Laboratory Protocols in Applied Life Sciences

1772 Pages·2014·448.027 MB·English

by  Prakash Singh Bisen

Life Sciences
Bisen
As applied life science progresses, becoming fully integrated into the biological, chemical,
LABORATORY
and engineering sciences, there is a growing need for expanding life sciences research
techniques. Anticipating the demands of various life science disciplines, Laboratory
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Protocols in Applied Life Sciences explores this development. n
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This book covers a wide spectrum of areas in the interdisciplinary fields of life sciences, A PROTOCOLS
pharmacy, medical and paramedical sciences, and biotechnology. It examines the B
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principles, concepts, and every aspect of applicable techniques in these areas. Covering
elementary concepts to advanced research techniques, the text analyzes data through pO
experimentation and explains the theory behind each exercise. It presents each in Applied Life Sciences
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experiment with an introduction to the topic, concise objectives, and a list of necessary iR
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materials and reagents, and introduces step-by-step, readily feasible laboratory protocols.
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Focusing on the chemical characteristics of enzymes, metabolic processes, product and
raw materials, and on the basic mechanisms and analytical techniques involved in life LT
science technological transformations, this text provides information on the biological i
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characteristics of living cells of different origin and the development of new life forms by
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genetic engineering techniques. It also examines product development using biological
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systems, including pharmaceutical, food, and beverage industries. S
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Laboratory Protocols in Applied Life Sciences presents a nonmathematical account
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of the underlying principles of a variety of experimental techniques in disciplines, e
including: P
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• Biotechnology cR
• Analytical biochemistry e
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• Clinical biochemistry s
• Biophysics
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• Molecular biology
• Genetic engineering
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• Bioprocess technology
• Industrial processes
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• Animal
• Plant
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• Microbial biology
• Computational biology
• Biosensors L
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Each chapter is self-contained and written in a style that helps students progress from
basic to advanced techniques, and eventually design and execute their own experiments

in a given field of biology.
K15259
ISBN: 978-1-4665-5314-9
90000
Prakash S. Bisen
9 781466 553149
K15259_COVER_final.indd 1 1/22/14 2:42 PM
L ABOR ATORY
PROTO COL S
in Applied Life Sciences

L ABOR ATORY
PROTO COL S
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
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© 2014 by Taylor & Francis Group, LLC
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Version Date: 20140114
International Standard Book Number-13: 978-1-4665-5315-6 (eBook - PDF)
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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.
© 2010 Taylor & Francis Group, LLC

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 10
1.10.1 Antibodies Can Be Used to Detect Specific Molecules
by Fluorescence Microscopy 10
1.11 Experiments on Microscopy 14
1.11.1 Introduction 14
1.11.2 Microscope Operation 15
1.11.3 Smear Preparation 16
1.11.4 Dyes 18
1.11.5 Staining Procedure 18
1.11.6 Microscopic Examination of Microorganisms 19
1.11.7 Hanging Drop Technique 19
1.11.8 Hemocytometer 20
1.11.9 Ocular Meter and Stage Micrometer for Micrometry 20
1.12 Electron Microscopy 21
1.12.1 Transmission Electron Microscopy 21
© 2010 Taylor & Francis Group, LLC vii
viii Contents
1.12.2 Scanning Electron Microscopy 24
1.12.3 Scanning Tunneling Microscope 25
1.13 Sample Preparation for Electron Microscopy 26
1.13.1 Preparation of Specimen Supports 27
1.13.1.1 Glow Discharge 29
1.13.1.2 Polylysine Procedure 29
1.13.2 Sample Preparation and Contrast Enhancement 30
1.13.3 Embedding, Sectioning, and Staining 30
1.13.3.1 Replica Formation 31
1.13.3.2 Freeze-Etching and the Critical Point Technique 31
1.13.3.3 Freeze-Fracture 32
1.13.3.4 Shadow-Casting 32
1.13.3.5 Negative Contrast Technique 34
1.13.3.6 Positive Staining 35
1.13.3.7 Kleinschmidt Spreading with Positive Staining
and Rotary Shadowing 35
1.13.3.8 Special Protocol Used with the Kleinschmidt Technique 38
1.13.4 Visualization of Nucleic Acid Protein Complexes on Polylysine Films 38
1.13.5 Improving the Quality of the Image and Image Reconstruction 38
1.13.6 Minimal Beam Exposure 39
1.13.7 Three-Dimensional Reconstruction of Molecular Structure from
Unstained Sample 39
1.13.8 Image Rotation and Rotational Filtering 40
1.13.9 Special Mechanisms of Image Formation 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 47
2.1 Introduction 47
2.2 Sectioning 47
2.3 Traditional Histology 48
2.3.1 Fixation, Dehydration, Embedding, and Staining 48
2.3.1.1 Fixation 49
2.3.1.2 Dehydration 50
2.3.1.3 Embedding 50
2.3.1.4 Staining 52
2.4 Ultramicrotome 52
2.5 Cryostat 53
2.5.1 Tissue Chilling 54
2.5.1.1 Apparatus and Materials 54
2.5.1.2 Protocol 54
© 2010 Taylor & Francis Group, LLC
Contents ix
2.5.2 Tissue Mounting 55
2.5.3 Preparation for Sectioning 55
2.5.4 Cryosectioning 56
2.5.5 Section Picking 60
2.6 Incubation 60
2.6.1 Methods of Incubation 60
2.6.2 Microcell Method 60
2.7 Electron Microscopy Technique 63
2.7.1 Special Preparation for Electron Microscope 63
2.7.1.1 Metal Shadowing 64
2.7.1.2 Freeze-Fracture and Freeze-Etch Electron Microscopy 65
2.7.1.3 Spectroscopy (Especially FTIR or Infrared Spectroscopy) 66
2.8 Botanical Microtomy 66
2.9 Processing of Tissue 67
2.9.1 Methods of Tissue Processing 67
2.9.2 Fixatives 68
2.9.3 Staining Procedures 69
2.9.4 Fixation, Dehydration, Embedding, and Staining 73
2.9.4.1 Introduction 73
2.9.4.2 Material Preparation 74
2.9.4.3 Material Required 74
2.9.4.4 Protocol 75
Suggested Readings 80
Important Links 80
3. pH and Buffers 81
3.1 Introduction 81
3.2 Acids and Bases 81
3.2.1 Definitions 81
3.2.1.1 Acids and Bases 81
3.2.1.2 Alkali 82
3.2.1.3 Ampholytes 82
3.2.2 Strength 83
3.2.2.1 Strong Acids or Bases 83
3.2.2.2 Weak Acids or Bases 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 K 84
w
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 86
3.4.1 pH Indicators 86
3.4.2 Measurement of pH 87
3.4.2.1 Glass Electrode 87
3.4.2.2 Calomel Electrode 87
© 2010 Taylor & Francis Group, LLC