Microbiological Techniques

456 Pages·2019·22.562 MB·English

by  N Murugalatha, Lali Growther, J Vimalin Hena, N Hema Shenpagam, R Anitha , D Kanchana Devi, G Rajalakshmi

Contents i
MICROBIOLOGICAL TECHNIQUES

MICROBIOLOGICAL TECHNIQUES
N. Murugalatha
Assistant Professor and Head
Lali Growther J. Vimalin Hena
Assistant Professor Assistant Professor
N. Hema Shenpagam R. Anitha
Assistant Professor Assistant Professor
D. Kanchana Devi G. Rajalakshmi
Assistant Professor Assistant Professor
Department of Microbiology and Biotechnology
Hindusthan College of Arts and Science
Coimbatore, Tamil Nadu
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Cataloguing-in-Publication Data
Microbiological techniques / by N. Muruga-
Latha ... [et al.].- chennai: MJP
Publishers, 2012.
xiv, 442 p.; 24 cm.
Includes Appendix, References.
ISBN 978-81-8094-107-8 (hb.)
1. Microbiology 2. Techniques-Microbiology.
I. Murugalatha, N ... [et al.].
579 dx22 MIC MJP 142
ISBN 978-81-8094-107-8 MJP PUBLISHERS
© Publishers, 2012 New No. 5, Muthu Kalathy Street
All rights reserved Triplicane
Printed and bound in India Chennai 600 005
Publisher : J.C. Pillai
CIP Data : Prof. K. Hariharan, Librarian
RKM Vivekananda College, Chennai.
This book has been published in good faith that the work of the author is original. All efforts have been taken to make the material error-free. However,
the author and publisher disclaim responsibility for any inadvertent errors.
PREFACE
Microbiological Techniques is designed for the students, to explore the world of microorganisms and
how the process of scientific discovery is carried out, with an ease. The study of microbiology is dynamic
because of the ubiquitous nature of the microbes and the variability inherent in every living organism. The
broad nature of the subject and diversity of topics from the fundamentals to its unique fields can make
the way of presentation a little difficult; but it is also a part of what makes microbiology an interesting
and challenging subject.
The book primarily focuses on the basic microbiological techniques with applications for undergraduate
and postgraduate students in diverse area of biological techniques. This book is the outcome of nearly a
decade of teaching and research experience. The manual comprises twelve parts in which exercises in first
three parts provide sequential developments of fundamental techniques. The remaining exercises are as
independent as possible to allow the instructor to select the desirable sequence. Exercises are pursued in a
normal scale providing maximum details so that one can perform the experiment independently and safely.
The style and simplicity of expression have been our twin objectives. All exercises have been thoroughly
tested in our laboratory by our students with wide variety of real talents and enthusiasm.
We wish to express our immense gratitude to the Management of Hindusthan College of Arts and
Science for allowing us to pursue this work, during our service in their Institution.
We have immense pleasure in expressing our deepest gratitude to our principal Dr. N. Baluswamy,
Hindusthan College of Arts & Science, for his valuable support which has been a great help in this endeavour.
We extend our gratefulness to all our colleagues in the Department of Biotechnology and Microbiology,
for encouraging us right through our work. We are most grateful to every soul involved in making up of
this manual spending their time, talent and interest in the work.
We look forward for the support, constructive ideas and valuable suggestions from the users of the book.
N. Murugalatha
Lali Growther
J. Vimalin Hena
N. Hema Shenpagam
R. Anitha
D. Kanchana Devi
G. Rajalakshmi

CONTENTS
1. Introduction to Microbiology 1
Introduction 1
Bacteria 1
Stages of bacterial growth 2
Factors affecting bacterial growth 3
Cyanobacteria 7
2. Tools of Microbiology 9
Microscopy 9
General principles of microscopy 9
Light microscopy 11
Electron Microscopy 18
Scanned-Probe Microscopy 20
Use and Care of Microscopes 22
Autoclave 26
Hot Air Oven 28
Incubator 29
Water Bath 31
BOD Incubator 32
Colony Counter 34
Haemocytometer 35
Oxygen Electrode 38
Lyophilizer 40
Laminar Air Flow (Vertical) 43
3. Fundamentals of Microbiology 45
Laboratory Precautions 45
Principles of Aseptic Techniques 46
Cleaning of Glassware 48
viii Contents
Safety 51
Control of Microorganisms 52
Sterilization 52
Disinfection 61
Culture Media Preparation 64
Different types of Media 65
Sterilization 68
Media Inoculation and Incubation 69
Pure Culture Techniques 71
Aerobic Culture Techniques 71
Anaerobic Culture Techniques 78
Robertson’s Cooked Meat (Rcm) Medium (Clostridium Sp.) 78
Anaerobic Jar (Total Anaerobes) 79
Wright’s Tube Method 82
Staining Techniques 84
Smear preparation 84
Types of staining techniques 85
Simple Staining 85
Gram Staining 86
Capsule Staining 89
Spore Staining (Schaeffer and Fulton Method) 90
Acid-Fast Staining (Ziehl–Neelsen’s Method) 92
Flagellar Staining 94
Negative Staining 96
Granule Staining 97
Giemsa stain for thin films (Blood) 97
Fontana’s stain for Leptospires 98
Periodic Acid—Schiff (PAS) Stain 98
Fungal wet mount—Lactophenol Cotton Blue Staining 99
Slide Culture Method 100
Micrometry 102
Micrometry and Measurement of Microorganisms 102
Motility Determination 105
Enumeration of bacteria, fungi and actinomycetes from soil 110
Phenol Coefficient Test 113
Maintenance and Preservation of Cultures 114
Contents ix
4. Microbial Physiology 117
Growth Curve 117
Direct Count 117
Viable Count 122
Turbidity Method 125
Biochemical Tests 128
IMViC Reactions 128
Indole Production Test 128
Methyl Red–Voges Proskauer Test 130
Citrate Utilization Test 131
Oxidase Test 133
Catalase Test 134
Urease Test 135
Hydrogen Sulphide Test 136
Triple Sugar Iron Agar Test 137
Nitrate Reduction Test 138
Polymer (Starch, Casein, Gelatin, Lipid) Degradation 140
Carbohydrate Fermentation Test 142
Amino Acid Decarboxylase Test 143
Phenylalanine Deaminase Test 145
Coagulase Test 146
Esculin Hydrolysis 147
ONPG ( O-Nitrophenyl β-d-Galactopyranoside) Test 149
OF Test for Carbohydrate Utilization 150
Malonate Utilization Test 152
Lecithinase Test 153
Pigment Extraction From Algae 154
Effect of Temperature on the Growth of Bacteria and Fungi 158
Effect of Osmotic Pressure on the Growth of Bacteria and Yeast 162
Effect of pH on the Growth of Bacteria and Fungi 164
5. Industrial Microbiology 167
Wine Production 167
Citric Acid Production 169
Production of Glutamic Acid 172
Protease Estimation 175
Production of Extracellular Cellulase by Solid-state Fermentation 180
x Contents
Mushroom Cultivation 183
Extracellular Enzyme Production—Amylase 185
Immobilization of Cells 186
6. Environmental Microbiology 189
Isolation of Nitrogen Fixers 189
Isolation of Symbiotic Nitrogen Fixers (Rhizobium Sp.) 189
Isolation of Free-living Nitrogen Fixers (Azotobacter Sp.) 190
Isolation of Phosphate Solubilizers 191
Standard Qualitative Analysis of Water by MPN Method 192
Quantitative Analysis of Water by Membrane Filter Technique 196
Dissolved Oxygen 200
Biological Oxygen Demand (BOD) 202
Chemical Oxygen Demand (COD) 204
Total Suspended Solids 206
Decolorization of Dye and Dye-containing Effluents 209
Nitrogen Cycle 210
Ammonification 211
Nitrification 212
Denitrification 213
Enumeration of Microorganisms from Wood and Paint 214
Assay of Microorganisms from Biomedical Waste 215
Isolation and Culture of Algae 216
Spraying 216
Single Cell/Colony/Filament Isolations 217
7. Food Microbiology 219
Methylene Blue Reduction Test 219
Phosphatase Test for Pasteurisation in Liquid Milk 221
Turbidity, Colony and Coliform Tests for Pasteurized Milk 223
Isolation of Food Spoilers 224
Analysis of Food Sample for Mycotoxin (Aflatoxin) 226
8. Genetics 229
Isolation of Chromosomal DNA from Bacteria 229
Isolation of Genomic DNA from Cauliflower by Supaquick Method 232
Isolation of Plasmid DNA 234
Agarose Gel Electrophoresis 235