds 1573 27.17.6.3 Development of Breed-Specific Signatures/Profiles 1573 27.17.6.4 Development of Breed Hybrid Indices/Profiles 1575 27.18 DNA-Based Signature of Plant Variety (Example, Basmati Rice)

1575

27.19 DNA-Based Bar-Coded Signature of Fishes

1576

27.20 Bioinformatics Tool and Protocol for SNP/STR Signatures 27.20.1 BioEdit 27.20.2 GeneClass2 27.20.3 Cleaver

1577 1579 1579 1580

27.21 FastPCR

1582

Suggested Readings

1584

Important Links

1585

Appendix A

1587

Appendix B

1615

Appendix C

1623

Appendix D

1625

Appendix E

1627

Appendix F

1629

Appendix G

1631

Glossary 1633 Index 1681


Preface

Laboratory Protocols in Applied Life Sciences presents the basic as well as latest techniques in wide areas of life sciences, namely, biotechnology, analytical biochemistry, clinical biochemistry, biophysics, molecular biology, genetic engineering, bioprocess technology, industrial processes, animal, plant, and microbial biology, computational biology, biosensors, etc., in an illustrative fashion. The book includes 27 chapters covered under 12 units with appendices, important links, and a glossary. This book analyzes information derived through real experiments and focuses on cutting-edge techniques in the field. Up-to-date protocols covering a range of frequently used methods are provided. The main focus in writing this book has been on developing highly doable experiments that will provide students and professionals with a successful learning experience. Basic as well as advanced methods available in wide-ranging areas of biology are covered and compiled in one book. Each experiment is presented with an introduction to the topic, concise objectives, a list of the necessary materials and reagents, and step-by-step, readily reproducible laboratory protocols. Each chapter is self-contained and written in a style that enables the student to progress from elementary concepts to advanced research techniques. Laboratory Protocols in Applied Life Sciences serves as a valuable tool for both beginner research workers and experienced professionals. I have taken utmost care to include information to help students to develop concept and to design and execute the experiment in a given field of biology. I hope that this book will be able to promote interest among students, teachers, and encourage beginners to the level of excitement. Constructive suggestions and comments are welcomed for the improvement of the book. Prakash S. Bisen Gwalior, India

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About the Book

Life sciences have been qualitative in nature, seeking to identify the mere presence of the molecules but seldom explain them quantitatively. Although the development of instrumentation techniques began as far back as the early part of the twentieth century, these technologies progressed at a slow pace, contributing only minimally to the field; it is only in the middle of the twentieth century that instrumentation began to make rapid strides, increasing its speed and accuracy. With the advent of molecular biology, studies in biological sciences have become increasingly dependent on new and emerging techniques, essentially based on well-known principles of physical and chemical sciences. For a number of years, it has been difficult for the students of biology to grasp the basic principles of these potential emerging techniques, particularly owing to their mathematical bias and an absence of a comprehensive book dealing with all the integrated techniques used for the qualitative as well as quantitative analysis of living systems. Laboratory Protocols in Applied Life Sciences identifies several important technologies now used that are critical to fulfilling the potential of life sciences based on the use of increasingly sophisticated instrumentation. The book attempts to present a nonmathematical account of the underlying principles of a variety of experimental techniques, including electron microscopy, spectroscopy, ultracentrifugation, radiation biology, biochemistry, bioinformatics, enzyme technology, separation technology, protein engineering, bioprocess engineering, environmental biology, etc., with the hope that it would be useful to all students of biology at graduate and postgraduate levels. This book is an endeavor toward that end. While standard laboratory books are available for such disciplines as genetics, biochemistry, microbiology, molecular biology, immunology, environmental biology, biochemical engineering, etc., students of interdisciplinary fields such as life sciences, pharmacy, medical and paramedical sciences, biotechnology, etc., find them too exhaustive and terse. Students of these fields require a single volume, integrating all branches with contents arranged and presented in a way comprehensible to them. The book deals with the principles, concepts, techniques, and applications used in life sciences in a very comprehensive and illustrative manner, which is particularly useful for all biology students.

lxxxv



Acknowledgments

I would like to express my gratitude to Dr. G.S. Davar and Puneet Davar, Tropilite Foods Pvt. Ltd, Gwalior, India, and to Rakesh Singh Rathore, Anil Singh, and Richa Verma, Vikrant Institute of Technology and Management, Gwalior, Madhya Pradesh, India, for their invaluable advice and moral support. My sincere thanks to Dr. Dinesh Kumar, senior scientist, Indian Statistical Agricultural Research Institute, Pusa, New Delhi, and Dr. Prem Prakash Dubey, Department of Animal Genetics and Breeding, Guru Angad Dev University of Veterinary and Animal Sciences, Ludhiana, for contributing a full chapter on bioinformatics and to Dr. Divya Shrivastava and Shweta Mittal, School of Life Sciences, Jaipur National University, Jaipur, for helping me in preparing the chapter on genetic engineering. I am grateful to Professor G.B.K.S. Prasad, School of Studies in Biochemistry; Professor Ishan K. Patro and Dr. Nisha Patro, School of Studies in Neurosciences, Jiwaji University, Gwalior; Dr. Ram P. Tiwari and Dr. Anubhav Jain, Diagnostic Division, RFCL Limited (formerly Ranbaxy Fine Chemicals Limited), Avantor Performance Materials, New Delhi, India; Dr. Ruchika Raghuvanshi, Bhagwan S. Sanodiya, Gulab S. Thakur, Rakesh K. Baghel, and Rohit Sharma, Tropilite Foods Pvt. Ltd., Gwalior; and Sumit Govil and Shailesh Kumar of Jaipur National University, Jaipur, India, for their valuable assistance in the preparation of this book. My sincere thanks to Devendra Singh, Avinash Dubey, and Rahul Jha, Gwalior, India for all the computational work involved in making this book presentable. The cover page of the book was designed by courtesy of Mr Rahul Jha. Thanks are also due to Michael Slaughter, Kari Budyk, and Michele Smith of CRC Press/Taylor & Francis Group, for their support in publishing this book in time and for their patience and enthusiasm. Finally, I would like to thank my wife Shashi Bisen for her constant support, cooperation, and understanding. I am grateful to the Council of Scientific and Industrial Research, New Delhi, for bestowing me with the title of Emeritus Scientist. Prakash S. Bisen Gwalior, India

lxxxvii



Author

Prakash S. Bisen received his PhD in 1972 and his DSc in 1981 from Jabalpur University, Jabalpur, India. He has guided 60 doctoral thesis students and has to his credit 200 publications in indexed international journals of repute as well as 7 international books on contemporary issues in biological sciences. He was a fellow of the UNESCO/UNDP/ICRO and Hungarian Academy of Sciences at the Institute of Plant Physiology, Biological Research Center, Szeged, Hungary; a U.S. National Science Foundation fellow in the Department of Biological Sciences at the University of Illinois at Chicago and in the Department of Bacteriology at the University of California, Davis; UNESCO/WHO fellow at the Institute of Microbiology, Czechoslovak Academy of Sciences, Budejovika, Praha, Czechoslovakia; Marie Curie fellow at the Institute of Environmental and Biological Sciences, University of Lancaster, United Kingdom; and DAAD (German Academic Exchange Fellowship) fellow at the University of Bonn, Germany. He was awarded the Life Time Professorship, the highest honor of Bundhelkhand University, Jhansi, India. Professor Bisen is currently pursuing his research in the field of medical biotechnology as emeritus scientist at Defence Research Development Establishment, Defence Research Development Organization, Ministry of Defence, Government of India, Gwalior, India. He has been elected fellow of the National Academy of Sciences, India, for his enormous contributions to the field of biology. The diagnostic technology developed by Professor Bisen is unique in several respects and has received recognition worldwide. The technology has been patented in India, the United States, Europe, and Japan. Other patents are under active consideration. He has published six international patents in the United States, Europe, Japan, and India. Professor Bisen is the external scientific advisor to a Barcelona-based multinational company, Biokit, which is involved in the manufacturing of diagnostic kits. He also serves as the chief of the research and development center of an upcoming industry, Tropilite Foods, based at Gwalior, which is involved in food biotechnology and develops several innovative products for the food industry.

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