A Practical Guide To Environmental Biotechnology-Compressed

Learning Materials in Biosciences
Jayanta Kumar Patra

Gitishree Das
Swagat Kumar Das
Hrudayanath Thatoi
A Practical Guide
to Environmental
Biotechnology
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More information about this series at http://www.springer.com/series/15430

Jayanta Kumar Patra • Gitishree Das • Swagat Kumar Das
Hrudayanath Thatoi
A Practical Guide to
Environmental
Biotechnology
Jayanta Kumar Patra Swagat Kumar Das
Research Institute of Biotechnology & Department of Biotechnology, College of
Medical Converged Science Engineering and Technology
Dongguk University Biju Patnaik University of Technology
Goyang-si, Korea (Republic of) Odisha, India
Gitishree Das Hrudayanath Thatoi

Research Institute of Biotechnology & Department of Biotechnology
Medical Converged Science North Orissa University
Dongguk University Odisha, India
Goyang-si, Korea (Republic of)
ISSN 2509-6125 ISSN 2509-6133 (electronic)

ISBN 978-981-15-6251-8 ISBN 978-981-15-6252-5 (eBook)
https://doi.org/10.1007/978-981-15-6252-5
© Springer Nature Singapore Pte Ltd. 2020

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V
Preface
Environmental biotechnology is the most important subject for the study of biotech-
nology. Environmental biotechnology is applied to and used to study the natural
environment and its conservation by application of biotechnological tools. The pres-
ent book entitled A Practical Guide to Environmental Biotechnology covers the whole
range of experiments related to environmental biotechnology. It also contains basic
laboratory safety guidelines followed in a laboratory. Each chapter starts with an
introduction or background into the basic approach followed by detailed methods
sections with easy-to-follow workable protocols and comprehensive troubleshooting
calculation. One of the important aspects of the book’s manual is the first part which
deals with general guidelines of laboratory safety, rules and regulations, different
symbols, glassware and equipments used commonly in an environmental laboratory.
The second part of the book deals with different experiments on basic and advanced
environmental studies.
This book is an indispensable tool for introducing advanced undergraduates and
beginning graduate and master’s students of Environmental Science and biotechnol-
ogy. There are few books available on practical environmental aspects targeting stu-
dents of the undergraduate and graduate levels. In this regard, this proposed book
will be of great help to the students as it will cover the major experiments as per the
curricula of Environmental Science and Biotechnology stream at both undergradu-
ate and graduate levels. The book will be of great use to researchers and scientists in
the relevant field of study.
We are honestly grateful to all the literature and search engines we referred to
write the chapters of this book. We are also thankful to Dr. Sue Lee, Editor, Medi-
cine & Life Sciences Journals, and Dr. Emmy Lee, Associate Editor, Medicine and
Life Sciences Books, and the team at Springer Nature Singapore Pte Ltd., Singapore,
for their generous cooperation at every stage of the book’s publication.
Jayanta Kumar Patra

Goyang-si, Republic of Korea
Gitishree Das
Goyang-si, Republic of Korea
Swagat Kumar Das

Bhubaneswar, Odisha, India
Hrudayanath Thatoi
Baripada, Odisha, India
VII
Contents
1 eneral Guidelines of Laboratory Safety, Calculations

G
Used in Laboratory Experiments, and Basic Laboratory
Glassware and Instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.1 General Guidelines of Laboratory Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.2 Fire Safety in a Laboratory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.3 Gas Safety in a Laboratory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1.4 Safety Signs in Biotechnology Laboratory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.5 General Calculations Used in Laboratory Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
1.6 Basic Laboratory Glassware and Instruments Used in an Environmental Lab . . . . . . . . . 16
References and Suggested Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
2 Water Quality Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

2.1 Electrical Conductivity of Waste Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
2.2 Alkalinity of Waste Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
2.3 Dissolved Oxygen Content in Water Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
2.4 Biochemical Oxygen Demand in Water Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
2.5 Chemical
Oxygen Demand in Water Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
2.6 Total
Suspended Solids (TSS) and Total Dissolved Solids (TDS) in Water Samples . . . . . 52
2.6.1 Total Dissolved Solids (TDS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
2.7 Chloride
Content in Water Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
2.8 Sulfate
Content in Water Samples (Turbidimetric Method) . . . . . . . . . . . . . . . . . . . . . . . . . . 54
2.9 Nitrogen
Content in Water Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
3 Soil Quality Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

3.1 Moisture Content in Soil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
3.2 pH of the Soil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
3.3 Particle Size in Soil Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
3.4 Organic Matter and Organic Carbon Content in Soil Samples . . . . . . . . . . . . . . . . . . . . . . . 70
3.5 Chloride
Content in Soil Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
3.6 Sulfate
Content in Soil Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
3.7 Nitrogen
Content in Soil Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
3.8 Potassium
Content in Soil Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
3.9 Phosphorus
Content in Soil Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
4 Isolation, Culture, and Biochemical Characterization of Microbes . . . . . . . . 83

4.1 Laboratory Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
4.2 Laboratory Etiquette . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
4.3 Cleaning Glassware . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
4.4 Sterilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
VIII Contents
4.5 Determination of pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
4.6 Study of the Effect of pH on Growth of Microbes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
4.7 Preparation
of Culture Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
4.7.1 Preparation of Nutrient Agar (NA) Slants and Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
4.7.2 Preparation of Potato Dextrose Agar (PDA) Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
4.8 Isolation of Cyanobacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
4.9 Techniques
for Isolation of Pure Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
4.9.1 Serial Dilution Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
4.9.2 Streak Plate Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
4.9.3 Pour Plate Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
4.9.4 Spread Plate Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
4.10 Staining Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
4.10.1 Simple Stains: Microbial Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
4.10.2 Negative Staining (Indirect Staining) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
4.10.3 Differential Stains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
4.10.4 Acid-Fast Staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
4.10.5 Special Staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
4.10.6 Fungal Staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
4.11 Microbial Growth Curve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
4.12 Isolation of Genomic DNA from Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
4.13 DNA Quantification and Quality Analysis by Agarose Gel Electrophoresis . . . . . . . . . . . 120
4.14 DNA Quantification by Spectrophotometric Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
4.15 PCR Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
4.16 Sequence Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
4.17 Sampling of Air Microflora . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
4.18 Antibiotic Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
4.19 Coliform MPN Test for Bacteriological Examination of Water . . . . . . . . . . . . . . . . . . . . . . . . 130
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
5 Plant Tissue Culture Techniques and Nutrient Analysis . . . . . . . . . . . . . . . . . . . . 135

5.1 Plant Propagation and Tissue Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
5.1.1 Preparation of Culture Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
5.1.2 Callus Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
5.1.3 Cell Suspension Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
5.1.4 Protoplast Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
5.1.5 Artificial Seeds/Synthetic Seeds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146
5.2 DNA
Isolation and Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
5.2.1 Isolation of DNA from Plant Sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
5.2.2 Agarose Gel Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
5.2.3 Spectrophotometric Quantification of DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
5.3 Plant Nutrient Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
5.3.1 Estimation of Protein Content in Plant Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
5.3.2 Estimation of Fatty Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
5.3.3 Estimation of Ammonia Nitrogen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156
5.3.4 Estimation of Phosphorus in Plant Sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
IX
Contents
5.3.5 Estimation of Potassium in Plant Sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158

5.3.6 Estimation of Sulfur . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
5.3.7 Estimation of Boron in Plant Sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160
5.3.8 Estimation of Zn, Cu, Mn, and Fe in Plant Sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
Supplementary Information
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
About the Editors
Jayanta Kumar Patra, M.Sc., Ph.D.,

is currently working as Assistant Professor at Dongguk University,
Republic of Korea. He has about 14 years of research and teaching
experience in the field of food, pharmacology and nano-biotechnology.
To his credit, he has published more than 140 papers in various national
and international peer-reviewed journals and around 30 book chapters
in different edited books. Dr. Patra has also authored 12 books in vari-
ous National and International publications.
Gitishree Das, M.Sc., Ph.D.,

is currently working as Assistant Professor at Research Institute of Bio-
technology & Medical Converged Science, Dongguk University, Repub-
lic of Korea. She has 12 years of research and teaching experience in the
field of rice molecular biology, plant breeding, endophytic bacteria and
green nanotechnology. Her current research is focused on the biosyn-
thesis of nanoparticles using food wastes and plant materials and their
applications in the biomedical and agricultural fields. To her credit, she
has published around 70 research articles in reputed National and Inter-
national journals and 16 book chapters in different books. Dr. Das has
also authored 6 books in various International publications.
Swagat Kumar Das, B.Pharm., M.Tech., Ph.D.,

is currently working as Assistant Professor in the Department of Bio-
technology at the College of Engineering and Technology, an autono-
mous and constituent College of Biju Patnaik University of Technology,
Rourkela, Odisha, India. He obtained his B.Pharm degree from BPUT,
Rourkela, and M.Tech. degree from Rajiv Gandhi Proudyogiki Vish-
wavidyalaya, Bhopal, and Ph.D. from Ravenshaw University, Cuttack,
India, and is a Fellow of Eurasian Academy of Environmental Sciences
(FEAES). He has more than 10 years of teaching and research experi-
ence and published more than 20 research and review papers in National
and International journals of repute along with 7 book chapters. He has
co-authored 2 practical books. His research activities involved phyto-
chemical analysis and drug development from mangrove plants for dia-
betes. His research area also focused on green synthesis of nanoparticles
and evaluation of their pharmacological potentials.
XI
About the Editors
Hrudayanath Thatoi, M.Sc., M.Phil, Ph.D.,

is currently working as Professor and Head of the Department of Bio-
technology at North Orissa University, Odisha, India. He has around
30 years of teaching and research experience. His research activities are
basically based on medicinal plants, bioremediation, biodiversity, ethno-
pharmacology, mangrove biology, etc. Prof. Thatoi obtained his M.Phil.
and Ph.D. from Utkal University, Odisha, India, and his Ph.D. research
work was based on N2 fixation in legume plants under dual inocula-
tion of Rhizobium and VAM fungi and contributed significantly towards
development of technology for mine waste reclamation. Prof. Thatoi
has handled many research projects from State Government and Cen-
tral Government organizations like DST, Govt. of Odisha, UGC-DAE,
Govt. of India, Department of Forest, Govt. of Odisha, etc. Around
15 students have obtained Ph.D. and another 6 students are continu-
ing their Ph.D. research under his guidance and supervision. Besides,
several M.Sc., M.Tech, and B.Tech. students have received his guidance
for their dissertation works. Prof. Thatoi has published more than 200
research papers in various National and International reputed journals
and around 30 book chapters. Prof. Thatoi has also authored around
15 books/manuals by different notable publishers like STUDIUM Press
LLC USA, IK International, Narosa publishers, Biotech Books, APH
Publication, CRC Press, Apple Publication, Springer and Elsevier pub-
lications, etc. He has also authored a textbook on Microbiology and
Immunology, published by India Tech Publication, New Delhi, for
M.Sc. and B.Sc. Students. Prof. Thatoi has contributed immensely in
the field of microbiology and biotechnology throughout his research
and teaching career.
1 1
General Guidelines
of Laboratory Safety,
Calculations Used
in Laboratory Experiments,
and Basic Laboratory
Glassware and Instruments
Contents
1.1 General Guidelines of Laboratory Safety – 2
1.2 Fire Safety in a Laboratory – 3
1.3 Gas Safety in a Laboratory – 5
1.4 Safety Signs in Biotechnology Laboratory – 9
1.5 eneral Calculations Used in Laboratory

G
Experiments – 11
1.6 asic Laboratory Glassware and Instruments Used in an

B
Environmental Lab – 16
References and Suggested Reading – 34

J. K. Patra et al., A Practical Guide to Environmental Biotechnology, Learning Materials in Biosciences,
https://doi.org/10.1007/978-981-15-6252-5_1
2 Chapter 1 · General Guidelines of Laboratory Safety, Calculations Used in Laboratory Experiments...
What You Will Learn in This Chapter

1 In this chapter, you will learn about the general laboratory safety guidelines that need to
be followed while working in a laboratory; about the good laboratory practices, chemical
laboratory safety education, types of hazards in a laboratory, fire safety, gas safety and
various safety signs used in a laboratory; and about the general calculations used in vari-
ous laboratory experiments together with information of basic laboratory glassware and
instruments.
1.1 General Guidelines of Laboratory Safety
Maintaining safety inside a laboratory is of utmost importance for scientific person-

nel for the betterment of the environment and human kind. The principal objective
of the laboratory safety standard is the safety of its staff against any kind of likely
dangerous materials or any infections. Additionally, it is vital to educate students/
researchers and laboratory personnel about the laboratory safety guidelines and also
about the hazardous chemicals, materials, instruments, etc., present in the laboratory
and their uses (. Figs. 1.1, 1.2, 1.3, and 1.4; . Table 1.1).
There are a number of issues that are to be followed for maintaining the safety of
a laboratory. They are as follows:
1. There should be a list of emergency contact numbers, such as those of lab in-
charge, safety manager, and fire service, which should be mentioned in a visible
area inside the laboratory.
2. The basic laboratory safety rules and guidelines should be displayed on the labo-
ratory wall in an appropriate place.
3. A first aid kit containing basic essential medicines should be kept in a laboratory.
4. Protective gears such as lab coats, gloves, and goggles should be kept in sufficient
numbers in a laboratory and should be properly used while working inside a
laboratory.
5. All the chemicals and reagents should be labeled properly with the date of prepa-
ration and the type of reagents.
6. All other activities such as eating and storing food should not be allowed inside
a laboratory.
7. All the instruments should be checked properly before and after use.
8. Electrical gears should be checked at regular intervals.
9. The instruments should be labeled properly with their date of manufacture,
instructions for use, etc..
10. Laboratory working place should be always kept clean and all the essential
chemicals and glassware should be placed in their right place properly arranged.
11. Excessive things such as instruments, chemicals, glassware, etc. should be avoided
in the working area.
12. The sink should be washed properly every day before and after use.
13. The gas openings and hoods should be used for specific chemicals and purpose
only.
14. The broken glassware should be castoff in the specific container and must not be
dumped anywhere.
15. Reagents and chemicals should never be dispensed down in the sink; it should be
kept in a specific container and discarded properly.
1.2 · Fire Safety in a Laboratory
3 1
Good Laboratory Practice (GLP):
Listen carefully to the safety instructions from the teaching staff at the start of
each practical.
• Read the Safety section (COSHH and safety considerations) at the start of each
practical schedule.
• Make sure you know where the nearest fire exit is.
• Make sure you know where the eyewash stations, first aid boxes and fire
extinguishers are located in each lab you use.
• Always wear a labcoat (correctly fastened) - remove the labcoat if you leave the
laboratory.
• Wear safety glasses for handling hazardous chemicals as instructed.
• Wear disposable gloves for handling hazardous chemicals or if you have a cut or
wound on your hand. Always remove gloves before opening doors, drawers, lab-
books or handling any instrument controls to avoid contaminating surfaces.
• No eating, drinking or application of cosmetics in labs.
• No open-toed footwear in the laboratory.
• No mouth pipetting - use automatic pipettes or pipetting devices.
• Dispose of chemicals and bacterial cultures in a safe manner as instructed.
• Report immediately any spillage of chemicals, bacterial cultures or breakages to
the person in charge.
• Do not put broken glass, pipette tips or needles in the normal waste - use the
SHARPS disposal bins provided.
• Switch off all electrical equipment and gas burners when you are finished in the
lab.
IF IN DOUBT, ASK - DON’T PUT SAFETY AT RISK !
.. Fig. 1.1 The rules of good laboratory practice in the laboratory. (Reproduced with permission from
Seiler 2005a)
1.2 Fire Safety in a Laboratory
Utmost care should be taken for fire safety in a laboratory. There are certain guide-
lines that need to be followed for proper maintenance of fire safety in a laboratory.
1. A smoke detector and fire alarm should be installed inside every laboratory.
2. There should not be any obstacle near the fire exit point.
3. A fire extinguisher should be kept in a laboratory and it should be checked at
regular interval for its functioning.
4. The laboratory personnel should be aware of how to use the fire extinguisher
properly.
5. All persons working in a laboratory should be educated with fire safety (. Figs. 1.5
and 1.6).
1 Corporate management
Management Management Management Corporate quality

America Europe Asia assurance
Management
finances
Management
research
Management
marketing
Management Quality
safety assurance
TFM chronic TFM repro- TFM TFM

toxicology toxicology carcinogenesis ecotoxicology
.. Fig. 1.2 Possible example of an organization chart of a large, multinational company with a number
of test facilities and several levels of management. (Reproduced with permission from Seiler 2005b)
1.3 · Gas Safety in a Laboratory
5 1
.. Fig. 1.3 Products of chemi-
cal/laboratory safety education. Chemical/laboratory safety education
(Reproduced with permission
from Hill Jr. 2019)
Knowledge Caring for

understanding safety
Critical
thinking
1.3 Gas Safety in a Laboratory
A laboratory working with different types of gases should take utmost care for its
safety.
1. The gases should be kept in specific cylinders and should be properly positioned.
2. The pipes used for the flow of gases in a laboratory should be properly selected
and maintained.
3. The type of gas and the direction of the flow of the gases should be clearly indi-
cated on the gas pipes.
4. The gas pipes should be placed at a small distance from the electrical wiring in a
laboratory.
5. The gas leak alarms should be connected to the gas cylinders.
6. While using toxic gases, gas masks should be properly used in a laboratory.
.. Fig. 1.4 Laboratory safety

1 clothes and accessories used in
a laboratory. (Reproduced with
permission from Patra et al.
2019)
1.3 · Gas Safety in a Laboratory
7 1
.. Table 1.1 Different types of hazards in a laboratory
Type Hazards
Physical hazards Runaway reactions
Housekeeping
Electrical hazards
High pressure/low pressure
Cryogenics
Lasers
Catalysts
Corrosives
Flammables
Explosives
Reactive/unstable chemicals
Incompatibles
Gases and gas cylinders
Toxic hazards Target organ toxicants
Teratogens
Lachrymators
Allergens (sensitizers)
Carcinogens
Asphyxiants
Poisons
Irritants
Biological hazards Human specimens/some animal specimens
Bacteria
Viruses
Fungal agents
Rickettsial agents
Biological toxins
Reproduced with permission from Hill Jr. (2019)

.. Fig. 1.5 Fire safety alarm

1 sensor connected in a laboratory
.. Fig. 1.6 Laboratory safety cabinet and fire extinguisher used in a laboratory. (Reproduced with
permission from Patra et al. 2019)
1.4 · Safety Signs in Biotechnology Laboratory
9 1
1.4 Safety Signs in Biotechnology Laboratory
Harmful or Irritant Flammable Explosives Oxidizer
Corrosive Environmental Respiratory Gloves Required

Hazard Protection
Eye or Face Protective Clothing Protective Footwear Eye Protection

Protection Required
Ear Protection Fire Extinguisher Fire Hose Safety Flammable Gas

Required
Fire Blanket Nonflammable Gas International Reactive Material

Biohazard
Prohibition Non-potable Water Do Not Touch No Open Flames
Do Not Eat or Do Not Enter Carcinogen Hazard Low Temperature

Drink (Health Hazard) Warning
Hot Surface Magnetic Field Optical Radiation Laser Warning

Warning
Compressed Gas Non-ionizing Generic Warning Ionizing Radiation

Radiation
Remote Control Biohazard Sign High Voltage Warning Toxic Chemical

Equipment Sign Symbol
1.5 · General Calculations Used in Laboratory Experiments
11 1
Toxic Substance Irritant Emergency Call Escape Route Sign

Button
Fire Triangle First Aid Safety Shower Eye Wash
1.5 General Calculations Used in Laboratory Experiments
There are different types of calculations such as molarity, normality, percent by

weight, etc., used in a laboratory and they are discussed in . Table 1.2.
.. Table 1.2 Concentration expression terms
Unit Symbol Definition Relationship
Molarity M Number of moles of solute per mol

liter of solution M=
liter
Normality N Number of equivalents of solute equivalents

per liter of solution N=
liter
Percent by wt% Ratio of weight of solute to wt solute ´100

weight (parts per weight of solute plus weight of wt % =
total wt
hundred) solvent × 100
wt/vol% Ratio of weight of solute to total wt solute ´100

volume × 100 wt / vol% =
total volume
Percent by vol% Ratio of volume of solute to vol of solute ´100

volume total volume × 100 vol% =
total volume
(continued)
1 .. Table 1.2 (continued)
Unit Symbol Definition Relationship
Parts per million ppm Ratio of solute (wt or vol) to mg solute

total weight or volume × ppm =
kg solution
1,000,000
m g solute
=
g solution
mg solute
=
L solution
m g solute
=
mL solution
Parts per billion ppb Ratio of solute (wt or vol) to m g solute

total weight or volume × ppb =
kg solution
1,000,000,000
ng solute
=
g solution
m g solute
=
L solution
ng solute
=
mL solution
Reproduced with permission from Tyl and Ismail (2017)
zz Serial dilutions
There are a number of processes for serial dilution and are discussed in . Figs. 1.7,
1.8, and 1.9.
.. Fig. 1.7 Dilution schemes

a
for a “dilute to” a and “dilute
with” b scenario. (Reproduced
with permission from Neilson
and O’Keefe 2017)
b
13 1
.. Fig. 1.8 Example standard curve dilution schemes for sequential dilutions a and parallel dilutions
b. (Reproduced with permission from Neilson and O’Keefe 2017)
.. Fig. 1.9 Diagram of a complex multistep dilution and concentration scheme. (Reproduced with
permission from Neilson and O’Keefe 2017)
zz Concentration of common commercial strength acids is given in . Table 1.3.
.. Table 1.3 Concentration of common commercial strength acids
Acid Molecular weight Concentration Specific gravity

(g/mol) (M)
Glacial acetic acid 60.05 17.4 1.05
Formic acid 46.02 23.4 1.20
Hydroiodic acid 127.9 7.57 1.70
Hydrochloric acid 36.5 11.6 1.18
Hydrofluoric acid 20.01 32.1 1.167
Hypophosphorous acid 66.0 9.47 1.25
Lactic acid 90.1 11.3 1.2
Nitric acid 63.02 15.99 1.42
Perchloric acid 100.5 11.65 1.67
Phosphoric acid 98.0 14.7 1.70
Sulfuric acid 98.0 18.0 1.84
Sulfurous acid 82.1 0.74 1.02
Reproduced with permission from Neilson et al. (2017)

15 1
zz Few examples of basic glassware and pipette used in a laboratory are shown in
. Figs. 1.10 and 1.11.
a b c d e
.. Fig. 1.10 Class A volumetric flask a and other types of non-class A volume-measuring glassware:
graduated cylinder b, Erlenmeyer flask c, beaker d, and bottle e. (Reproduced with permission from
Neilson et al. 2017)
a b c d e
.. Fig. 1.11 Class A volumetric pipette a and non-volumetric pipettes: adjustable pipettors b, reed
pipettor c, serological pipettes d. (Reproduced with permission from Neilson et al. 2017)
1.6 asic Laboratory Glassware and Instruments Used

B
1 in an Environmental Lab
zz Glassware
Glassware Picture Description Uses
Beaker A cylindrical con- These are used

tainer with a flat for various
bottom base and purposes such
spout on one top as for prepar-
side. It is made up ing mixture
of glass borosilicate solution, for
and sometimes plas- storing liquids
tic, polypropylene, for later use,
etc. It is available in for performing
various sizes (e.g., reactions, for
10 ml, 50 ml, 100 ml, titration exper-
250 ml, 500 ml, iments, for
1000 ml, and heating liquids
5000 ml etc.). It has or chemicals,
a measuring mark in etc.
Glass beaker
its side to measure
the quantity of
liquid.
Plastic beaker
1.6 · Basic Laboratory Glassware and Instruments Used in an Environmental…
17 1
Conical flask It is otherwise It is used for

(Erlenmeyer known as Erlen- preparing
flask) meyer flask. It has a reagents, mix-
flat bottom base ing and storing
which narrows chemicals, per-
toward the mouth forming experi-
and is available with ments, and also
a ring in the neck or in preparing
without a ring. It is microbial cul-
made up of glass, ture. It is used
borosilicate, and in titration
sometimes plastic, experiment.
polypropylene, etc.
It is available in var-
ious sizes (e.g.,
10 ml, 50 ml, 100 ml,
Glass flask 250 ml, 500 ml,
1000 ml, and
5000 ml). It has a
measuring mark in
its side to measure
the quantity of
liquid.
Plastic flask
Round- These types of flask They are used

bottom flask have a round bottom for heating,
instead of flat bot- boiling liquids,
tom as in the case of in the distilla-
conical flask. They tion process, in
are made up of glass the extraction
(heat-resistant boro- of compounds,
silicate). They have a etc.
tubular section
called neck, with an
opening. Depending
upon the uses, there
are two- to three-
necked round bot-
tom flasks. They
come in a variety of
sizes starting from
5 ml, 10 ml, 50 ml,
100 ml, 250 ml,
500 ml, 1 liter to 20
liter in industrial
scale.

1
Burette It is a graduated It is used in the
glass tube with a tap titration exper-
at one end. The iment.
stopcock is used to
dispense a known
volume of liquid
slowly and as per
the requirement. It
is made up of glass
or plastic.
Measuring It is a narrow gradu- It is used to

cylinder ated cylinder and is measure the
available in various volume of a
sizes. It is made up liquid.
of glass or polypro-
pylene. It comes in
various sizes
(10 ml–5 liter).
Funnel It is a tube with It is used for

wide mouth at one dispensing liq-
end and narrow uid or solid
mouth at the other chemicals from
end. It is made up of one container
stainless steel, glass, to another with
aluminum, or the small
plastic. opening.
19 1
Crucible It is a ceramic or a It is used for

metal container melting or
which is basically heating any
inert and is used for chemical or
melting any sub- metal in a lab-
stance (metal or oratory.
chemicals). It is
made up of high-
temperature-resis-
tant materials
depending upon the
purpose of use.
Mortar and The mortar is a It is used for

pestle bowl made up of mixing, grind-
ceramic, wood, ing, or crush-
metal, or granite ing the
stone. It has a pestle materials.
which is made up of
the same material as
that of the mortar.
The pestle is heavy
and blunt club
shaped/ rod shaped,
which is easy to hold
in the hand. The
substance to be
crushed is put in the
mortar and then
crossed using the
mortar.
Test tubes It is cylindrical nar- It is used for

row tube with round biochemical
closing at the bot- experiments.
tom and an opening
with a rim at the
top. It is made up of
borosilicate glass
material.

1
Culture tubes It is similar to that It is used in
of a test tube, but it microbiologi-
has a stopper or cal culture
closing cap in the experiments.
mouth.
Centrifuge It is cylindrical nar- It is used in

tubes row graduated tube centrifugation
with conical bottom process.
and a screw capped
mouth. It is made
up of plastic or
polypropylene mate-
rials.
Petri plates It is also known as It is used in

Petri dish or culture microbiologi-
dish or plates. It is a cal culture
shallow/flat cylindri- experiments.
cal glass with a lid
to cover it. It is
made up of borosili-
cate glass or plastic.
Glass pipette It is a narrow, grad- Its main pur-

uated thin cylinder pose is to
used to transport transport
liquids or media. required
They come in vari- amount of liq-
ous sizes and shapes. uid from one
container to
another.
21 1
Dropper It is similar to the Its main pur-

pipette, but has a pose is to
plastic bulb at one transport
end to suck liquid required
inside the glass/plas- amount of liq-
tic tube. uid from one
container to
another.
Pipette bulb It is placed at the It is an aid to

top of a pipette and the pasture
is used to create vac- pipette.
uum by pulling the
air inside and then
the desired liquid
into the pipette.
Volumetric It is a type of labo- It is used for

flask ratory flask cali- accurate dilu-
brated to contain a tion and prepa-
particular amount ration of
of liquid in it. It standard solu-
comes in various tions.
sizes and is normally
made up of glass. It
is a pear-shaped flat-
bottom flask with a
long neck and a
stopcock.

1
Wash bottle It is made up of It is used to
plastic or polyethyl- squeeze out
ene. It has a screw water forcibly.
cap with a long nar-
row hole at the top
in order to squeeze
out water.
Volumetric It is made up of It allows the

pipette glass. It allows the actual mea-
actual measurement surement of a
of a solution. It is solution.
calibrated to contain
a specific amount of
liquid. It has a large
bulb in the middle
with a long narrow
mouth at the bot-
tom.
Test tube It is made up of a It is used to

holder wooden holder with hold test tubes,
an iron frame and a when they are
clamp. hot.
23 1
Test tube It is made up of It is used to

stand wood, iron rod, or hold upright a
thermostable plastic. multiple num-
It comes in a variety ber of test
of shapes, sizes, tubes at a time
compositions, and at one place.
materials.
Micropipette It is a sophisticated It is used to

type of pipette used hold small
to hold small quan- quantity of liq-
tity of liquid uid with preci-
(0.001 μl). Its basic sion.
structure includes a
nozzle button, a tip
ejector button, vol-
ume adjustment
dial, volume display,
tip ejector, and
shaft. There are dif-
ferent types of
micropipettes avail-
able in the market,
based on the types
of uses, design,
weight, and preci-
sion.
Eppendorf It is made up of It is used in

tubes polypropylene and it molecular
comes in various experiments to
sizes ranging from hold small
10 μl to 2 ml quantity of
capacity. liquid.

1
Reagent bot- It is made up of It is used for
tles borosilicate glass or storing
polypropylene and reagents and
comes in various chemicals.
colors such as color-
less, white, or amber.
It comes in various
shapes and sizes.
Beaker It is made up of It is used to

holder stainless steel, iron, handle hot
and sometimes plas- beakers firmly.
tic. It comes in dif-
ferent shapes and
sizes.
Tripod stand It is made up of iron It is used to

or wooden frame. hold the bea-
ker or conical
flask for heat-
ing purpose.
25 1
Burner, spirit It comes in different Used to get fire

lamp shapes and sizes in a laboratory
depending upon the for heating
source of fire to be purpose.
used.
Spreader It is made up of It is used in

glass or heat-resis- microbiology
tant plastic. It has a experiment for
definite shape. spreading the
culture uni-
formly on a
Petri plate.
Microplates It is a specially It is used for

designed plate, made holding small
up of plastic or amount of liq-
polypropylene. It is uids multiple
transparent or number of
opaque depending times in the
upon its uses. It has wells (24 or 96
multiple number of wells)
wells (24, 96 wells).
Forceps It is made up of For holding

stainless steel. It is a certain things.
hand-held instru-
ment used to hold
delicate things. It
comes in various
shapes and sizes as
per the requirement.

1
Spatula It is made up of Used for lifting
stainless steel. It is a solid chemicals
hand-held instru- for weighing
ment used for mix- purpose.
ing, spreading, or
lifting solid chemi-
cals.
Burette stand It has a long rod, It is used for

with a base and a holding the
clamp attached to it burette at the
to hold the burette required posi-
firmly. tion and to
carry out the
titration exper-
iment.
Thermom- It is made up of It is used for

eter thermostable glass; measuring the
it has a temperature temperature of
sensor (mercury any liquid or
bulb) to sense the reagent.
temperature and a
measuring scale in
Centigrade or Fahr-
enheit to measure
the temperature.
Capillary It is made up of Used for capil-

tubes plastic or glass. Rod lary experi-
shaped, hollow ments.
inside.
27 1
zz Instruments
Instruments Picture Description Uses
Weighing A variety of bal- It is used to

balance ances are available measure any
depending on uses chemical accu-
and application. rately.
Magnetic It has a hot plate It is used to

stirrer with the thermo- mix any type
stat where we can of chemical or
control the heating liquid.
temperature. The
magnetic stirrer
is also connected
with a magnet and
a magnetic bead is
used to properly
mix the solute in
the solvent to make
a uniform solvent.
Bacterio- The incubator is Normally it is

logical incu- a double-walled used for bacte-
bator modular structure rial cell culture
with insulation. purpose.
Basically, its inte-
rior is made up of
stainless steel. The
inside part is con-
nected to a heating
apparatus and a
thermostat to con-
trol the heat. It has
two doors: inside
is a glass door and
outside is the main
door with secure
gasket, heavy-duty
door hinges and
latches to main-
tain a secure and
uniform seal. The
racks and trays
are all made up of
stainless steel with
adjustable height.
It has both heating
and cooling sys-
tems to control the
temperature.

1
Shaker A number of shak- Normally it is
ers are used in a used to mix,
laboratory: vortex combine, or
shaker, platform stir various
shaker, orbital substances in
shaker, and incuba- a tube or flask,
tor shaker depend- by shaking
ing upon their them.
specific purpose
and uses.
Oven The oven consis- It is used for

tently and evenly heating mate-
distributes heat rials such as
throughout the glassware, lab-
entire surface of oratory uten-
the mold placed sils and to dry
inside the oven. certain things
The exteriors have like plant
a hard, scratch- materials, and
resistant finish, other samples
normally light gray that need to be
in color. The inte- dried.
rior is made up of
heavy steel double
wall and doors
are insulated. The
interior is made
up of corrosive-
resistant aluminum
steel. There is a
temperature con-
trol thermostat to
control the tem-
perature along with
a thermometer to
show the interior
temperature.
pH meter A highly essential It is used to

instrument in a measure the
basic scientific acidity or alka-
laboratory. It has linity of a solu-
two parts, a pH tion based on
electrode and a the hydrogen-
measuring stand ion activity of
with digital setup the solution
to display the mea- (water based).
surements.
29 1
Atomic Atomic absorption It is used to

absorption spectroscopy is determine the
spectros- used for qualitative concentration
copy determination of of a particular
chemical elements element in a
using the absorp- sample, for
tion of optical example, the
radiation (light) by determination
free atoms in the of various ele-
gaseous state. It is ments in the
based on absorp- soil samples;
tion of light by free used in num-
metallic ions. ber of fields
such as geol-
ogy, minerals,
metallurgy,
steel, non-
ferrous metals;
environmental
analysis: air,
water, soil,
and solid
waste quality;
petrochemical
industry and
light industry
products, crude
oil and other
products; food,
biomedicine,
and health
products;
building mate-
rials (glass,
ceramics,
paints, etc.).

1
Spectropho- Spectrophotometer It is used for
tometer is an instrument quantitative
that uses the Beer– measure-
Lambert law to ment of the
measure the inten- light intensity
sity of any particle transmitted as
in the sample based a function of
on the transmis- wavelength.
sion of light pass- It is normally
ing through it. It used in a
deals with visible biochemistry
light, ultraviolet laboratory for
light, and infrared biochemical
light. There are experiments.
different types of
spectrophotometer
depending upon
the capacity of
light source it uses
for the measure-
ment.
Deep It comes in a vari- It is used for

freezer ety of temperature quick freezing
ranging from −20 and storing
to −80 °C etc. It chemicals
has quick freeze and samples
functions. (animal, plant,
DNA, RNA,
protein, blood
samples, clini-
cal and medi-
cal samples,
vaccines, etc.)
in low temper-
ature in order
to preserve
their proper-
ties.
Autoclave It is a pressure It is used for

chamber connected sterilization
to heating coil. of samples at
It is made up of higher temper-
stainless steel and ature and pres-
connected with a sure (121 °C
temperature and and 15 lb pres-
pressure setup sure).
to maintain the
temperature up to
121 °C and 15 lb
pressure.
31 1
Micro- It contains a stain- Used for

biological less steel hood microbiologi-
safety cabi- connected to a cal or cellular
net high-efficiency analysis pur-
particulate air filter pose.
(HEPA) to provide
the flow of puri-
fied air in outward
(horizontal) or
downward (verti-
cal) direction. It
has an ultraviolet
light source for
sterilization of
the working area.
There are differ-
ent types of hood
depending upon
the uses.
Sonication It is an instrument It is used in

machine used to agitate par- the extraction
ticles in the sample of multiple
using the sound compounds
energy. It uses from plants
ultrasonic frequen- and for mixing
cies (>20 kHz) for a particular
its applications. compound in a
solvent.
Centrifuge It is an instru- It is used to

machine ment used to spin separate a
an object around solute from a
a fixed axis by solvent in the
applying a perpen- solution.
dicular force. It
uses sedimentation
principle, by which
due to the applica-
tion of centrifugal
force, the denser
substances are
moved outward in
the radial direc-
tion or settle at
the bottom of the
container. It comes
in various sizes and
capacities for spe-
cific purposes and
applications.

1
Vortex It is a simple elec- It is used to
mixer tric device with mix reagents
a motor, a drive and chemicals
shaft, and a rubber inside a test
piece of different tube, vial, or
shape to hold the micro tube.
test tubes or micro
tubes. When the
switch is turned on,
the motor moves in
a circular motion.
It has a speed con-
troller to control
the speed of the
movement of the
motor.
Colony It is an electric It is used to

counter machine used in a count the num-
microbiology labo- ber of the bac-
ratory to count the terial colony in
number of bacte- a petri plate.
rial colonies grown
in a Petri plate. It
has a flat circular
surface with small
lines, squares
drawn, over which
the Petri plate is
kept; then it is illu-
minated from the
bottom. A digital
pen is connected
to the instru-
ment, and when
the digital pen/
electronic counter
points toward or
softly touches the
bacterial colony
grown in the plate,
it is counted and
the number is dis-
played on a small
display attached to
the machine.
33 1
Microscope It is an instru- It has various

ment used to see applications
smaller objects in and uses in sci-
a highly magnified ence. Its main
form. There are objective is to
different types of visualize tiny
microscope avail- objects in a
able depending highly magni-
upon their uses, fied version
magnification, and which is visible
purpose. How- to the naked
ever, basically it is eye.
divided into two
types, light or elec-
tron microscope. A
simplified micro-
scope contains
various parts such
as base, arm, tube,
and eyepiece with
different types of
lens.
Turbidity It is an instrument It is used for

meter used for determin- measuring the
ing the concentra- turbidity of a
tion of suspended solution.
particles in a
sample of water
on the basis of
the incident light
scattered from the
tube containing the
solution at right
angle, which is cap-
tured by the sensor
in the form of pho-
tons and recorded
as electronic signals
by the machine.

1
Sieving It is an instrument It is used for
machine used for determin- measuring the
ing the particle particle size of
size of soil samples the soil sample.
based on their size
and texture. It has
different layers
of sieves with dif-
ferent sizes that
separate the soil
samples based on
the size of the par-
ticles.
Flame pho- It is used for the It is used for

tometer estimation of determination
potassium content of potassium
in the soil and content in
water samples soil and water
based on the emis- samples.
sion values.
Take-Home Messages
55 Maintaining laboratory safety inside a laboratory is of utmost important for scien-
tific personnel and for betterment of the environment and human kind.
55 The basic laboratory safety rules and guidelines should be displayed on the labora-
tory wall in an appropriate place.
55 All the instruments should be checked properly before and after use.
55 Laboratory working place should be always kept clean and all the essential chemi-
cals and glassware should be placed in their right place in an arranged manner.
55 Reagents and chemicals should never be dispensed down in the sink; it should be
kept in specific container and discarded properly.
References and Suggested Reading

Basic Laboratory Safety. https://shp.utmb.edu/ClinicalLaboratorySciences/clsHandbook.asp. Accessed
on 20th May 2019.
Guidelines for Chemical Laboratory Safety in Secondary Schools. American Chemical Society
Committee on Chemical Safety. 2016. https://www.acs.org/content/dam/acsorg/about/governance/
committees/chemicalsafety/publications/acs-secondary-safety-guidelines.pdf ?logActivity=true.
Accessed on 9th May 2019.
Hill RH Jr. Recognizing and understanding hazards — the key first step to safety. J Chem Health Saf.
2019;26(3):5–10.
35 1
Keith Furr A. CRC handbook of laboratory safety. Boca Raton: CRC Press; 2000. ISBN 9780849325236.
Laboratory Safety Handbook. Sabanci University, Istanbul. https://fens.sabanciuniv.edu/sites/fens.
sabanciuniv.edu/files/lab-safety/labsafety_web.pdf. Accessed on 20th May 2019.
Laboratory Safety Manual. https://case.edu/cse/eche/daigroup/Lab%20Safety%20Manual.pdf. Accessed
on 20th May 2019.
Neilson AP, O’Keefe SF. Dilutions and concentrations. In: Food analysis laboratory manual. Cham:
Springer International Publishing; 2017. p. 33–47.
Neilson AP, Lonergan DA, Nielsen SS. Laboratory standard operating procedures. In: Food analysis
laboratory manual. Cham: Springer International Publishing; 2017. p. 3–20.
Patra JK, Das SK, Das G, Thatoi HN. General aspects of pharmacology laboratory. In: A practical
guide to pharmacological biotechnology. Learning materials in biosciences. Singapore: Springer;
2019.
Seiler JP. What is good laboratory practice all about? In: Good laboratory practice — the why and the
how. Berlin, Heidelberg: Springer Berlin Heidelberg; 2005a. p. 1–57.
Seiler JP. How is good laboratory practice regulated? In: Good laboratory practice — the why and the
how. Berlin, Heidelberg: Springer Berlin Heidelberg; 2005b. p. 59–358.
Thatoi HN, Dash S, Das SK. Practical biotechnology, principle and protocols. New Delhi: I. K.
International Pvt. Ltd; 2017.
Tyl C, Ismail BP. Preparation of reagents and buffers. In: Food analysis laboratory manual. Cham:
Springer International Publishing; 2017. p. 21–32.
37 2
Water Quality Analysis

Contents
2.1 Electrical Conductivity of Waste Water – 38
2.2 Alkalinity of Waste Water – 40

2.3 Dissolved Oxygen Content in Water Samples – 43
2.4 Biochemical Oxygen Demand in Water Samples – 46
2.5 Chemical Oxygen Demand in Water Samples – 49
2.6 otal Suspended Solids (TSS) and Total Dissolved Solids
T
(TDS) in Water Samples – 52
2.6.1 Total Dissolved Solids (TDS) – 52
2.7 Chloride Content in Water Samples – 53
2.8 ulfate Content in Water Samples (Turbidimetric

S
Method) – 54
2.9 Nitrogen Content in Water Samples – 56

38 Chapter 2 · Water Quality Analysis

In this chapter, you will learn about the easy-to-use protocols for determining electrical
conductivity, alkalinity of waste water, dissolved oxygen content, biochemical oxygen
2 demand, chemical oxygen demand, total suspended solids and total dissolved solids, chlo-
ride content, sulfate content, and nitrogen content in the water samples. You will also learn
about how to calculate and interpret the results.
2.1 Electrical Conductivity of Waste Water

zz Theory
Electrical conductivity is a material’s ability (water or any other liquid) to accom-
modate the transportation of an electric charge to conduct an electric current. The
standard international derived unit for the measurement of electrical conductiv-
ity of water is Siemens per meter or Siemens per centimeter (named after Werner
von Siemens) or, more simply, S.m−1 or S.cm−1. The electrolytes present in a solu-
tion generally separate into both positive (cations) and negative (anions) ions and
impart conductivity. Normally, salts or other types of chemical constituents that
are present in water in dissolved form are broken down into both positively or nega-
tively charged ions; these free ions are accountable for the conduction of electricity
in the water sample. At constant temperature, the electrical conductivity of water is
equivalent to the concentration of ions present in it. It is influenced by the tempera-
ture of the water also. Thus, the higher the concentration of electrolytes present in
the water, the more the electrical conductivity. Conductivity affects the occurrence
of various aquatic plants and animals in the water; some types of aquatic organ-
isms are sensitive to the increased conductivity in water, while some are tolerant to
it. It is an indicative parameters to determine the increase in pollution in the water
system. Distilled water is an insulator as it almost does not conduct any electric-
ity, whereas salt water is a good conductor of electricity. The majority of ions that
are responsible for high electrical conductivity in water are sodium, magnesium,
calcium, potassium, etc., whereas ions like chloride, carbonate, bicarbonate, and
sulfate are negative conductors.
zz Materials Required
Glassware, such as measuring cylinder, beaker, conical flask, and glass pipette, and
instruments, such as conductivity meter, conductivity cell, thermometer, and differ-
ent water samples, are taken for analysis
2.1 · Electrical Conductivity of Waste Water
39 2
.. A sample conductivity meter setup
zz Procedure
1. The procedure for testing the electrical conductivity of any water sample largely
depends upon the type and quality of the instrument used. However, common
procedure is as follows.
2. Carefully study the operational manual of the conductivity meter and then regu-
late the temperature compensation knob of the conductivity meter equivalent to
the temperature of the sample.
3. Keep the selector switch to ×1000 and calibrate to CAL (calibration) mark. Dip
the conductivity cell in the sample contained in a beaker and connect the cell
terminals to the sockets provided in the instrument. If the meter shows negligible
deflection, disconnect the cell terminal. Move the selector switch to ×100 and
calibrate to CAL mark.
4. Reconnect the cell terminal and note the deflection. If it is still negligible, discon-
nect the cell, move the selector switch to ×10, and calibrate to CAL mark. Recon-
nect the cell and note the deflection (dial reading).
5. Switch off the cell after the use and wash it with distilled water.
6. However, there are different protocols for different types of instruments used for
the analysis of electrical conductivity.
zz Results
Sl. no Temperature compensation knob Selector switch Deflection
2 1.
2.
3.
4.
zz Calculation
EC ( S) = DR × SS
Where ES is the electrical conductivity; DR is the dial reading; and SS is the value
of selector switch.
2.2 Alkalinity of Waste Water
(Carbonate, bicarbonate, hydroxide, and total alkalinity of water samples)
zz Theory
The alkalinity of water is sometimes referred to as the acid-neutralizing ability of the
water at a given circumstance. It is the capability of the water sample to neutralize a
strong acid or resist the changes in the pH value of the water with increasing concen-
tration of the acids in the solution. It is characterized by the existence of hydroxyl
(OH−) ions capable of coalescing with hydrogen (H+) ions. A number of bases such
as hydroxides, carbonates, bicarbonates, phosphates, nitrates, silicates, and borates
add up to the alkalinity of a solution and are therefore stated as the total alkalinity
or alkalinity due to distinct bases. It is stated that the higher the alkalinity of the
water, the greater its ability to neutralize the acids. The fundamental procedure for
the measurement of water alkalinity is observing the changes in the pH value by
adding an acid drop by drop to the water. In general, the pH of natural waters is
in the range of 6–9, while most of the surface water has a pH that ranges from 7.5
to 8.0. However, water found in the deserts or sandy area has acidic pH. In case of
the natural water, most of the alkalinity is triggered due to the presence of carbon
dioxide (CO2). The free CO2 usually combines with the water sample partly in order
to form carbonic acid (H2CO3), which is further dissociated into hydrogen (H+) and
bicarbonate (HCO3−) ions. The HCO3− ions thus formed further dissociate into H+
and carbonate (CO3−) ions.
CO 2 + H 2 O  H 2 CO3
H 2 CO3  HCO3− + H +
HCO3−  CO3− + H +
2.2 · Alkalinity of Waste Water
41 2
The carbonate and bicarbonate ions present in the water samples further yield
hydroxyl (OH−) ions that increase the pH of the water sample. In highly productive
waters, the alkalinity is ought to be over 100 mg/L.
zz Materials Required
Laboratory glassware such as measuring cylinder, beaker, conical flask, pipette,
burette, and burette stand and specific chemicals such as sulfuric acid, phenolphtha-
lein indicator, and methyl orange indicator are required.
1. Sulfuric acid (0.02N): About, 2.8 mL of concentrate sulfuric acid is diluted to
1 L using distilled water. Further, 200 mL of this stock solution (0.1N) is diluted
to 1 L using distilled water to prepare 0.02N sulfuric acid titrant. The reagent is
standardized prior to use.
2. Phenolphthalein indicator: About. 1 g of phenolphthalein is dissolved in 100 mL
of ethyl alcohol and then 100 mL of distilled water is added to it and mixed prop-
erly. Then, NaOH solution (0.22N) is added to it drop by drop till a faint pink
color appears.
3. Methyl orange indicator: About 0.1 g of methyl orange is dissolved in 200 mL of
distilled water and the solution is mixed properly.
.. A complete setup for titration process for carrying out the alkalinity test
zz Procedure
1. Different types of water samples are collected away from the shore and at least
1 feet under the surface of water in a clean glass container and immediately trans-
2 ferred to the laboratory for further analysis.
2. Take about 50 mL of the water sample in a conical flask and to it add 2–3 drops
of phenolphthalein indicator and mix properly.
3. If the solution becomes slight pinkish in color, then it is due to phenolphthalein
alkalinity (due to hydroxide or carbonate present in the water sample). If a pink
color does not appear in the water sample after the addition of phenolphthalein
indicator, then it is due to the presence of free CO2 but absence of phenolphtha-
lein alkalinity.
4. Then, titrate the water solution against sulfuric acid until the solution becomes
colorless to attain the end point.
5. Note the reading as “p” and further add 2–3 drops of methyl orange indicator
in the same flask and then continue to titrate against the remaining sulfuric acid
until yellow color of the solution turns to orange color to attain the end point.
6. Then, immediately stop the titration process and then note the reading as “t”
which is the volume of titrant used for both the titration.
7. Calculate the alkalinity of the sample according to the following formula.
zz Tabulation
Sl. no Volume of sample used Phenolphthalein alkalinity (p) Total volume of Mean
titrant used
IBR FBR
1.
2.
3.
IBR initial burette reading, FBR final burette reading
zz Calculation
Phenolphthalein alkalinity
P × 10000
Phenolphthalein alkalinity ( as calcium carbonate in mg/L ) =
S
Total alkalinity
T × 1000
Total alkalinity ( as calcium carbonate in mg/L ) =
S
Where, P is the volume of the titrant used against the phenolphthalein indicator in
mL; S is the volume of the sample in mL; and T is the total volume of the titrant used
for both the titrations in mL.
2.3 · Dissolved Oxygen Content in Water Samples
43 2
The value of the different forms of alkalinity (hydroxide, carbonate, and bicar-
bonate) in the water samples in terms of CaCO3 (mg/L) can be calculated as follows:
Different forms of alkalinity in terms of calcium carbonate
Forms Hydroxide Carbonate Bicarbonate
P=0 0 0 T
P < 0.5T 0 2P T-2P
P = 0.5T 0 2P 0
P > 0.5T 2P-T 2(P-T) 0
P=T T 0 0
The concentrations of various ions such as OH−, CO3−, and HCO3 in the water sam-
ple are calculated as follows:
Hydroxide ions in mg/mL = Valueof alkalinity of hydroxide × 0.34
Carbonate ions in mg/mL = Valueof alkalinity of carbonate × 0.60
Bicarbonate ions in mg/mL = Valueof alkalinity of bicarbonate × 1.22
2.3 Dissolved Oxygen Content in Water Samples

zz Theory
The quantity of oxygen present in water is termed as dissolved oxygen (DO) concen-
tration of the water. It is extremely inclined by the water temperature; for example, the
colder the water (lower the temperature), the more oxygen it can hold, since gases like
oxygen are more easily dissolved in cool water. The concentration of dissolved oxygen
is always lower during the night than during the day because the water is much cooler
during the nighttime than during the daytime. The quantity of dissolved oxygen plays
an imperative role in defining the type of organisms that live in the water. It is mea-
sured by using chemical analysis methods such as Winkler’s titration method.
zz Principle
Estimation of dissolved oxygen content in the water sample is a vital factor for deter-
mining the extent of pollution caused by waste materials in water samples. DO is
also essential for all types of aerobic natural wastewater treatment procedures. The
modified Winkler test for dissolved oxygen involves oxidation of Mn(II) to Mn(IV)
state. The Mn(IV) will then oxidize iodide to iodine. The synthesized iodine is then
titrated against sodium thiosulfate with starch as an indicator. In the fresh water, the
atmospheric oxygen solubility ranges from 14.6 mg/L at 0 °C to about 7.0 mg/L at
35 °C under one atmospheric pressure.
The reactions involved are as follows:
zz Reactions
2 MnSO4 ( solid ) + O 2 ( aqueous ) → 2 MnO ( OH )2 ( solid )

2 4 Mn ( OH )2 ( solid ) + O 2 ( aqueous ) + 2 H 2 O → 4 Mn ( OH )3 ( solid )
Mn ( SO 4 )2 + 2 I − ( aqueous ) → Mn 2 + ( aqueous ) + I 2 ( aqueous ) + 2 SO 4 2 − ( aqueous )
2 S2 O32 − ( aqueous ) + I 2 → S4 O6 2 − ( aqueous ) + 2 I − ( aqueous )
.. A setup for estimating dissolved oxygen in water sample
zz Requirements
Glassware such as measuring cylinder, beaker, dissolved oxygen specific bottle, ther-
mometer, pipette, burette, burette stand, and chemicals and reagents.
1. Alkaline iodine: About 500 g of sodium hydroxide and 150 g of potassium iodide
are dissolved in about 350 mL of distilled water separately in two beakers. Sodium
hydroxide solution is allowed to cool down and then both the solutions are mixed
2.3 · Dissolved Oxygen Content in Water Samples
45 2
together. Then 10 g of sodium azide (NaN3) is dissolved in 40 mL of distilled
water in another beaker and then it is added to the previous mixture and the
whole mixture solution is made up to 1 L.
2. Manganese sulfate (MnSO4): This reagent can be prepared using different forms
of manganese sulfate as stated below.
About 480 g of MnSO4·4H2O or 400 g of MnSO4·2H2O or 365 g of
MnSO4·H2O is dissolved in 750 mL of distilled water and then boiled if required
and the final volume of the solution is made up to 1 L with distilled water.
3. Sodium thiosulfate (0.025 M): About 6.205 g of sodium thiosulfate (NaSO3·5H2O)
is taken in a conical flask containing distilled water. To it, 0.4 g NaOH (solid) is
added and the final volume is made up to 1 L with distilled water.
4. Starch solution: To prepare starch solution, 2 g of soluble starch is dissolved in
100 mL of hot distilled water, mixed properly, and kept in a reagent bottle. For
long-term preservation of the solution, 0.2 g of salicylic acid is added to it.
zz Procedure
1. Water sample were collected in speacially made Biochemical Oxygen Demand
(BOD) bottles during day time at a depth of 1 foot deep.
2. Prudently fill the 300 mL glass biochemical oxygen demand (BOD) stoppered
bottles with the sample water. Care should be taken that no water bubble is left
inside the bottle.
3. Add 2 mL of manganese sulfate solution immediately to the sample collection
bottle by injecting a pipette just underneath the surface of the water sample.
4. If the manganese sulfate solution is added above the surface of the water sample,
then extra oxygen will enter into the water sample. Then the pipette is squeezed
slowly so that no air bubbles enter the sample bottles through the pipette.
5. Then add 2 mL of alkali-iodide-azide reagent solution in the same manner into
the water sample bottles.
6. Stopper the bottles containing the water samples carefully so that no additional
air enters into it.
7. Then, mix the chemicals in the water sample bottles carefully by overturning it
3–4 times.
8. The bottle is then checked for the presence of any more air bubbles. If still it
contains air bubble, then discard it and start with fresh sample repeating the steps
from the beginning.
9. If extra oxygen is present in the water sample bottles, then a brownish-orange
cloud of precipitate will appear inside the bottle. When this precipitation slowly
settles down to the bottom of the bottle, mix the water sample again by whirling
it upside down 3–4 times and then leave it to settle down again.
10. Then, add 2 mL of the concentrated sulfuric acid into the BOD bottles contain-
ing the water samples with the help of a glass pipette just above the surface of the
water sample.
11. Then, the stopper is carefully fixed and the sample is mixed by making the bottle
upside down 3–4 times in order to dissolve the precipitation formed.
12. At this point, the water sample and the dissolved oxygen present in it are fixed
and the samples can be stored for up to 8 h at this stage in a refrigerator.
13. The BOD bottles containing fixed water samples can be wrapped tightly with
aluminum foil to avoid unnecessary damage to the water sample while storing.
14. To analyze the DO content in the water sample, required amount of the fixed
water sample is taken in a glass flask and titrated against sodium thiosulfate
using a titration setup as shown in the figure above.
2 15. The titration process was initiated by recording the initial burette reading fol-
lowed by addition of few drops of starch solution so that a blue color is formed.
16. Then the titration process is continued with slowly dropping the titrant solution
from the burette into the flask continuously with continuous stirring of the water
sample till it reaches the end point of clear and colorless solution.
17. When the titration process approaches the end point, it will take only one more
drop of the titrant to remove the blue color.
18. Utmost care should be taken in the titration process – the titrant should be added
slowly, drop by drop to the water sample, and it should be mixed properly, before
adding the next drop to the solution.
19. For better visualization of the color change, a white paper sheet can be placed
just below the conical flask containing the water sample.
20. When the sample reaches the end point, the final burette reading is recorded.
21. The concentration of the dissolved oxygen present in the water sample is calcu-
lated using the following formula.
zz Calculation
The dissolved oxygen concentration can be calculated based on the amount of titrant
added to the sample. The formula used is:
( FBR − IBR ) × N × 8000

DO =
Sample volume
Where, IBR is the initial burette reading; FBR is the final burette reading; N is the
normality of the manganese sulfate solution. Sodium thiosulfate solution is used to
titrate the sample.
zz Tabulation
Sl. no Volume of the water sample used in mL Volume of the titrant used in mL
IBR FBR
1.
2.
3.
2.4 Biochemical Oxygen Demand in Water Samples

zz Theory
Biochemical oxygen demand (BOD) is defined as the rate of removal (i.e., con-
sumption) of oxygen distributed in the form of dissolved oxygen or even particulate
organic matter in the water bodies by microorganisms through aerobic degradation.
BOD is used as an indicator of serious organic pollution in the water bodies. BOD
2.4 · Biochemical Oxygen Demand in Water Samples
47 2
is estimated by determining the concentration of dissolved oxygen present in the
water sample before and after incubation period for 5 days in dark and at 20 °C
temperature. When the BOD of water increases and exceeds the available DO, the
DO is gradually reduced or exhausted, which is very much harmful to the aquatic
organisms and sometimes leading to the death of aquatic fish and other organisms.
zz Requirements
1. BOD incubators, BOD bottles, laboratory glassware
2. Alkaline iodine: About, 500 g of sodium hydroxide and 150 g of potassium iodide
are dissolved separately in 350 mL of distilled water in two different beakers. The
sodium hydroxide solution was allowed to cool down to room temperature and
then both the mixture solutions are mixed together. Then dissolve 10 g of sodium
azide (NaN3) in 40 mL of distilled water and this solution is mixed with the previ-
ous two solutions and the whole content is made up to 1 L with distilled water.
3. Manganese sulfate (MnSO4): This reagent can be prepared using different forms
of manganese sulfate as stated below.
About 480 g of MnSO4·4H2O or 400 g of MnSO4·2H2O or 365 g of
MnSO4·H2O is dissolved in 750 mL of distilled water; it is then boiled if required
and the final volume of the solution is made up to 1 L with distilled water.
4. Sodium thiosulfate (0.025 M): About 6.205 g of sodium thiosulfate (NaSO3·5H2O)
is taken in a conical flask containing distilled water; to it 0.4 g NaOH (solid) is
added and the final volume is made up to 1 L with distilled water.
5. Starch solution: To prepare starch solution, 2 g of soluble starch is dissolved in
100 mL of hot distilled water, mixed properly, and kept in a reagent bottle. For
long-term preservation of the solution, 0.2 g of salicylic acid is added to it.
6. BOD-free water: Deionized distilled water is passed through the activated carbon
column and then redistilled again.
7. Phosphate buffer solution: About 33.4 g of Na2HPO4·7H2O, 21.75 g of K2HPO4,
8.5 g of KH2PO4, and 1.7 g of NH4Cl are dissolved properly in about 500 mL of
distilled water and the total volume is made up to 1 L with distilled water and the
pH is adjusted to 7.2.
8. Calcium chloride solution: About 27.5 g of CaCl2 is dissolved in distilled water
and the total volume is made up to 1 L.
9. Ferric chloride solution: About 0.25 g of FeCl3·6H2O is dissolved in distilled
water and the total volume is made up to 1 L.
10. Sulfuric acid (1N): About 2.8 mL of concentrated sulfuric acid is slowly added to
100 mL of distilled water and mixed properly.
11. Sodium hydroxide solution (1N): About 4 g of NaOH is mixed in 20 mL of dis-
tilled water and the total volume is made up to 100 mL.
12. Allylthiourea solution: About 500 mg of allylthiourea is dissolved in about
1000 mL of distilled water.
zz Procedure
1. For the preparation of dilution water, aerate the BOD-free water samples in a
large-size conical flask for about 30 min.
2. Add 1 mL each of phosphate buffer solution, magnesium sulfate solution, cal-
cium chloride solution, and ferric chloride solution per liter of the dilution water
solution.
3. Adjust the pH of sample to 7.0, using sulfuric acid or sodium hydroxide as per the
requirement.
4. For exhaustion of all the oxygen during the incubation process, the samples are
2 diluted with dilution water at the rate as shown in the table below.
5. Two sets of BOD bottles are filled with the above-prepared water samples and to
them 1 mL of allylthiourea solution is added.
6. The dissolved oxygen content (DO) in one set is determined immediately follow-
ing Wrinkle’s method and the other set of the BOD bottle is incubated at 20 °C
for 5 days in a BOD incubator.
7. The dissolved oxygen content of the stored bottle is determined after 5 days of
incubation.
8. Calculate the BOD according to the following formula.
.. A setup for estimating biochemical oxygen demand in water sample

2.5 · Chemical Oxygen Demand in Water Samples
49 2
Table for dilution of samples as required for various ranges of expected BOD values
Expected BOD value in mg/L Volume of the water sample Dilution factor
taken in mL
0–6 1000 1
4–12 500 2
10–30 200 5
20–60 100 10
40–120 50 20
100–300 20 50
200–600 10 100
zz Calculation
BOD5 = ( D0 − D5 ) × Dilution factor
Where D0 is the initial dissolved oxygen content in the sample in mg/L at 0 day of
incubation and D5 is the dissolved oxygen content left out in the sample after 5 days
of incubation.
Table for determination of DO
Sl. no Volume of sample used in mL Volume of titrant used in mL
IBR FBR
1.
2.
3.
2.5 Chemical Oxygen Demand in Water Samples

zz Theory
For the evaluation of the quality of the water, it is crucial to know the quantity
of oxidizable organic substances present in it. It is usually determined by three
different aspects which are: (1) estimation of total organic carbon content, (2) esti-
mation of dissolved organic carbon contents, and (3) estimation of the chemical
oxygen demand (COD) of the water sample. COD is defined as the measurement of
dissolved oxygen required to oxidize the organic compounds present in the water
samples. During the estimation of COD, chemicals such as potassium dichromate
and potassium permanganate are used. In simplified words, it is the quantity of
dissolved oxygen abridged by the action of chemical reactions in the water sample.
The estimation of COD in water samples is of utmost importance with respect to
the water samples which have adverse growth of microorganisms and presence of
2 highly toxic chemicals and where the estimation of the BOD of the water samples
is not accurate.
.. A COD reflux unit for COD estimation
zz Requirements
1. COD reflux unit: It consists of 250 mL/500 mL Erlenmeyer flasks with ground
glass.
2. 30 cm straight jacket Liebig condenser with 24/40 ground-glass joint (30 cm).
3. Hot plate water bath.
4. Potassium dichromate solution (0.25N): About 12.259 g of K2Cr2O7, previously
dried at 150 °C for 2 h, or dried powder is dissolved in 500 mL of distilled water
and then the final volume is made up to 1 L.
5. Silver sulfate: dried and powdered.
6. Mercuric sulfate (HgSO4): crystals or powder.
7. Sulfuric acid: concentrated.
8. Ferroin indicator solution: About 0.695 g of ferrous sulfate and 1.485 g of 1,
10-phenonthorline are dissolved in 50 mL of distilled water and the volume is
made up to 100 mL.
2.5 · Chemical Oxygen Demand in Water Samples
51 2
9. Ferrous ammonium sulfate solution (0.25N): About 98 g of Fe(NH4)2(SO4)2·6H2O
is dissolved in 500 mL of distilled water and to it 20 mL of concentrated H2SO4
is slowly mixed, and the mixture solution is cooled and diluted to 1000 mL with
distilled water.
Further, for the standardization of the solution, 25 mL of potassium dichro-
mate solution is taken in a conical flask and the volume is made up to 250 mL
with distilled water followed by addition of 90 mL of concentrated sulfuric acid
and cooled. Then 5–6 drops of ferroin indicator solution are added to it and
titrated against ferrous ammonium sulfate solution. The color changes from blue
green to reddish blue at the end point.
zz Procedure
1. 20 mL of the water sample is taken in a flask of efflux unit and to it 10 mL of
potassium dichromate solution is added, followed by a pinch of silver sulfate and
mercuric sulfate, along with 30 mL of sulfuric acid.
2. The Liebig condenser is then attached to the mouth of the flask and kept on a hot
water bath or a heating mantle for about 2 h to reflux the contents.
3. Afterward, the flask is allowed to cool, detached from the unit, and its content is
diluted to about 150 mL by adding distilled water.
4. Further, 2–3 drops of ferroin indicator solution are added to it and titrated
against ferrous ammonium sulfate solution.
5. Change in color of the reaction mixture from blue green to reddish blue is the end
point.
6. Distilled water blank is run simultaneously in a similar manner and the blank is
adjusted.
Table for determination of COD
Sl. no Volume of sample Volume of the titrant used Volume of the titrant used
used in mL against the sample in mL against the blank in mL
IBR FBR IBR FBR
1.
2.
3.
zz Calculation
( Z − Y ) × N ×1000 × 8
COD =
V
Where Y is the volume of the titrant used against the water sample in mL; Z is the
volume of the titrant used against the blank in mL; N is the normality of the titrant
(0.25); and V is the volume of the water sample in mL.
2.6 otal Suspended Solids (TSS) and Total Dissolved Solids (TDS)
T
in Water Samples
2 2.6.1 Total Dissolved Solids (TDS)
zz Theory
A huge amount of salts including calcium, iron, magnesium, manganese, potassium,
chlorides, carbonates, bicarbonates, nitrates, phosphates, sodium, and sulfate are
found in dissolved form in natural water. A higher amount of these dissolved sub-
stances present in the water samples results in the elevation of water density, thereby
influencing the osmoregulation of freshwater organisms and also reducing the solubil-
ity of the gases and finally the usability of water for drinking, irrigation, and industrial
purposes. It is an important parameter used to in analyze saline lakes, estuarine, etc.
zz Requirements
Glassware, such as beaker, measuring cylinder, glass pipette, conical flask, and evap-
orating dish, and instruments, such as weighing balance, desiccator, hot water bath,
and filter paper, are required
.. Evaporating disc used for TDS analysis
zz Procedure
1. Take a clean and dry evaporating dish of suitable size and pre-weigh it before use.
2. Filter out about 250–500 mL of the selected water sample using a filter paper
(Whatman No. 4) and take the filtrate in the pre-weighed evaporating dish.
3. Vaporize the whole water sample by placing the evaporating dish containing the
required water sample on the hot water bath.
4. After complete drying of the water samples on the evaporating dish, the con-
tainer is allowed to cool to room temperature inside the desiccator and then its
final weight is measured in a weighing balance.
5. Calculate the amount of the total dissolved solids present in the water sample
using the following formula.
2.7 · Chloride Content in Water Samples
53 2
zz Calculation
( A − B)
TDS = × 100
V
Where TDS is the total dissolved solids of the water sample; A is the final weight of
the evaporating dish after complete drying of the water samples; B is the initial pre-
weight of the evaporating dish before putting the water sample in it, and V is the total
volume of the water sample taken for the analysis.
Note: if total solids, instead of total dissolved solids, are to be computed, do not
filter the sample.
2.7 Chloride Content in Water Samples

zz Theory
Chloride is a major ion present in both salt and natural water. It is formed from the
dissociation of sodium and calcium salts. In normal fresh water, the higher amount of
chlorides content is taken as an indicator of pollution due to the presence of organic
waste of animal origin (animal excreta contain high amount of chloride and nitrogenous
wastes) in the water samples. Industrial effluents may also increase the chloride content in
natural waters. The measured chloride ions can be utilized as an indicator of salinity for
various water bodies. If the water samples comprising chlorides are titrated against silver
nitrate, then the chlorides present in water usually precipitate to white color silver chloride.
Potassium chromate solution which is used as an indicator for the experiment basically
reacts with the silver chloride to form silver chromate which is reddish brown in color.
zz Requirements
1. Laboratory glassware such as beaker, conical flask, burette, burette stand, glass
pipette, measuring cylinder, and reagents.
2. Silver nitrate solution (0.025N): About 3.397 g of silver nitrate is dissolved in
distilled water and the final volume is made up to 1 L with distilled water. The
solution is stored in dark glass bottles in order to avoid reaction with light.
3. Potassium chromate indicator: About 10 g of potassium chromate is dissolved in
about 20 mL of distilled water and to it few drops of 0.025N silver nitrate solu-
tion are added in order to produce a red precipitate. It is allowed to stand for 12 h,
filtered, and then further diluted to 1 L with distilled water.
4. Sodium chloride (0.02N): About 1.170 g of sodium chloride is dissolved in
900 mL of distilled water and the volume is made up to 1 L in distilled water.
zz Procedure
1. Take 10 mL of the water sample in a conical flask and to it add 5–6 drops of
potassium chromate indicator and mix properly. The color of the water sample
will become yellow.
2. Titrate the water samples against silver nitrate solution using burette setup until a
persistent brick red color appears which is the end point of the reaction.
3. Care is taken that the silver nitrate solution is added to the water sample drop by
drop, and after addition of each drop, the water sample is mixed properly.
4. A white sheet is placed below the conical flask containing the water sample in the
burette setup in order to better visualize the color change.
5. Calculate the concentration of chloride in the water sample using the following
2 formula.
Table for chloride content
Sl. no Volume of the water sample taken for Volume of the silver nitrate titrant used against
the experiment in mL the water sample in mL
IBR FBR
1.
2.
3.
zz Calculation
(V × N × 35.475 ×1000 )
Chloride content in mg / mL =
S
Where, V is the total volume of the titrate used in mL, N is the normality of the
titrant (0.02), 35.475 is the equivalent weight of chlorine, and S is the volume of
sample in mL.
2.8 Sulfate Content in Water Samples (Turbidimetric Method)

zz Theory
Sulfate is present in higher quantity in natural water and soil samples, more significantly
where the salt content is more. These are completely soluble in the water providing hard-
ness to it. Domestic sewage and industrial effluent, besides biological oxidation, may
add to the sulfate content of the water and soil. It is the second largest amount of min-
eral content present in the seawater. However, they are considered to be a serious con-
cern to the water bodies as they are reduced to form hydrogen sulfide under the absence
of oxygen followed by increase in odor and corrosion. Determination of sulfate content
in the sample is based on the precipitation of sulfate after adding barium chloride.
zz Requirements
1. Laboratory glassware, test tube, flask, beaker, measuring cylinder, spectropho-
tometer, magnetic stirrer, filter paper (Whatman No. 1).
2. NaCl–HCl solution: About 250 g of sodium chloride is dissolved in a little dis-
tilled water, followed by addition of 20 mL of HCl to it, and then the total volume
is made up to 1 L with distilled water.
3. Glycerol–ethanol solution: About 50 mL of glycerol is added to 100 mL of ethyl
alcohol and shaken well in a reagent bottle.
4. Barium chloride: Dry crystals of barium chloride are used for the experiment.
2.8 · Sulfate Content in Water Samples (Turbidimetric Method)
55 2
5. Standard sulfate solution: About 0.147 g of anhydrous sodium sulfate is dis-
solved in distilled water and the total volume is made up to 1 L with distilled
water. Standards of various concentration 0, 5, 10, 15, 20, 25, 30, 35, and 40 mg/L
are prepared with the stock solution.
zz Procedure
1. The water sample to be tested is filtered through a filter paper and from it 50 mL
is taken in an Erlenmeyer flask.
2. To it, 10 mL of NaCl–HCl solution, 10 mL of glycerol–ethanol solution, and
0.15 g of barium chloride are added.
3. Stir the sample for about an hour with the help of a magnetic stirrer.
4. Measure the absorbance(s) of the sample at 420 nm using a spectrophotometer
against the distilled water blank.
5. Make a standard curve, taking different concentration of standard sulfate solu-
tion in similar manner.
6. Plot a standard graph with strength (mg/L) on one axis and absorbance on the
other and calculate the sulfate content of the unknown water sample from the
standard graph.
1.8
1.6
1.4
1.2
Absorbance values
1.0
0.8
0.6
0.4
0.2
0
0 5 10 15 20 25 30 35 40
Concentration of standard Sulphate
.. A sample standard calibration curve for understanding
Table for determination of sulfate content in water sample
Sl. no. Sample of different strength in mg/L Absorbance at 420 nm
1.
2.
3.
4.
5.
2.9 Nitrogen Content in Water Samples

zz Theory
2 Presence of total nitrogen content in the water sample is considered as a chemical
evidence of occurrence of current organic pollution due to the animals in the water
body. Estimation of nitrogen in various forms in the water is carried out to assess its
bacteriological quality.
The process of analyzing nitrogen content is based on the principle, that in the
presence of sulfuric acid and mercuric sulfate, catalyst organically bound to nitrogen
and gets converted into ammonium sulfate. The ammonia that then gets liberated is
absorbed by boric acid and the absorbed ammonia is then estimated either by titra-
tion process or by direct Nesslerization process.
zz Requirements
1. Glassware like beaker, measuring cylinder, conical flask, and glass pipette; instru-
ments like digestion apparatus which consists of a Kjeldahl flask (with a capacity
of 500 or 800 mL), still head along with a provision of suction of fumes and a
heating device, a complete distillation apparatus with a condenser and collector,
Nessler tubes (100 mL capacity), and UV-visible spectrophotometer.
zz Reagents
1. Preparation of digestion reagent
About 134 g of K2SO4 is dissolved in 650 mL distilled water followed by addi-
tion of 200 mL of concentrated H2SO4. Further, a solution of 2 g of HgO in
25 mL of 6N H2SO4 is also added to it with continuous stirring. Finally the whole
solution is made up to 1000 mL with distilled water and stored at room tempera-
ture (around 25 °C or higher) to prevent crystallization.
2. Phenolphthalein indicator: About 1 g of phenolphthalein is dissolved in 100 mL
of ethyl alcohol and to it 100 mL of distilled water is added. Further NaOH solu-
tion (0.22N) is added drop by drop till a faint pink color appears.
3. Sodium hydroxide–sodium thiosulfate reagent: About 500 g of NaOH and 25 g
of Na2S2O3 are dissolved and diluted to make up the volume to 1000 mL.
4. Mixed indicator: About 200 mg of methyl red is dissolved in 100 mL of 95% ethyl
alcohol in a beaker and 100 mg of methylene blue is dissolved in 50 mL of 95%
ethyl alcohol in another beaker. Finally the two solutions are mixed properly.
5. Boric acid: About 20 g of H3BO3 is dissolved in distilled water and to it 10 mL
of the mixed indicator is added and the total volume is made up to 1000 mL with
distilled water.
6. Sodium hydroxide 6N: About 240 g of NaOH is dissolved in 1 L of ammonia-free
distilled water.
7. Standard sulfuric acid: About 2.8 mL of concentrated sulfuric acid is diluted
to 1 L with distilled water. And 200 mL of this stock solution (0.1N) is further
diluted to 1 L using distilled water to prepare 0.02N sulfuric acid titrant. Stan-
dardize the solution.
2.9 · Nitrogen Content in Water Samples
57 2
zz Procedure
A. Digestion Process
1. Take appropriate volume of well-mixed water sample in a Kjeldahl flask.
2. Add 50 mL of the digestion reagent to the water sample. If the concentration
of the suspended solids is high, then large amount of the reagents are added
to it to maintain the salt to acid ratio as 0.8.
3. Digestion process is continued for around 30 min till the sample appears to be
clear to ensure complete decay/destruction of the organic matter.
4. Then it is cooled to the room temperature and diluted with distilled water to
150 or 300 mL depending on the capacity of the flask.
B. Distillation Process
1. Place the conical flask containing the digested water samples in an appropriate
position in the distillation apparatus and turn on the heat.
2. Then to it, around 0.5 mL of the phenolphthalein reagent is added followed
by addition of sodium hydroxide–sodium thiosulfate reagent till the pH of the
mixture solution rises just above 8.3.
3. Then it is distilled and around 200 mL of the distillate is collected in a 500 mL
Erlenmeyer flask containing 50 mL of boric acid solution. Extend the tip of
the condenser well below the level of boric acid solution.
4. When the distillation process is complete, the flask containing the distillate
is removed first and then the heat system is put off in order to avoid back
suction.
5. The concentration of ammonia is measured by the process of Nesslerization.
The distillate is titrated with 0.02N H2SO4 till the indicator turns to a pale
lavender color.
6. A blank is run throughout the procedure in all steps to make necessary
correction.
zz Calculation
( A − B ) × 280
Organic N =
Sample ( mL )
Where A is the mL of 0.02N H2SO4 required for the sample and B is the mL of 0.02N
H2SO4 required for the reference blank.
Take-Home Messages
55 Electrical conductivity is a material’s ability (water or any other liquid) to accom-
modate the transportation of an electric charge to conduct an electric current.
55 The alkalinity of water is referred to as the acid-neutralizing ability of the water
at a given circumstance.
55 In highly productive waters, the alkalinity is ought to be over 100 mg/L.
55 The quantity of oxygen present in water is termed the dissolved oxygen (DO) con-
centration of the water.
55 The quantity of dissolved oxygen plays an imperative role in defining the type of
organisms that live in the water.
2 55 Biochemical oxygen demand (BOD) is defined as the rate of removal (i.e., con-
sumption) of oxygen distributed in the form of dissolved oxygen or even par-
ticulate organic matter in the water bodies by microorganisms through aerobic
degradation.
55 For evaluating the quality of the water, it is crucial to know the quantity of oxidiz-
able organic substances present in it.
55 The estimation of COD in the water samples is of utmost importance with respect
to the water samples which have adverse growth of microorganisms and presence
of highly toxic chemicals and where the estimation of the BOD of the water sam-
ples is not accurate.
55 Total dissolved solid is an important parameter used to analyze the salinity of
lakes estuarine, etc.
55 Chloride is a major ion present in both salt and natural water.
55 Sulfate is present in higher quantity in natural water and soil samples, more signifi-
cantly where the salt content is more.
55 Presence of total nitrogen content in the water sample is considered as a chemical
evidence of occurrence of current organic pollution due to the animals in the water
body.

AWWA, WEF, APHA. Standard methods for the examination of water and wastewater. Washington,
DC: American Public Health Association; 1998.
Bisen PS. Laboratory protocols in applied life sciences. Boca Raton: CRC Press; 2014.
Bureau of Indian Standards. Indian standard drinking water – specification (IS 10500). New Delhi:
Bureau of Indian Standards; 2012. http://cgwb.gov.in/Documents/WQ-standards.pdf. Accessed
3 June 2019.
Dunnivant FM. Environmental laboratory exercises for instrumental analysis and environmental chem-
istry. Hoboken: Wiley-Interscience; 2004.
Fresenius W, Quentin KE, Schneider W. Water analysis. Berlin, Heidelberg/New York: Springer-Verlag;
1988. ISBN-13: 978-3-642-72612-5.
FSSAI. Manual of methods of analysis of foods – water. New Delhi: Food Safety and Standards
Authority of India, Ministry of Health and Family Welfare, Government of India; 2015. https://
old.fssai.gov.in/Portals/0/Pdf/Draft_Manuals/WATER.pdf. Accessed 3 June 2019.
Government of India & Government of The Netherlands. Standard analytical procedures for water
analysis. 1999. http://indiawrm.org/Website/Webpages/PDF/-download-manuals-WaterQuality-
TechnicalPapers-StandardAnaly.pdf. Accessed 3 June 2019.
http://egyankosh.ac.in/bitstream/123456789/13529/1/Unit-2.pdf. Accessed 5 July 2019.
http://egyankosh.ac.in/bitstream/123456789/51189/3/Block-2.pdf. Accessed 5 July 2019.
Lange ED. Manual for simple water quality analysis. Amsterdam, the Netherlands: International Water
Tribunal Foundation; 1994. ISBN 907080302X.
Patel H, Vashi RT. Chapter 2 – Characterization of textile wastewater. In: Patel H, Vashi RT, editors.
Characterization and treatment of textile wastewater. Boston: Elsevier; 2015. p. 21–71.
59 2
Patra JK, Das SK, Das G, Thatoi H. A practical guide to pharmacological biotechnology. Singapore:
Springer; 2019.
Sawyer CN, McCarty PL, Parkin GF. Chemistry for environmental engineering. 4th ed. New York:
McGraw-Hill, Inc.; 2000.
Thatoi HN, Dash S, Das SK. Practical biotechnology, principle and protocols. New Delhi:
I.K. International Pvt. Ltd; 2017.
61 3
Soil Quality Analysis

Contents
3.1 Moisture Content in Soil – 62
3.2 pH of the Soil – 62
3.3 Particle Size in Soil Samples – 64

3.4 rganic Matter and Organic Carbon Content
O
in Soil Samples – 70
3.5 Chloride Content in Soil Samples – 72

3.6 Sulfate Content in Soil Samples – 73
3.7 Nitrogen Content in Soil Samples – 74
3.8 Potassium Content in Soil Samples – 77
3.9 Phosphorus Content in Soil Samples – 79

62 Chapter 3 · Soil Quality Analysis

In this chapter, you will learn about the easy-to-use protocols used for the determina-
tion of moisture content, pH, particle size, organic matter and organic carbon content,
chloride content, sulfate content, nitrogen content, potassium content, and phosphorus
content in the soil samples. You will also learn about the easy-to-use laboratory protocols
and how to calculate and interpret the results.
3
3.1 Moisture Content in Soil
zz Theory
The moisture content in soil refers to the amount of water present in the soil. Since
most of the estimation of soil characteristics is carried out on a dry weight basis,
the moisture content of the soil needs to be determined immediately after sampling
before any further analysis.
zz Requirements
1. Container for storing the soil (tins or flask with lids)
2. Hot air oven
zz Procedure
1. Moisture tin or flask to be used for moisture analysis is cleaned and dried properly.
2. Around 5–10 g of the soil sample is collected in a clean and dry moisture tin or
a flask with lid and weighed using a weighing balance machine and the initial
weight of the container containing the soil sample is recorded.
3. The sample was then dehydrated inside a hot air oven at a temperature of 105 °C
for overnight with the lids open.
4. After heating, the sample was removed from the oven with the lids closed.
5. Further it was allowed to cool to room temperature inside a desiccator, followed
by taking the final weight of the container containing the sample.
6. The moisture content in the selected soil sample was then calculated with the help
of the following formula.
zz Calculation
A− B
Moisture content =
B − ( tare the weight of the container used)
Where, A is the weight of the container with sample before heating and B is the
weight of the container with sample after heating and cooling.
3.2 pH of the Soil

zz Theory
The determination of pH of the selected soil samples is an important factor for the
characterization of soil sample. Normally, the pH of any sample ranges from 1 to
14; however, in the soil science, it ranges from 1 to 12. Soil samples with very high
pH are normally influenced by the various salts present in them. The preliminary
3.2 · pH of the Soil
63 3
measurement of pH of soil is carried out by mixing the collected soil sample in a
neutral solvent and measuring the pH of the mixture solution. The pH denotes the
physicochemical environment of a soil at a particular location.
The classification of the soil pH as per the data presented by Pansu and
Gautheyrou (2006a, b, c) is given below:
pH range Classification
Lower than 3.5 Hyperacidic
Between 3.5 and 5.0 Very acidic
Between 5.0 and 6.5 Acidic
Between 6.5 and 7.5 Neutral
Between 7.5 and 8.7 Basic
Greater than 8.7 Basic
Source: Pansu and Gautheyrou (2006a, c). Data are presented with suitable permission from the
original source
zz Procedure
There are different methods for the measuring the soil pH. These methods includes,
measuring the pH of the saturated paste extract; 1:5 dilution of soil: water ration; or
by 1:5 dilution of soil: Calcium chloride solution.
1. The soil water dilution method is the more suitable method and is often used in a
soil science laboratory.
2. Around 10 g of soil is taken and is mixed in a beaker containing 50 mL of water.
3. It is mixed properly and its pH reading is recorded using a pH meter.
.. A digital pH meter is used for the measurement of pH of any sample

3.3 Particle Size in Soil Samples

zz Theory
The separation of different contents in the soil samples basing on their size along
with their specific quaintly refers to the particle size in a soil sample, which is essen-
tial for various fields such as agronomy, pedology, sedimentology, and road geotech-
3 nics. Soil is a systematized standard that includes the accumulation of different types
of inorganic and organic particles that belong to an incessant dimensional series. The
soil is classified into a number of standard categories in various countries, such as
fine clays, silts, coarse silts, very fine sands, fine sands, and coarse sands (. Fig. 3.1;
Pansu and Gautheyrou 2006a, b, c). The particles in the soil sample are separated
with particle size less than 2 mm. The most important step in the separation process
is the pretreatment process where the primary particles completely get rid of calcium
carbonate and other organic matters present in the soil. The size of the particle in the
soil samples are based on the measurements that link them to their physical features
0.1 µm 1 2 µm 1020 50 100 200 µm 500 Fine earth Coarse elements

NC
SC
0.002 0.02 0.1 0.2 0.5 1 mm 2 mm 20 mm 75 mm 250 mm
0.0002 0.005 0.05 0.10 0.25 0.50 1.00 2 00

Very
CSI
VFS
Coarse Coarse
FSI Medium Fine
CSSC
FC MS coarse Gravels Stones 10
clays silts sands sands
sands
0.002 0.02 0.05 0.10 0.20 0.50 1.00 2 00
Fine Coarse Coarse
France
Fine FS sands CS sands sands
CSI
Clays 8
silts (1) (2) (1) (2) (3)
0.002 0.05 0.10 0.25 0.50 1.00 2 00
Very Gravels
USDA
Fine 7
VFS
Clays Silts MS Coarse Stones

coarse
sands sands Fine Coarse
sands
Gravels
AFNOR
Fine Coarse 5
CSI
Clays Fine
silts sands sands
0.002 0.02 0.20 2 00
Fine sands (III) Coarse sands (IV) ISSS

Silts
ISSS
Clays Gravels 4
Atterberg
CSI
(I) (II) FS1 FS2 CS1 CS2 CS3
Fines
ASTM
FG
Fine Medium sands Stones 3

CS
(silts and clays) CG

sands
200 35 10 1/2 3 Inch or standard
75 µm 500 µm 2 mm ASTM
.. Fig. 3.1 Ranges of particle size used for soils (NC number of classes; FSi fine silts, CSi coarse silts;
FS, VFS, CS fine, very fine and coarse sands, respectively; FC fine clays; FG, CG fine gravels and coarse
gravels); from top to bottom: CSSC Canadian Soil Survey Committee (1978): 10 particle size ranges
<2 mm; France (before 1987): 8 ranges; USDA United States Department of Agriculture (1975): 7
ranges; AFNOR Association Française de Normalisation (1987): 5 ranges; ISSS = International Soil
Science Society (1966): 4 ranges; ASTM = American Society for Testing Materials (1985): 3 ranges.
(Reproduced with permission from Pansu and Gautheyrou (2006a, b, c))
3.3 · Particle Size in Soil Samples
65 3
0.1 0.2 1 2 5 µm 10 50 µm 100 200 500 µm 1 mm 2 mm 20 mm
0.01 Divers Sedimentation Sieving ultra-sons Sieving Manual shifting
1–150 µm Elutriation
1–150 µm Sedimentation balances
0.001–5 µm Malvern Zetasizer

3 nm–3 µm Malvern 4600
0.01–3 µm
0.5–560 µm Malvern Autosizer
Malvern 3600 E
0.1–2000 µm 80 130 600
Malvern Mastersizer X (4 ranges)
0.1–600 µm Malvern Mastersizer X (3 ranges)
1.2–564 µm
Malvern 2600 short
1.2–1880 µm
Malvern 2600 short
3 nm–3 µm
Malvern HiC Coulter N4
0.04–3 µm Coulter NaNoSizer
0.6–500 µm Coulter TAII
3–570 µm Coulter LS130
0.004–2000 µm
CILAS-ALCATEL (1996)
0.7–400 µm
0.1–500 µm CILAS 920
CILAS (1993)
10 nm–5 µm Niconp TC100
1–200 µm –500 µm
Analysette 20 Fristsh
0.1–300 µm
Micromeritics Sedigraph 5100
0.04–1000 µm Micromeritics Sedigraph 5000 E
Moriba LA-900
0.02–50 µm –100 µm
Moriba Capa 500
0.1–150 µm
Shimadzu SA CI210-SACI220
0.01–30 µm Joyce-Loebl Mark III
0.01–60 µm
Joyce-Loebl DC-3
X-ray UV VISIBLE
Infra-Red Micro-waves
0.01 µm 0.4 µm 1 µm 1 mm
.. Fig. 3.2 Some automated instruments to measure particle sizes and their ranges. (Reproduced with
permission from Pansu and Gautheyrou (2006a, b, c))
of the suspension of the soil samples after the dispersion process (. Figs. 3.2 and
3.3a and b; Pansu and Gautheyrou 2006a, b, c).
zz Requirements
Instruments and glassware
Hot plate, hot air oven, water bath, shaking machine, sieving machine (. Fig. 3.4),
different sets of sieves, funnel with stand, small sieves, container to determine the
moisture content, timer, beaker, measuring cylinder, conical flask, pipette, etc.
zz Reagents
55 Hydrogen peroxide: 30% hydrogen peroxide is used in the experiment.
55 Dispersing agent: About 40 g of sodium hexametaphosphate (4%) and 1% of
sodium carbonate solution are dissolved in 800 mL of distilled water and the
volume is then made up to 1 L in a volumetric flask. Before use, both the chemicals
are dried properly at 105 °C for overnight to remove any suspended water particles
from it.
55 Calcium chloride solution: 1 M solution of calcium chloride is prepared by
dissolving 147 g of calcium chloride in 800 mL of distilled water and then the
volume is made up to 1 L in water.
a
– destruction of organic matter (H2O2, Na hypochlorite), hypobromite...
individualization
– destruction of cements (AI, Fe, Si): – acid or basic media
of particles
– reducer or complexing media
– in water
desaturation (elimination cations2+)
chemical acid medium (some andosols)
choice of pH basic medium: NaOH, NH4OH, pyrophosphate, hexametaphosphate
– preliminary treatments various surfactants
suspension
ultrasounds
3 (dispersion)
physical
mechanical agitation (disintegration: 40 reversals/min)
limiting concentrations
– choice of concentration
wall-attachment effects
separation – techniques used – size phase 1990
principle advantages drawbacks
measurements range recovery firms
measurement by
fragility of sieves,
separation on sieve
mesh defects,
2 mm with vibration with or
measurement of dry mesh obtrusions,
– without ultrasonic
1. sieving etc. Saulas
0.050 yes waves simple
particles wet when dry, fine Tamisor, etc
(5 µm) weighing of the
powders stick to
fractions
coarse ones
(discontinuous)
measurement of
weight of a molecular
flim (nitrogen,
EGME...) retained at
2. surface measurement (for the surface of the internal and
≤ 2 µm no difficult to measure micromeritics
memorandum) particles external surface
(preliminary separation
of phases)
separation very slow

measurement inverse used for sediment
dry
of sedimentation: studies
density breakdown of gravity
1–
3. elutriation
150 yes force by a gaseous or
soils = 2.65 µm liquid flow Per
wet Ascensum
(discontinuous)
radioactivity
for labelled elements
(for memorandum)
calibrated hole,
by fluctuation no
distribution of given hole calibration, Coulter
of conductivity
dry particles by elimination of obturation
(or photometrics) no
or proportional changes density effect Quantimat 720
wet 0.5 – in displaced electrolyte Micro-
4. counting
samples 500 or proportional Videomat
µm extinction (Zeiss)
by microscopic Integramat
shape, habitus equipment is
measurement: no
counting on image expensive (Leitz)
optical, MEB, MET...
analyzer or manual
and image analysis
counting
Mettler,
gravimetric
1– Becker, Cahn,
sedimentation determination by
150 no very slow analysis Sartorius,
balances cumulating vs. time
µm Prolabo
.. Fig. 3.3 a and b Various systems of characterization of particulate for particle size distribution of
soils. (Reproduced with permission from Pansu and Gautheyrou (2006a, b, c))
67 3
b
separation techniques used size phase 1990
principle advantages drawbacks
– measurement range recovery firms
pipette
sedimentation at
methods volumetric sampling of
fixed level vs.
(Robinson, 1– fine particles.
time. Critical
Andreasen) 150 yes sieving for silts and
concentration per
reference method µm sands
unit of volume
AFNOR standardization
simple gravity
photo- influence of shape
sedimentation of colour of
optical method:
(turbidimetry) particles on
absorption, diffusion or laser: high
diffusion extinction Stanton
1 – 20 dispersion of light intensity lighting
diffraction coefficient, Malvern...
– 50 no (white or of small volume
influence of Cilas
laser. µm monochromatic light or low concentation
wavelength
laser)
diffraction effect for
small particles
5. sedimentation
X-ray soft 0.1 – measurement by concentration

g attenuation Micromeretics
only one 100 no electromagnetic good detection higher than
5100...
neutron µm radiation –X-ray or g photometry
scattering
influence of
method cannot density bouyoucos: densimeter density,
variable 1 – 50 measurement of (Casagrande, temperature,
be used for depth µm densimeter depth ASTM...) viscosity,
verticality of
cylinders
hydrometer De Leenher: surface

constant densimeter with chain evaporation
depth
Shimadzu
increase in gravity
continuous Capa 500
force by centrifugation
centrifugation Horiba
all force. Change of
0.02 –
concentration at a
300
500 No Horiba
classes of given depth,
5’. centrifugation µm
photo detection
700
Horiba
Beckman
discontinuous
centrifugation
– weighing of
Stokes law: speed of solid
cumulated deposits or
fractination: one static liquid
measurement of
phase – one solid mobile
height of deposits
phase, no deformation, no Sharples
particles – possible
reaction with liquid phase, 0.02 – Simcar
= yes approximate
homogeneously dispersed. 2 µm ...
separation of phases
Effect of temperature (viscosity,
– pipette centrifuge
convection flows),
analyser
effect of density, particle
diameter. Repulsive
electrostatic effect of particles
mobility of
the frequency of
particles,
6) M Brownian movement 0.003 displacements is
effective surface fine particles only Malvern
Zeta potential – 2 µm inversely proportional
charge
to particle size
.. Fig. 3.3 (continued)

.. Fig. 3.4 A sieving machine

used in the particle size
determination of soil samples
zz Procedure
The method for determining the particle size of the selected soil samples is a com-
bination of a number of steps such as organic matter oxidation, carbonate removal,
dispersion, and separation of different fractions of soil using different sieve size.
Each of the steps is described in detail below.
1. Organic matter oxidation
(a) About 20 g of the soil sample is taken in a 1 L beaker, and to it 15 mL each of
water and H2O2 (30%) is added and kept as such for overnight with a glass cover.
(b) Then the following day, the beaker is placed in a water bath at 80 °C and
5–10 mL of H2O2 (30%) is further added to it at regular intervals for com-
plete decomposition of the organic matter, as evident from the change in the
color of the mixture solution to colorless.
(c) Further, the volume of the mixture solution is made up to 300 mL by add-
ing more water to it and then it is placed on a hot plate to remove any H2O2
(30%) from it.
(d) The beaker is cooled and is centrifuged at 5000 rpm to remove the soil from
the mixture solution.
2. Removal of carbonate
(a) To the residues collected, around 25 mL of 1 M HCl is added for each 4 mL
of the sample and the total volume is made up to 250 mL with addition of
more water.
69 3
(b) Further, the mixture solution is again heated in a water bath at 80 °C for
15 min with occasional stirring.
(c) The mixture is removed and is allowed to stand for overnight and then centri-
fuged.
3. Dispersion
(a) The residue suspension obtained from the above step is transferred to a 1 L
polythene bottle.
(b) To it, 20 mL of the dispersing agent is added and the volume of the mixture
solution is made up to 400 mL with distilled water and in an air-tight con-
tainer and shaken continuously overnight in a shaker at lower speed.
4. Separation of fractions
(a) The mixture sample thus obtained from the above step is passed through a
50 μm sieve in a sedimentation cylinder and then the total volume is made up
to 1 L with water and is kept for determination of silt and clay. The residue
which is left over the sieve is taken for the determination of sand fraction.
5. Determination of sand
(a) The residue thus collected from the 50 μm sieve is basically the sand particles.
(b) It is taken for analysis using a sieving machine as shown in . Fig. 3.4.
(c) The sample is kept on the top sieve of the machine which contains sieves of
different sizes from top to bottom with mesh sizes of 1000, 500, 250, 100, and
50 μm. The machine is set to run for 10 min at 3000× per minute.
(d) The sample at each sieve is collected in a weighing dish and is weighed.
6. Determination of silt and clay
(a) The remaining solution which is left over after separation of fractions, is
passed over for the determination of silts (<50, <20, <2 μm) and clay using
fractionation procedure of different size sieves.
(b) Most commonly, the pipette method is used, which is based on sampling a 1 L
sample suspension with a 20 mL of pipette.
(c) To eradicate possible error in the experiment, blank pipetting is done with
only water and the value is termed as X.
(d) The sample in the sedimentation cylinder is closed with a rubber stopper and
shaken well. It is allowed to stand, and 20 mL of the sample is collected with
a pipette in a moisturized tin container. Then the solution is dried at 105 °C
in a water bath followed by cooling in a desiccator and then weighed.
(e) This step is repeated for each sample collected in various sieves (<20, <2 μm)
and weighed.
(f) The calculation is done according to the following formula.
zz Calculations
The sample is dried in a desiccator and is weighed and the calculation is as follows:
Clay ( < 2 µm ) = ( O × 50 ) − ( X × 50 )
Silt ( 2 − 20 µm ) = ( N × 50 ) − ( X × 50 ) − K
Silt ( 20 − 50 µm ) = ( M × 50 ) − ( X × 50 ) − K − L
Sand ( > 50µm ) = A + B + C + D + E
Sample weight is K + L + U + V
Where, E is the weight of the individual sand samples; M is the weight of 20 mL

pipette sample of <50 μm; N is the weight of 20 mL pipette sample of <20 μm; O
3 is the weight of 20 mL pipette sample of <2 μm; X is the weight of 20 mL pipette
sample of blank; 50 is the calculation constant (1 L of sample and 20 mL of aliquot
pipette is considered 50 times).
3.4 Organic Matter and Organic Carbon Content in Soil Samples

zz Theory
Organic matter present in the soil and sediments is extensively dispersed throughout
the earth surface. Soils and sediments comprise a large diversity of organic materials
that extend from simple sugars and starches and polysaccharides to the more com-
plex fats, waxes, proteins, and organic acids. The organic matter plays a momentous
role in the formation of soil and it can amend the physical, chemical, and biological
nature of the soil to prodigious extent. Imperative features of the organic matter
comprise its ability to form water-explicable and water in-explicable complexes with
the metal ions and hydrous oxides; relate with the clay minerals and bind the particles
together; sorb and desorb both the naturally occurring and anthropogenically intro-
duced organic compounds; absorb and discharge the plant nutrients; and hold water
in the soil. On account of these features, the estimation of the total organic carbon
in the soil sample is an indispensable part of any site description, since its existence
or nonappearance can distinctly influence how the chemicals will react in the soil or
sediment. Estimation of the total organic carbon (TOC) of the soil and sediment
samples is characteristically demanded with pollutant analyses as part of an ecologi-
cal risk assessment data package. The total organic content in the soil and sediments
are basically used to assess the nature of the sampling location site qualitatively and
it may be also used to standardize the portions of the analytical chemistry data set.
zz Requirements
1. Laboratory glassware such as beaker, funnel, burette, magnetic stirrer, conical
flask, and measuring cylinder and reagents
2. Potassium dichromate solution (1N): About 49.08 g of potassium dichromate is
dissolved in distilled water and the volume is made up to 1 L.
3. Sulfuric acid: concentrated.
4. Phosphoric acid: concentrated.
5. Sodium fluoride: dry and powdered.
6. Diphenyl amine indicator: About 0.25 g of diphenylamine is dissolved in 10 mL of
distilled water and gradually 50 mL of concentrated sulfuric acid is added to it.
7. Ferrous ammonium sulfate (0.5N): About 20 mL of concentrated sulfuric acid is
added to 800 mL of distilled water and 196.1 g of ferrous ammonium sulfate is
dissolved in it. Further, distilled water is add to make the volume of the solution
to 1 L.
3.4 · Organic Matter and Organic Carbon Content in Soil Samples
71 3
zz Procedure
1. The soil samples are air-dried and about 0.5 g of them are taken in an Erlenmeyer
flask.
2. Add 10 mL of potassium dichromate solution to the soil sample and then gradu-
ally add 20 mL of concentrated sulfuric acid to it, and wait for about 30 min.
3. Further, add 200 mL of distilled water to the mixture solution, followed by addi-
tion of 10 mL of phosphoric acid, 0.2 g of sodium fluoride, and 1 mL of diphe-
nylamine indicator.
4. Titrate the whole content of the mixture against ferrous ammonium sulfate solu-
tion. The initial dull green color will change to turbid blue to brilliant green at the
end of the titration process.
5. Run distilled water simultaneously as blank and adjust it during calculation.
.. Titration experiment setup for the determination of organic matter content in soil sample
Table for organic carbon content

Sl. no Volume of sample Volume of the titrant used Volume of the titrant used against
used (mL) against the sample (mL) the blank, distilled water (mL)
IBR FBR IBR FBR
3 1.
2.
3.
zz Calculation
6.791  T1 
Organic matter content = 1 −  × 10
W  T2 
6.791  T1 
Carbon content ( % ) = 1 − 
W × 1.724  T2 
Where, W is the weight of the soil sample in g; T1 is the volume of the titrant used
against the soil sample in mL; and T2 is the volume of the titrant used against the
distilled water blank in mL.
3.5 Chloride Content in Soil Samples

zz Theory
The accumulation of chlorine in the soil occurs mainly due to the deposition of
Cl from various sources such as rainwater, irrigation waters, fertilizer applications
(KCl), dust, and air pollution. High content of chlorides in the soil sample is consid-
ered as pollution, due to the animal excreta and other industrial effluents. Chlorine
deposits in the soil samples near the sea or salt lakes are extremely high. Further
application of various fertilizers to the soil also increases the chlorine contents in the
soil samples.
zz Requirements
1. Laboratory glassware and reagents such as glass bottles, beakers, measuring cyl-
inder, pipette.
2. 0.02N Sodium chloride solution: About 1.170 g of NaCl is taken in a beaker and
the volume is made up to 1 L with distilled water.
3. 0.025N Silver nitrate solution: About 3.397 g of AgNO3 is dissolved in distilled
water and the volume is made up to 1 L in an amber color glass bottle. The
reagent is standardized with sodium chloride solution and then kept in dark until
further use as silver nitrate is sensitive to light.
4. Potassium chromate indicator: About 10 g of K2CrO4 is dissolved in 20 mL of dis-
tilled water, and to it, few drops of 0.025N AgNO3 solution is added to produce
3.6 · Sulfate Content in Soil Samples
73 3
a red precipitate. It is then allowed to stand for 12 h followed by filtration and
dilution of the whole solution to 1 L in distilled water.
zz Procedure
1. About 10 g of the soil sample is taken after determination of its moisture content
(drying at 105 °C for overnight and then cooling in a desiccator). The soil sample
is mixed with 100 mL of distilled water in an Erlenmeyer flask. To it, few drops
of K2CrO4 indicator are added and mixed properly till the color of the sample
becomes yellow.
2. The mixture solution is then titrated against the AgNO3 solution until a persistent
brick red color appears to reach the end point.
Table for chloride content

Sl. no Volume of sample used (mL) Volume of titrant used against sample (mL)
IBR FBR
1.
2.
3.
zz Calculation
V × N × 35.453 × 1000
Chloride ( mg / mL ) =
S
Where, V is the volume of the titrate in mL; N is the normality of titrant (0.02); S
is the volume of the sample in mL; and 35.453 is the molecular weight of chlorine.
3.6 Sulfate Content in Soil Samples

zz Theory
Sulfates are found in considerable amount in the soil, mainly high in the arid and
semi- arid regions where the salt content is comparatively high. Domestic sewage and
industrial effluent, besides biological oxidation, may add to the sulfate content of
the soil. The salt is effortlessly hydrated and dehydrated under the effect of climatic
changes, which results in the high accumulation of sulfate contents in the soil sample.
Determination of sulfate content in the sample is based on the precipitation of sul-
fate after adding barium chloride.
zz Requirements
1. Laboratory glassware such as conical flask, beaker, measuring cylinder, funnel,
glass crucible, etc. Instruments like weighing balance, shaker, water bath, muffle
furnace along with filter paper (Whatman no. 1).
2. Concentrated hydrochloric acid.
3. Barium chloride (10%): Dissolve 10 g of barium chloride in 100 mL of distilled

water.
4. Phenolphthalein indicator: Dissolve 1 g of phenolphthalein in 100 mL of ethyl
alcohol and add 100 mL of distilled water. Add NaOH solution (0.22N) drop by
drop till a faint pink color appears.
3 zz Procedure
1. The soil sample is taken, crumbled to small size, and then dried at 105 °C over-
night.
2. The sample is then crushed to fine powder using a mortar and pestle.
3. Around 10 g of the crushed soil sample is taken in a conical flask and 100 mL of
distilled water was added to it and dissolved properly with occasional shaking for
around 2 h.
4. The sample was then filtered using a Whatman no. 1 filter paper and then 50 mL
of the filtrate was taken in a conical flask.
5. The solution is checked for its alkalinity using phenolphthalein indicator, and if
required, it is neutralized using concentrated hydrochloric acid.
6. Then another 4 mL of the concentrated hydrochloric acid is added to it to make
it acidic.
7. The mixture solution is then boiled, and to it hot barium chloride solution is
added with constant stirring, till precipitation is developed.
8. Then the container is kept in a water bath for around 4 h and the precipitation
was allowed to settle.
9. Further the precipitate is passed through an ashless filter paper and washed to
make it free from chlorine, dried, and then transferred to a pre-weighed glass
crucible.
10. It is then heated using a muffle furnace at 600–700 °C for 30 min.
11. Afterward, it is cooled using a desiccator and then its weight is recoded.
12. Calculation of sulfate content is done as per the following equation:
zz Calculation
41.15 × M 1
Sulfate% =
M2
60.85 × M 1
Sodium sulfate% =
M2
Where M1 is the weight of the precipitate in g and M2 is the total weight of the soil
sample taken for the precipitation process.
3.7 Nitrogen Content in Soil Samples

zz Theory
Nitrogen is an essential element present in the atmosphere and it is highly essential
for the growth of plants. Besides, it is also required by animals, but not in its current
form, but in association with other elements such as oxygen and hydrogen by the
3.7 · Nitrogen Content in Soil Samples
75 3
process of nitrogen fixation. The process of nitrogen fixation in the soil is carried
out by free living bacteria or the bacteria present in the roots of the plants. There are
various methods for determining nitrogen content in the soil sample, but the most
common is the Kjeldahl method. It consists of three steps: digestion, distillation, and
volumetric analysis.
zz Requirements
1. Digestion apparatus: Kjeldahl flask with a capacity of 500–1000 mL, still head
together with a facility of suction of fumes and a heating device.
2. Distillation apparatus: Used for the distillation process.
3. Spectrophotometer: Used at 400–425 nm with a light path 1 cm or more for anal-
ysis purpose.
4. Nessler tubes, capacity 100 mL, beaker, conical flask, measuring cylinder, etc.
zz Reagents
1. Digestion reagent: About 134 g of K2SO4 is dissolved in 650 mL of distilled water
and 200 mL of conc. H2SO4 followed by the addition of mercury oxide solution
(dissolve 2 g of HgO in 25 mL of 6N H2SO4), with continuous stirring. Then the
whole mixture is made up to 1000 mL and stored at ambient temperature to pre-
vent crystallization.
of ethyl alcohol and to it 100 mL of distilled water is added followed by the addi-
tion of NaOH solution (0.22N) drop by drop till a faint pink color appears.
3. Sodium hydroxide–sodium thiosulfate reagent: About 500 g of NaOH and 25 g
of Na2S2O3 are dissolved in water and made up to a volume of 1000 mL.
4. Mixed indicator: About 200 mg of methyl red is dissolved in 100 mL of 95% ethyl
alcohol, and 100 mg of methylene blue is dissolved in 50 mL of 95% ethyl alcohol
in two separate beakers and then both the solutions are mixed properly.
5. Indicating boric acid: About 20 g of H3BO3 is dissolved in distilled water and
10 mL of mixed indicator is added and the solution is made up to 1000 mL.
6. Sodium hydroxide 6N: Dissolve 240 g of NaOH in 1 L of ammonia-free distilled
water.
7. Standard sulfuric acid: Dilute 2.8 mL of concentrated sulfuric acid to 1 L using
distilled water. Dilute 200 mL of this stock solution (0.1N) to 1 L using distilled
water to prepare 0.02 N sulfuric acid titrant. Standardize the solution.
zz Procedure
A. Digestion
1. Around 50 g of the soil sample is taken in a 500 mL of Kjeldahl flask.
2. To it, 50 mL of the digestion reagent is added. If the concentration of the
suspended solids is high, then more amount of the reagent is added to the soil
sample in order to keep the salt to acid ratio at 0:8.
3. The digestion process is continued at low flame first and gradually the temper-
ature is increased till the sample appears clear green color to ensure complete
breakdown and to avoid damage of the organic matter.
4. The mixture is allowed to cool and then it is diluted with distilled water to 150
or 300 mL as per the requirement and is transferred to the distillation flask.
B. Distillation
1. The flask is placed at the proper position in the distillation apparatus and the
heating system is turned on.
2. About 0.5 mL of phenolphthalein reagent is added to it followed by addition
of sodium hydroxide and sodium thiosulfate reagent till the pH of the mixture
rises above a value of 8.3.
3 3. The distillation process is completed and about 200 mL of the distillate is
collected with 50 mL of boric acid in a conical flask. Normally, plain boric
acid is used for colorimetric estimation, and indicating boric acid is used for
the titrimetric estimation. The tip of the condenser is extended well below the
level of boric acid solution.
4. When the distillation is completed, then the flask containing the distillate is
removed first, and then the heat system is put off in order to avoid back suc-
tion.
C. Volumetric analysis
1. The concentration of ammonia in the distillate is determined by Nessleriza-
tion process or titrate process.
2. Around three drops of the mixed indicator are added to the distillate and they
are titrated with 0.02N H2SO4 till the indicator turns to a pale lavender color.
3. A blank reference is also carried out in all the steps to adjust for necessary
correction.
.. Complete Kjeldahl method setup for the determination of nitrogen content in soil sample
zz Calculation
( A − B ) × 0.28 ×1000
Organic N ( mg / L ) =
S
Where, A is the mL of 0.02N H2SO4 required for the sample to reach the end point
and B is the mL of 0.02N H2SO4 required for the blank reference to reach the end
point.
3.8 · Potassium Content in Soil Samples
77 3
3.8 Potassium Content in Soil Samples
zz Theory
Water-soluble potassium is present in most of the soil sample except saline or sodic
soils. These exchangeable K is commonly used for the evaluation of the soil K status
or in the determination of the requirement of K for the crops. Potassium plays a
substantial role in the growth of the plants.
zz Requirements
1. Beaker, conical flask, volumetric flask, funnel, pipette, measuring cylinder, flame
photometer, shaking machine, Whatman filter paper.
2. Ammonium acetate solution: About 57 mL of glacial acetic acid is taken in
a 2000 mL conical flask and to it 800 mL of distilled water is added followed
by gradual addition of concentrated ammonium hydroxide solution till the pH
of the solution changes to 7.0. The volume of the whole mixture is made up to
1000 mL with distilled water.
3. Standard potassium solution: A stock solution is prepared by taking 1.90 g of potas-
sium chloride in distilled water and the volume is made up to 1 L. The concentra-
tion of the stock solution is 1 g K/L or 1000 ppm K or 1000 mg K/1000 mL. From
this stock solution, different concentration of the potassium solution standard is
prepared with distilled water.
zz Procedure
1. The soil sample collected for analysis is air-dried and from it 50 g is taken in a
conical flask. To it, about 100 mL of 40% of ethyl alcohol is added and shaken
vigorously and then kept for about 10 min. After 10 min, the mixture solution is
filtered to another flask using a Whatman filter paper (no. 50). The residues left
over the filter paper are again washed with 40% ethyl alcohol and finally in abso-
lute alcohol to filter out any trace of other elements present in the soil sample.
The whole residue is then transferred to a beaker and to it 100 mL of ammonium
acetate solution is added and stirred vigorously and then allowed to stand over-
night. The mixture solution is again filtered using Whatman filter paper (no. 50)
and the filtrate is collected as the soil extract for further analysis.
2. Read the manual of flame photometer thoroughly before use.
3. Initially the instrument is turned on for 5–10 min. The compressor is started and
the burner is turned on.
4. The distilled water is aspirated and the meter value is adjusted to 0.
5. Then the highest concentration of standard potassium solution is aspirated and
it is adjusted to read the full value of the emission on the scale.
6. Then different concentrations of standard potassium solution ranging from 0 to
100 mg/L is fed to the aspirate one after another and the emission value for each
is recorded.
7. Then the solution of the unknown sample is aspirate and its value is also recorded.
8. After the completion of the test, the system is cleaned five times by aspirating
distilled water for five times.
9. A standard curve is plotted between the concentration and emission of standard
potassium solution and from the standard curve the concentration of the
unknown sample is calculated.
.. A complete setup for flame photometer used in the determination of potassium content in soil samples
1.8
1.6
1.4
1.2
Emission value
Emission values
1.0 of unknown soil

sample
0.8
0.6
Corresponding
0.4 concentration of
unknown soil
0.2 sample
0
0 25 50 75 100
Concentration of standard potassium
.. A representation of standard curve plotted to calculate the concentration of potassium in an

unknown soil sample
3.9 · Phosphorus Content in Soil Samples
79 3
zz Calculation
A ×V
Potassium ( mg / g ) =
W × 1000
Where, A is the potassium content of the soil extract (mg/L); V is the total volume of
the soil extract (mL); and W is the weight of dry soil (g).
3.9 Phosphorus Content in Soil Samples

zz Theory
Phosphorus is a vital necessity for the growth of plants and, therefore, it is applied
in the form of fertilizer to the soil during agriculture process. Knowledge on the
quantity of phosphorus present in the soil particularly the cultivated soil is of utmost
important to farmers.
zz Requirements
1. Spectrophotometer, laboratory glassware such as beaker, conical flask, measuring
cylinder, funnels, pipettes, hot plate, Whatman filter paper.
2. Perchloric acid: 70%
of ethyl alcohol and to it another 100 mL of distilled water is added.
4. Sodium hydroxide solution (1N): About 4 g of sodium hydroxide is dissolved in
100 mL of distilled water.
5. Extraction solution: About 15 mL of the ammonium fluoride solution (37 g of
ammonium fluoride is dissolved in distilled water and the volume is made up to
1 L) and 25 mL of 0.5N hydrochloric acid (20.2 mL of concentrated hydrochloric
acid is mixed in water and the volume is made up to 500 mL) are mixed and the
volume is made up to 450 mL with distilled water and stored in a container.
6. Ammonium molybdate solution: About 62 mL of concentrated sulfuric acid is slowly
added to 80 mL of distilled water and it is allowed to cool. About 5 g of ammonium
molybdate is separately dissolved in 35 mL of distilled water and mixed with sulfu-
ric acid solution and the volume is made up to 200 mL with distilled water.
7. Stannous chloride solution: About 0.5 g of stannous chloride is dissolved in 2 mL of
concentrated hydrochloric acid and it is then diluted to 20 mL with distilled water.
8. Standard phosphate solution: About 4.38 g of dried anhydrous potassium hydro-
gen phosphate is dissolved in distilled water and the volume is made up to
100 mg/L. From it, 10 mL is taken and distilled water is added to make 1 L of
stock solution containing 1 mg P/L solution. Standard solution of different con-
centration was prepared (0–1 mg P/L).
zz Procedure
1. The collected soil sample is initially air-dried and from it around 10 g of the soil
sample is taken in a 250 mL conical flask.
2. To it, 100 mL of the extraction solution is added and the mixture is shaken vigor-
ously for about 5–10 min.
3. After 10 min, the mixture is filtered through Whatman filter paper and the filtrate
is used for phosphorous determination.
4. From the filtrate, about 25 mL of sample is taken in an Erlenmeyer flask and is

evaporated to dryness.
5. It is cooled and the residue is dissolved in 1 mL of perchloric acid.
6. The flask is heated slowly to make it colorless.
7. It is then cooled and to it 10 mL of distilled water is added followed by two drops
of phenolphthalein indicator.
3 8. The whole mixture is then titrated against sodium hydroxide solution until a
slight pink color appears.
9. Then the volume of the mixture solution is made to 25 mL by adding distilled
water.
10. Then 1 mL of ammonium molybdate solution is added to it followed by three
drops of stannous chloride solution. A blue color develops.
11. The mixture is kept for 10 min as such and then its absorbance is recorded at
690 nm using a spectrophotometer.
12. Distilled water blank is simultaneously run at every step of the procedure.
13. Further the standard curve is also plotted taking different concentrations of
standard phosphorus solution.
14. The concentration of phosphorous in the soil sample is calculated from the stan-
dard plot in mg P/L.
Sl. no Sample of different concentration Absorbance at 690 nm
1.
2.
3.
0.9
0.8
0.7
0.6 OD value of
unknown soil
0.5 sample
OD values
0.4
0.3
Corresponding
0.2 concentration of
unknown soil
sample
0.1
0
0 2.5 5.0 7.5 10.0
Concentration of standard phosphorous
.. A representation of standard curve plotted to calculate the concentration of phosphorous in the
unknown soil sample
81 3
Take-Home Messages
55 The moisture content in soil refers to the amount of water present in the soil.
55 The determination of pH of the selected soil samples is an important factor for the
characterization of soil sample.
55 The separation of different contents in the soil samples basing on their size along
with their specific quantity refers to the particle size in a soil sample, which is
essential for various fields such as agronomy, pedology, sedimentology, and road
geotechnics.
55 The total organic contents in the soil and sediments are basically used to assess
the nature of the sampling location site qualitatively and it may be also used to
standardize the portions of the analytical chemistry data set.
55 Accumulation of chlorine in the soil occurs mainly due to the deposition of Cl
from various sources such as rainwater, irrigation waters, and fertilizer applica-
tions.
55 Sulfates are found in considerable amount in the soil, mainly high in the arid and
semi-arid regions where the salt content is comparatively high.
55 Nitrogen is an essential element present in the atmosphere and it is highly essential
for the growth of plants.
55 Potassium plays a substantial role in the growth of the plants.
55 Phosphorus is a vital necessity for the growth of plants and, therefore, it is applied
in the form of fertilizer to the soil during agriculture process.

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phates. New Delhi: Bureau of Indian Standards; 1977.
Follain S, Schvartz C, Denoroy P, Villette C, Saby NPA, Arrouays D, Lemercier B, Walter C. A method
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Schumacher BA. Methods for the determination of total organic carbon (TOC) in solids and sediments.
Cincinnati: Ecological Risk Assessment Support Center, Office of Research and Development, US
Environmental Protection Agency; 2002. https://pdfs.semanticscholar.org/597f/563a7b3191491480
0379e61218ce89e83f87.pdf.
Van Reeuwijk LP. Procedures for soil analysis. Wageningen, The Netherlands: International Soil
References and Information Centre; 2002. https://www.isric.org/sites/default/files/ISRIC_
TechPap09.pdf. Accessed 3 June 2019.
3
83 4
Isolation, Culture,
and Biochemical
Characterization of Microbes
Contents
4.1 Laboratory Safety – 85
4.2 Laboratory Etiquette – 86
4.3 Cleaning Glassware – 87
4.4 Sterilization – 89
4.5 Determination of pH – 90
4.6 Study of the Effect of pH on Growth of Microbes – 91
4.7 Preparation of Culture Media – 92

4.7.1 Preparation of Nutrient Agar (NA) Slants and Plates – 94
4.7.2 Preparation of Potato Dextrose Agar (PDA) Medium – 96
4.8 Isolation of Cyanobacteria – 97
4.9 Techniques for Isolation of Pure Cultures – 99

4.9.1 Serial Dilution Method – 99
4.9.2 Streak Plate Method – 100
4.9.3 Pour Plate Technique – 102
4.9.4 Spread Plate Technique – 104
4.10 Staining Techniques – 105

4.10.1 Simple Stains: Microbial Morphology – 106
4.10.2 Negative Staining (Indirect Staining) – 107
4.10.3 Differential Stains – 108
4.10.4 Acid-Fast Staining – 110

4.10.5 Special Staining – 112
4.10.6 Fungal Staining – 113
4.11 Microbial Growth Curve – 115
4.12 Isolation of Genomic DNA from Bacteria – 116
4.13 NA Quantification and Quality Analysis by Agarose

D
Gel Electrophoresis – 120
4.14 NA Quantification by Spectrophotometric

D
Method – 122
4.15 PCR Amplification – 123

4.16 Sequence Analysis – 125
4.17 Sampling of Air Microflora – 126
4.18 Antibiotic Assay – 128
4.19 oliform MPN Test for Bacteriological Examination
C
of Water – 130
Bibliography – 133
4.1 · Laboratory Safety
85 4
You will learn how to isolate microorganisms, prepare their pure culture, investigate
microbial growth in a suitable medium, study the morphology of microorganisms based
on staining technique, prepare culture media for microbial growth, perform serial dilution
of the sample collected from the environment and isolation, and purify microorganisms
based on pour plating and serial dilution techniques. You will also learn the techniques on
how to isolate different microorganism using enrichment medium. Evaluation of antimi-
crobial activity through antibiotic assays, effect of pH on microbial growth, most probable
number (MPN) test for detection of coliform bacteria, sampling of air microflora. Besides,
you will also learn the protocol for isolation of genomic DNA from bacteria and its quan-
tification and quality assessments, PCR amplification of DNA, and sequence analysis of
isolated DNA.
zz Microbial Techniques
A microbiology laboratory is a place to grow and study microbes which are too
small to be seen with the naked eye. The Environmental Microbiology Laboratory
is concerned with investigation of microorganisms from the natural environments.
Personnel working in a microbiology laboratory remain directly or indirectly at risk
from biological exposure. Therefore, a microbiology laboratory requires special prac-
tices and containment facilities in order to properly protect persons working with
microorganisms. Containment refers to safe methods for managing infectious mate-
rial in the laboratory environment. Persons who work in a microbiology laboratory
may handle infectious agents in additional to other hazards such as chemicals and
radioactive materials. Therefore, safety in the laboratory is the primary concern. The
laboratory procedures must be read carefully prior to attending the laboratory session.
4.1 Laboratory Safety
Biosafety is important as many materials used in the laboratory could be harmful

and infectious and accidental exposure can have serious consequences. Laboratory
should have material safety data sheet (MSDS). MSDS offers important information
about chemicals that someone wants to use. It becomes an important document and
guideline in case of an accident. It is important to arrange the laboratory workplace
carefully to ensure safe and effective operations. Laboratory should be provided with
such equipment necessary for conducting microbiological practical procedures such
as laminar air flow and sterilization of equipment, UV lights, etc. Besides, provision
should be there for proper disposal of infectious and hazardous materials. The labo-
ratory should have safety equipment such as safety glasses, lab coats, and gloves for
the protection of workers.
There are some important Do’s and Don’ts rules in laboratories for the students
to follow.
55 Students without shoes should not be allowed in the laboratory.
55 They should bring lab coats and permanent markers while working in the
laboratory.
55 Students should always wear full sleeves clothes. They should wear safety glasses
and gloves when recommended.
86 Chapter 4 · Isolation, Culture, and Biochemical Characterization of Microbes
55 They should leave bags and coats in designated areas.

55 Eating, drinking, playing, or applying cosmetics is not allowed in the laboratory
as it can lead to unintentional poisoning.
55 Receive proper instruction and thereafter practice good microbiological tech-
nique (GMT).
55 Be aware of the routes by which pathogenic microorganisms can enter the human
body and cause infections. Treat all microbial cultures, especially unknown cul-
tures, as if they could be pathogens.
4 55 All materials that have come in contact with a microbial organism should be con-
sidered hazardous waste and must be treated/sterilized before their disposal. The
waste disposal protocol in the microbiology laboratory is designed to separate the
non-infectious from the infectious waste.
55 The infectious waste needs to be disposed of in a manner that minimizes the risk
to both staff and students and facilitates the recycling of reusable material. After
finishing the experiment, the area must be disinfected. Rooms must be decon-
taminated by using fumigants such as β-propiolactone or formalin.
55 Many biological techniques will require you to sterilize loops and spreaders by
flaming. After sterilization, the loop toward the flame must not be touched.
55 Mouth pipetting in the laboratory is strictly forbidden. Cover any open cuts on
hands and other exposed skin surfaces.
55 If a live culture is spilled, cover the area with a disinfectant such as mercuric chlo-
ride for 15 min and then clean it. Hands should be properly washed after contact
with hazardous materials and before leaving the laboratory.
55 Hand washing prevents the spread of biological and chemical contaminants and
protects you from accidental exposure. There are some good commercial anti-
bacterial soaps that both clean and disinfect your hands. Even if you wear gloves,
this simple step should not be skipped.
55 Unnecessarily, don’t move around in the laboratory with sharp objects like nee-
dle, razor, scissor, cover slips, glass slide, etc. Don’t carry flasks, beakers, tubes,
and other glassware in hands.
55 Tray should be used to transport laboratory materials.
55 Decontaminate contaminated materials, specimens, and cultures before disposal
or cleaning. Laboratory should be kept neat and clean and should be free of
unnecessary materials. Any laboratory materials should not be tested orally. Don’t
keep or store any eatables in the laboratory. Don’t smell any lab materials directly.
4.2 Laboratory Etiquette
Lab etiquette informs us on how to behave properly in the lab by developing safe
practices and habits, as well as effective responses to potentially hazardous activities
and outcomes. Knowledge and awareness of rules for personal and biological safety
are highly important for working in the laboratory. Biological hazards arise while
working with cultures of microorganisms, most of which are pathogens. Infection
can occur from a cut or by ingestion or by inhalation of the particles from an aero-
sol. It requires that we should be able to identify potential hazards and prevent or
manage them properly without compromising our safety and the safety of others. It
4.3 · Cleaning Glassware
87 4
is necessary to return all chemicals and supplies to the proper location after use. It
is necessary to take chemicals from reagent bottles, pour out slightly more than the
amount of chemical needed into a clean beaker. Never pour a chemical back into a
reagent bottle. One should clean up the working place for the next person. At the
end of each laboratory period, all used glassware must be cleaned and be placed on
a rack to dry. Always be careful and sincere of your lab mates and their workspace.
Avoid distracting and obstructing lab mates when carrying out an experiment that
would be unsafe to them and their experiments. No experiment is complete until the
laboratory is cleaned.
4.3 Cleaning Glassware

zz Background
Proper cleaning of the glassware is key to get the correct result in any laboratory
experiments because carefully executed work may give an erroneous result if dirty
glassware is used. In all instances, glassware must be physically and chemically clean
and sterile. All glassware must be absolutely grease-free. Autoclaving glassware is
absolutely necessary if working with microorganisms. It is much easier to clean the
glassware before residues in them become dry and hard. If the glassware is dirty and
cannot be washed immediately, it should be put in water to soak. Handle glassware
carefully while cleaning it as most breakage occurs at this time. Glassware such as test
tubes, Petri dishes, conical flasks, beakers, pipettes, and measuring cylinders are often
used in microbiological experiments. It is essential to clean these glassware properly
and then sterilize them before starting the experiment.
zz Objective
To clean and prepare glassware for conducting microbiological experiment
zz Requirements
Preparation of chromic acid solution
Chromic acid wash is prepared by mixing 63 g of potassium chromate in 350 mL dis-
tilled water by heating followed by cooling and adding distilled water to make a paste
of chromate salt. Add 300 mL of concentrated H2SO4 to the paste and make the final
volume to 1 L. Sulfuric acid should be added along the side of the flask slowly. The
solution should be carefully used and rubber gloves and apron should be worn while
using it. The mixture should be used until it turns green in color. It may be necessary
to let heavily soiled items soak overnight.
zz Procedure
1. Cleaning of glassware is done with a brush using some detergent (liquid/solid)
and water. During the washing, all parts of the glassware should be thoroughly
scrubbed with a brush. For cleaning glassware, special laboratory cleaning prod-
ucts such as alconox, teepol, lab wash, and lab clean may be used. After the
glassware are thoroughly cleaned, they are rinsed with tap water and finally with
deionized water to prevent the possible contamination later. Then they are put in
inverted position to drain out water. Finally glassware are dried in an oven. Most
new glassware is slightly alkaline in reaction. For precision chemical tests, new
glassware should be soaked several hours in acid water (a 1% solution of hydro-
chloric or nitric acid) before washing.
2. If glassware becomes unduly clouded or dirty or contains coagulated organic
matter, it must be cleansed with chromic acid cleaning solution as it is a very
powerful cleaner. Special types of precipitates may require removal with nitric
acid, aqua regia, or fuming sulfuric acid. The dichromate should be handled with
extreme care because it is a powerful corrosive and carcinogen. Due to the corro-
4 sive nature of chromic acid, the use of this procedure has been eliminated except
for highly contaminated or soiled glassware.
3. A safer alternative is the use of NoChromix® cleaning solution, containing hydro-
gen peroxide, which is also made up of sulfuric acid. Any glassware that is stained
should be soaked in a concentrated sulfuric acid-potassium dichromate acid. For
cleaning pipettes, chromic acid solution should be used. Chromic acid solution is
prepared by slowly dissolving potassium dichromate in sulfuric acid.
4. Tall cylinders are filled with this solution and used pipettes are dipped in this
solution for overnight. Then the pipettes are washed with distilled water in the
end. The time of the glassware allowed to stand in chromic acid solution depends
on the amount of contamination present in the glassware. Relatively clean glass-
ware may require only a few minutes of exposure; if debris is present, such as
blood clots or more contamination, then it may be necessary to let the glassware
stand overnight.
5. After cleaning, rinse the glassware with running tap water. It is imperative
that all soap, detergents, and other cleaning fluids be removed from glassware
before use as slight traces of detergents will interfere with serologic and cultural
reactions.
6. Rinsing with tap water is followed by rinsing with distilled/deionized water.
7. The glassware should be placed on drying rack to air-dry or can be kept in hot air
oven.
zz Preparation of Glassware for Sterilization

1. Before proceeding for sterilization, pipettes should be plugged at the mouth end
with a little bit of cotton.
2. These pipettes are placed in a pipette-can (stainless steel) and are sterilized.
3. Similarly, Petri dishes can be placed in Petri cans and test tubes plugged with cot-
ton can be placed in wire baskets and are sterilized.
4. The flasks are usually plugged before their sterilization.
zz Precautions
55 A chromic acid wash can hydrolyze and oxidize a biological molecules to make
it chemically decomposed. Follow all normal safety precautions when using con-
centrated acids and acid solutions. Acids can severely burn the skin. Dispose of
all acids properly.
55 The use of chromate-based cleaning solutions is not recommended in many
research laboratories because the chromium ions are highly toxic to the environ-
ment and pose a severe waste disposal problem.
4.4 · Sterilization
89 4
4.4 Sterilization
zz Background
Sterilization means destruction or removal of all forms of life, including viruses,
bacteria, and fungi and their spores that are both pathogenic and nonpathogenic.
Sterilization is a routine microbial technique that is performed to make the glass-
ware and media free from all the living organisms without damaging or altering the
substances being sterilized. Several methods can be employed to sterilize various
materials required for microbiological experiments: physical, chemical, and filterable
methods which aim in the complete prevention of contamination. The method of
sterilization is chosen on the basis of materials to be sterilized and types of microor-
ganisms involved.
zz Objective
To learn different methods of sterilization
zz Principle and Procedure

Dry heat sterilization
Heat sterilization is the most widely used and reliable method of sterilization, involv-
ing destruction of enzymes and other essential cell constituents. This method of ster-
ilization can be applied only to thermostable products. Sterilization of all glassware
are done by using dry heat. The glassware wrapped in either paper or aluminum foils
are kept in a hot air oven maintained at 160–170 °C for 2–3 h for sterilization. After
sterilization, the oven should not be opened immediately. It should be allowed to
slowly cool down and then remove the materials to be used.
Moist heat sterilization
Moist heat sterilization is done by means of autoclave. Different liquid substances
like agar media, broth, and liquids are usually sterilized by moist heat in an auto-
clave maintained at 15 lbs steam pressure and 121 °C temperature kept for about
15–20 min. Moist heat sterilization has better penetrating power than dry heat ster-
ilization. The steam under pressure can kill all microorganisms including bacterial
endospores. Autoclaving is also ideal for sterilizing biohazardous wastes, surgical
dressings, etc. However, certain items such as plastics and thermolabile chemicals
(heat sensitive) which cannot withstand autoclaving should not be sterilized in an
autoclave. High pressure enables steam to reach high temperature.
Sterilization by filtration
Heat-labile fluids like antibiotics, sugars, amino acids, and vitamins are sterilized by
passing through filters of 0.22 μm which retain bacteria. Membrane filters are com-
monly used which are made of usually cellulose acetate or polycarbonate that con-
tains small pores. The solutions are usually forced through the previously sterilized
filters by pressure or suction and the filtrate is collected in a sterile container.
Sterilization by radiation (UV radiation)
The working area in the inoculation chamber is routinely sterilized by ultraviolet
radiation. These lamps are called UV lamps which emit radiation chiefly at a wave
length of 230 nm. The radiation emitted by UV lamps causes damage to the cells by
thymine dimer formation and finally produces the lethal effect.
Sterilization of other materials

Heat to redness in Bunsen burner flame, e.g., wire loop.
Flaming in alcohol (70% industrial methylated spirit).
4.5 Determination of pH
zz Background
4 The pH of a solution is a measure of the concentration of hydrogen ions in the solu-
tion. The pH scale extends from very acidic (0) to very alkaline (14) with 7 being
the “neutral” point. A pH meter is used to measure the acidity of a solution. When
preparing media for growing microbes, it is important to have the proper pH balance.
If the pH is too low or too high, the organism might not grow properly. The pH can
influence the solubility of ions in nutrient media, the ability of agar to gel, and the
subsequent growth of cells. Thus accurate determination of culture media is neces-
sary.
zz Principle
The pH value is determined by measuring the electromotive force of a cell con-
sisting of an indicator electrode immersed into the test solution and a reference
electrode. Contact between the test solution and the reference electrode is usually
achieved by means of a liquid junction which forms part of the reference electrode.
The electromotive force is measured with a pH meter, i.e., a high impedance volt-
meter calibrated in terms of pH. The glass electrode in combination with calomel
electrode is generally used with reference potential provided by saturated calomel
electrode. The glass electrode system is based on the fact that a change of 1 pH
unit produces an electrical change of 59.1 mV at 25 °C. The active element of glass
electrode is a special glass membrane. The membrane forms a partition between two
liquids of differing hydrogen ion concentration and a potential is produced between
the two sides of the membrane which is proportional to the difference in the pH
between the liquids.
zz Requirements
Sample to be analyzed (liquid media) and a pH meter
zz Procedure
1. The liquid media is taken in a test tube.
2. The pH of the media in the tubes was measured by immersion of the electrode
into the media to a depth of approximately 1 inch (2.54 cm).
3. The tip should touch the culture medium and no gap should be present in between
them.
4. The pH is recorded and temperature displayed on the meter once stable.
5. Electrodes were rinsed with distilled water, and excess water was gently removed
with paper lab-wipes between readings.
6. All pH measurements were made at room temperature with each electrode cali-
brated against standard pH 7.0 buffer before use.
7. Flat-surface pH electrodes are commonly used for measuring pH of culture
media, especially agar plates.
4.6 · Study of the Effect of pH on Growth of Microbes
91 4
zz Precaution
55 The pH electrode should be kept clean.
55 Protein residues from pH electrode can be removed by using HORIBA 250 clean-
ing solution and/or warm water.
4.6 Study of the Effect of pH on Growth of Microbes

zz Background
Each bacterial species has an ability to grow within a specific pH range, which may
be broad or limited, with the most rapid growth occurring within a narrow optimum
range. Most natural environments have pH values between 5 and 9, and organisms
with pH optima in this range are most common. The pH optimum for most bacteria
is between 6.5 and 7.5. Fungi, molds, and yeast prefer an acidic environment, with
optimum activities at a pH of 4–6. Only a few species can grow at pH values of less
than 2 or greater than 10. Initial pH of the media affects bacterial growth, or the
production of certain metabolites, during microbial growth. The optimum growth
pH is the most favorable pH for the growth of an organism. The lowest pH value that
an organism can tolerate is called the minimum growth pH and the highest pH is the
maximum growth pH.
zz Objective
To study the effect of pH on bacterial cell growth.
zz Principle
The optimum growth pH for E. coli is near neutral. E. coli can grow within the pH
ranges (from pH 5.5 to 8.5. Extreme pH beyond this range will significantly decrease
the growth rate of E. coli and may sometimes even cause cell death. The minimum
and maximum growth pH for E. coli are 4.4 and 9.0, respectively. E. coli cells appear
to tolerate a low pH better than a high pH. In fact, extended exposure of E. coli cells
to a high pH causes cell lysis. The medium pH is determined by medium composi-
tions, buffers, cellular metabolites, and aeration conditions. E. coli cells use sugars
such as glycerol and glucose as carbon or energy source. When E. coli cells use these
sugars as carbon source, they will produce acetic acid and therefore lower the pH
medium. Carefully balancing the phosphate buffer, organic buffers, sugar contents,
and aeration conditions can maintain the culture medium near E. coli optimum
growth pH or within the range of three pH units.
zz Requirements
55 LB broth – 1%
55 (LB broth is prepared by adding 4 g of powdered LB broth in 396 mL distilled
water followed by autoclaving the media at 15 psi pressure for 15 min at 121 °C.)
55 Flasks
55 Laminar air flow
55 Microbial sample (E. coli)
55 Spectrophotometer
55 Cuvettes
55 Acetic acid
55 NaOH
55 pH meter
55 Dropper
55 Cotton plug
55 Autoclave
zz Procedure
Calibration of pH meter
4 1. The pH electrode is prepared according to the instruction manual.
2. At least three pH buffers (usually of pH 4.01, 7.00, or 10.01) are selected.
3. Then small amount of fresh pH buffers is poured into beakers for calibration.
4. The tip of pH electrode is rinsed with distilled or deionized water and blot dried
with soft tissue.
Sample preparation and measurement
1. The tip of the flat pH electrode is placed on the surface of the culture medium
sample to measure the pH so that the tip touched the culture medium and no gap
is there in between them.
2. The pH and temperature displayed on the meter are recorded once stable.
3. The literature is referred for the desired pH range of the culture medium.
4. Before measuring another sample, the tip of the pH electrode is rinsed with dis-
tilled or deionized water and blot dried with soft tissue.
5. The sample is discarded after testing.
Effect of pH on growth of microbes
1. 1% LB media (50 mL) after autoclaving is poured into flask aseptically.
2. The media is inoculated with E. coli in the laminar air flow cabinet.
3. The media was provided with different pH, i.e., 4, 7, and 9.
4. The pH of the media is set to 4 by adding glacial acetic acid to the media and to
9 by adding NaOH.
5. The absorbance of each sample is recorded at 600 nm after every 30 min.
zz Result
E. coli bacteria grow best at a near neutral pH of 6–8.
They do not grow at a more acidic pH of 4 or 5 and shows maximum growth at pH 9.
zz Precaution
55 Inoculation should be done under sterile conditions.
55 pH should be set accurately.
55 Absorbance should be noted carefully and should be recorded at 600 nm.
4.7 Preparation of Culture Media

zz Background
Culture media are the nutrient solutions/substances used to grow microorganisms.
Microorganisms like all other living organisms require basic nutrients for their
growth. Nutrient requirements of different microorganisms are different. Hence, dif-
ferent types of nutrient media are available to grow different microorganisms. In fact,
4.7 · Preparation of Culture Media
93 4
many microorganisms cannot grow in any known culture medium and are known to
be uncultivable. The cultivation or growth of microorganisms is necessary on a cul-
ture medium for their subsequent isolation, identification, and for their experimental
or industrial uses. All media should provide carbon and nitrogen sources in addition
to minerals and other growth factors for the growth of microorganisms.
Preparation of medium is a routine microbiological work. Different media with
different chemical compositions are used for cultivation/culture of different microor-
ganisms. Media of many types can be obtained premixed, in an often dehydrated and
powdered state. Such media are called prepared media. It is often possible to obtain
sterilized media that are ready to use. However, in most of the laboratory practices,
media are prepared by adding ingredients to distilled water.
zz Types of Media
Media can be broadly classified into three main types
55 Natural media
55 Synthetic media
55 Semisynthetic media
zz Natural Media
Natural medium does not contain any specific nutrients and also is not chemically
defined for which this medium is designated as a complex medium. Substrates of
natural origin that favor microbial growth are employed in this media. Examples
include milk, vegetable juice, diluted blood, and peptone. In this type of medium, the
exact chemical composition is not known.
zz Synthetic Media
A synthetic media is chemically defined or in the other words a synthetic medium is
one in which all the ingredients are mixed in a definite proportion. They are gener-
ally used to study the specific nutritional requirements of different microbes (e.g.,
minimal medium).
zz Semi-Synthetic Medium
There is also some medium in which the chemical composition is partially known
called semi-synthetic medium. Examples include potato dextrose agar (chemical
composition of potato is not known), Czapek-dox agar, nutrient agar, and beef
extract agar medium.
Based on their functional properties, media can also be classified as follows:
zz Simple Media
Simple non-synthetic medium is capable of providing the nutrient with relatively few
factors required for the cultivation of micro organisms. Example includes nutrient agar.
zz Complex Media
Complex media, on the other hand, supplies additional types of growth factors. Complex
non-synthetic media are consequently able to support the growth of more fastidious
microorganisms compared to simple non synthetic media. For example, beef extract
used in nutrient medium is chemically complex and its composition is not known.
zz Differential Media
These types of media are employed to distinguish bacteria based on their spe-
cific properties. These media contain dye and color indicators to give biochemical
response. Examples include EMB agar (eosine methyl blue agar), starch agar, blood
agar, and MacConkey agar.
zz Selective Media
These types of media selectively allow the growth of a particular type of organism
4 but suppress the growth of other microbes. Examples include nutrient agar with 0.1%
(W/V) crystal violet that favors growth of the gram positive bacteria but suppresses
the growth of gram negative bacteria.
zz Enrichment Media
Enrichment medium contains special nutrients that allow the growth of a particular
organism that might not otherwise be present in sufficient numbers to allow it to be
isolated and identified. Unlike a selective medium, an enrichment medium does not
suppress other organisms.
zz Assay Media
This medium is used to assay antibiotics, amino acid, vitamins, etc.
zz Reducing Media (Oxygen Scavenging Media)

Reducing medium is employed for growing obligate anaerobes. Reducing media par-
ticularly contains chemicals (reducing agents) that deplete molecular oxygen.
4.7.1 Preparation of Nutrient Agar (NA) Slants and Plates

zz Objective
To prepare nutrient agar (NA) slants and plates.
zz Principle
Nutrient media are basic culture media used for routine cultivation and maintenance
of microorganisms. Nutrient agar is ideal for microbiological examination of variety
of materials and is also recommended by standard methods. It can be used for the
cultivation and enumeration of bacteria which are not particularly fastidious. This
medium also favors the growth of some fungi in nutrient plates. It is a semi-syn-
thetic medium composed of peptone, beef extract, NaCl, and agar. The beef extract
contains water-soluble substances including carbohydrates, vitamins, organic nitro-
gen compounds, and salts. Peptones are the principal sources of organic nitrogen,
particularly amino acids and long-chain peptides. Sodium chloride ensures osmotic
balance. Agar is the solidifying agent. Nutrient medium is a general basic culture
medium for less fastidious microorganisms. Nutrient medium is prepared either as
broth (without adding agar) or as solid medium containing agar powder. It is fre-
quently used for isolation and purification of bacteria.
4.7 · Preparation of Culture Media
95 4
zz Requirement
55 Conical flask
55 Cotton plug for conical flask
55 Measuring cylinder
55 Beaker
55 Test tubes
55 Long glass rod
55 Magnetic stirrer
55 Heater
55 Autoclave
55 Nutrient medium
i Peptone 5g
Beef extract 3g
NaCl 3g
Agar 15
Distilled water 1L
pH is adjusted to 7.0.
zz Procedure
1. About 500 mL distilled water is taken in a 500 mL conical flask.
2. About 1.5 g of beef extract, 2.5 g of peptone, and 1.5 g of NaCl are weighed and
dissolved in 500 mL of water by proper agitation.
3. The pH of the medium is adjusted to 7.2 with 0.1N NaOH or 0.1N HCl using a
pH meter. The medium is sterilized and used as broth.
4. 3.5 g of agar is then added to the conical flask and slowly heated over heater/
flame so that agar will melt.
5. Then the flasks are plugged with cotton plug.
6. The flasks are then sterilized at 15 lb pressure for 20 min using an autoclave.
7. 50 mL of the broth is taken and 0.75 g of agar is added and melted.
8. Then the melted content is distributed 5 mL each to another set of 10 test tubes
for making agar slants.
9. Test tubes containing the agar medium are allowed to cool down to about
45–50 °C by keeping them in a slanting position on the table.
10. The test tubes are left in this position until the medium has cooled to room tem-
perature and solidified to look opaque.
11. Then, these tubes are stored at 4 °C for later use.
zz Preparation of Agar Plates

1. The Petri plates are labeled and organized inside a laminar air flow.
2. Then, conical flask containing sterile agar medium in liquid form is gripped with
right hand.
3. The left hand palm side is turned up and cotton plug is clamped between two
fingers.
4. The plug is then removed and the mouth of the conical flask is flamed.
5. The lid of the Petri dish is then lifted with the help of cotton plug and about
20–25 mL of sterilized nutrient agar medium is poured while opening the lid of
Petri dish partially.
6. This procedure is carried out near the flame of a spirit lamp or Bunsen burner
placed in the laminar air flow.
7. Then the lid is immediately replaced.
8. The mouth of the flask is flamed and poured to the next plate in the same way.
9. The plates are allowed to cool at room temperature (agar medium becomes
4 opaque) and the plates are stored.
zz Results
1. The liquid and solid media are observed after 2 days.
2. Properly sterilized medium will remain clean. Use only media that are properly
sterilized for work.
4.7.2 Preparation of Potato Dextrose Agar (PDA) Medium

zz Background
Potato dextrose agar medium is used for the cultivation of fungi. Potato dextrose
agar is composed of dehydrated potato infusion and dextrose that encourage luxuri-
ant fungal growth. Potato dextrose agar is also used for stimulating sporulation, for
maintaining stock cultures of certain dermatophytes, and for differentiating typical
varieties of dermatophytes on the basis of pigment production. Potato dextrose agar
(PDA) is a general purpose medium for molds and yeasts that can be supplemented
with acid or antibiotics to inhibit bacterial growth.
zz Objective
To prepare potato dextrose agar (PDA) medium for isolation and cultivation of fungi
zz Principle
PDA can also be used for growing clinically significant yeast and molds. Agar is
added as the solidifying agent. The nutritionally rich base (potato infusion) encour-
ages mold sporulation and pigment production in some dermatophytes. Adjust
the pH of the medium by tartaric acid to inhibit the bacterial growth. Heating the
medium after acidification should be avoided as it may hydrolyze the agar which can
render the agar unable to solidify.
zz Requirements
55 Knife
55 Cheese cloth
55 Heater
55 Beaker (1 L capacity)
55 Conical flask (2) (500 mL capacity)
55 Petri dish
55 Autoclave
55 PDA medium
4.8 · Isolation of Cyanobacteria
97 4
i Potato tubers 200 g
Dextrose 20 g
Agar-agar 15 g
HCl 1N
NaOH 1N
Distilled water 1L
zz Procedure
1. About 200 g potato tubers are weighed and peeled off.
2. The tubers are chopped off into small pieces with the help of a knife
3. The chopped potatoes are transferred into a beaker containing about 100 mL of
distilled water.
4. The contents are then boiled with the help of a heater for about 20 min.
5. The supernatant is decanted and filtered with fourfold of the cheese cloth and
filtrates are collected in a beaker. This filtrate is called potato extract.
6. About 20 g dextrose and 15 g agar are transferred to the extract and heated gently
and shaken well to dissolve the ingredients. Finally, the medium is transferred to
a measuring cylinder of 1 L capacity and the volume is made up to 1 L by adding
more distilled water.
7. The pH of the medium is measured and adjusted to 5.6 by using 1N HCl or
NaOH in drops.
8. The medium is poured into two or more Erlenmeyer flasks, cotton plugged, cov-
ered with aluminum foil, and autoclaved at 15 lb pressure and 121 °C for 20 min.
9. When the temperature cools down, the flasks are taken out and used if required
or stored at room temperature for later use.
10. For isolation and enumeration of fungi, PDA medium is transferred into few
Petri dishes and the medium is allowed to solidify.
11. Before transferring to Petri dishes, antibiotic (1 mL) is added to the molten
medium to restrict the growth of bacteria.
12. Then, inoculums are added or exposed to air by opening the lid of Petri dishes
for 10 min.
13. The Petri dishes are incubated at 28 ± 2 °C for 72 h.
zz Results
1. Fungi will grow as filamentous colonies of various colors.
2. Yeasts will grow as creamy to white colonies.
3. The numbers of colonies are counted and the fungal or yeast count per gram or
milliliter of material is recorded.
4.8 Isolation of Cyanobacteria

zz Background
Cyanobacteria (blue-green algae) are the members of Cyanophyceae. They are
photo-autotrophic nitrogen-fixing microorganisms found in most of the aquatic
habitats. They are polymorphic, prokaryotic microorganisms which may be single
celled, colonial, or filamentous, and they resemble the bacteria in cellular organiza-
tion and green plants in oxygenic photosynthesis. Some of the cyanobacterial taxa
are capable of fixing atmospheric nitrogen and produce nitrogen-rich environment

in their surroundings. Soil is the most important non-aqueous habitat of N2-fixing
cyanobacteria.
zz Objective
To isolate Cyanobacteria from paddy field
zz Principle
4 The rice field ecosystem represents a favorable environment for the growth of cyano-
bacteria. Cyanobacteria found in rice field soils belong to genera Anabaena, Nostoc,
Scytonema, Stigonema, Lyngbya, Oscillatoria, etc.
zz Requirements
55 Soil sample from a rice field
55 Chu’s medium No. 10/BG 11 medium
55 Pipette
55 Conical flask
55 Growth chamber
55 Inoculation needle
55 Polythene bags
55 Burner
55 Sterile distilled water
55 Composition of Chu’s medium No. 10 (alternatively BG 11 medium can be used)
i Ca(NO3)2 0.004%
MgSO4.7H2O 0.0025%
K2HPO4 0.0005–0.001%
Na2CO3 0.002%
Na2SiO3 0.0025%
FeCl3 0.00005%
55 Composition of BG11
i Ingredients g/L
Sodium nitrate 1.500
Dipotassium hydrogen phosphate 0.0314
Magnesium sulfate 0.036
Calcium chloride dihydrate 0.0367
Sodium carbonate 0.020
Disodium magnesium EDTA 0.001
Citric acid 0.0056
Ferric ammonium citrate 0.006
Final pH after sterilization (at 25 °C) 7.1
4.9 · Techniques for Isolation of Pure Cultures
99 4
zz Procedure
1. Collect soil samples in polythene bags or colonies of blue-green algae floating on
water surface in rice field in glass bottles and bring them to the laboratory.
2. Prepare Chu’s medium No. 10/BGA11 and autoclave the medium at 121 °C for
30 min. Then allow the medium to cool and transfer 20 mL into 100 mL conical
flasks.
3. Serially dilute the soil/algal sample in sterile distilled water as described in serial
dilution technique.
4. Pour aliquots of 1 mL of diluted soil/algal samples into 20 mL of sterilized
medium in flasks.
5. Incubate the flasks at 30 ± 1 °C for 2–3 weeks in an alternate light and dark
regime of 12 h. Algal colonies appear in broth medium.
6. Soon pick up individual colonies and transfer onto fresh agar slants and incubate
as above.
7. Several colonies of cyanobacteria will grow on slants. They secret copious amount
of mucilage.
zz Result
1. Take a small amount algae grown on the medium and transfer to a clean slide.
2. Observe it under microscope according to the physiological identification manual
(Komárek and Anagnostidis, 1989). Filaments of various colors and shapes are
observed.
3. These filaments may be heterocystous or non-heterocystous, and branched or
unbranched.
4.9 Techniques for Isolation of Pure Cultures

zz Background
Generally, in nature, microorganisms exist in mixed population. However, to study
the microorganisms and their processes, pure cultures are needed. Therefore, in the
laboratory, mixed populations are separated into pure single colonies. Colonies are
individual, macroscopically visible masses of microbial growth on a solid medium
surface, each representing the mass of a single organism type. These cultures are
suitable for studying their cultural, morphological, and biochemical properties. The
techniques commonly used for obtaining discrete colonies initially require that the
number of microorganisms in the inoculum be reduced. Techniques such as serial
dilution agar plate, streak plate, pour plate, and spread-plate methods are used to
accomplish this necessary dilution.
4.9.1 Serial Dilution Method

zz Objective
To isolate and enumerate microbes from the given sample by serial dilution agar plate
method.
zz Principle
The principle of serial dilution agar plate method is that when microorganisms are
grown in agar plates, possibly all live microorganisms and their spores grow and
develop as individual colonies. In serial dilution agar plate method, a known amount
of soil (1 g) or water (1 mL) sample is suspended into a known volume of sterile
water so that the total volume of microbial suspension becomes 100 mL. This gives
a dilution of 10−2. Then, 1 mL volume of suspension from 100 mL is transferred
to additional test tube containing 9 mL of sterile water to get a serially diluted sus-
4 pension of 10−3. Similarly, further dilution can produce suspension of dilutions of
10−4, 10−5, etc. Aliquot of 1 mL suspension is poured on the Petri plates and molten
medium prepared for the said purpose is poured on the plates. Then these Petri plates
are shook thoroughly to mix the inoculums uniformly with the medium. Based on
serial dilution agar plate method, bacteria, fungi, actinomycetes, etc., are isolated as
discrete colonies on agar plates.
zz Requirements
55 Test tubes
55 Petri plates (20 mL)
55 Sterile pipettes (1 mL)
55 Nutrient agar (5 × 100 mL)
55 Inoculation loop
55 Burner
55 Marker pen
55 Sterile chamber
55 Laminar air flow
zz Procedure
1. Exactly 1 mL of the given sample was added to 9 mL of distilled water in a test
tube. This solution will be labeled as 10−1.
2. From this test tube, take 1 mL of solution and add to another test tube contain-
ing 9 mL of distilled water, mix evenly, and label as dilution 10−2. Continue this
process until 10−10 dilutions are obtained. Dilutions of 10−3 and 10−4 are taken for
fungal isolation and 10−6 dilution for bacterial isolation.
3. For soil samples, take 1 g of soil in a conical flask and add 100 mL of water. This
is known as stock solution. This solution is labeled as 10−2. Then serial dilution as
mentioned above has to be performed to obtain the desired dilutions (. Fig. 4.1).
4.9.2 Streak Plate Method

zz Background
The streak plate method offers a most practical method of obtaining discrete colo-
nies and pure cultures. It was originally developed by two bacteriologists, Loeffler
and Gaffkey, in the laboratory of Robert Koch. In this method, a sterilized loop or
transfer needle is dipped into a suitable diluted suspension of organisms which is
then streaked on the surface of an already solidified agar plates to make series of
parallel non-overlapping streaks.
101 4
.. Fig. 4.1 Serial dilution technique. (Reproduced with permission from Al-Dhabaan and Bakhali
2017)
zz Objective
To isolate and enumerate microbes from the given sample by streak plate method.
zz Principle
The streak plate method is a rapid qualitative isolation method. It is essentially a
dilution technique that involves spreading a loop full of culture over the surface of
an agar plate such that the population of bacterial inoculums get reduced with subse-
quent streaking. Although many types of procedures are performed, the four-way, or
quadrant, streak is described here. The progressive dilution of bacteria or yeast over
the surface of solidified agar medium in a Petri dish by subsequent streaking makes
easier to find the plates with single isolated colonies.
zz Requirements
55 Mixed culture
55 24 or 48 h nutrient broth culture
55 Tripod stand and wire guaze
55 Bunsen burner
55 Beaker of water
55 Inoculation loop
55 Nutrient agar medium

55 Sterile Petri dish
55 95% Ethyl alcohol
zz Procedure
1. Pour the Petri dish with 25 mL of molten nutrient agar and allow it to solidify.
2. When the nutrient agar plate is solidified, place a loop full of culture on the agar
surface (in area 1).
4 3. Flame and cool the loop and then drag it rapidly several times across the surface
of area 1.
4. Reflame and cool the loop, and turn the Petri dish to 90 °C. Then, touch the loop
to a corner of the culture in area 1 and drag it several times across the agar in area
2. The loop should never enter the area 1 again.
5. Reflame and cool the loop, and again turn the dish to 90 °C. Streak area 3 in the
same manner as area 2.
6. Without reflaming the loop, again turn the dish to 90 °C and drag the culture
from the corner of area 3 across area 4, using a wider streak. Don’t let the loop
touch any of the previously streaked areas.
zz Result
The isolated colony of desired microbes at the site of last streak on the plate will
be observed. A confluent growth will be seen where the initial streak was made, the
growth is less dense away from the streak, and discrete colonies farthest away from
the streak (. Fig. 4.2).
zz Precautions
1. Avoid pressing the loop or needle too firmly against the agar surface as this dam-
ages it.
2. Inoculation loop should be cooled by touching the agar surface away from the set
of streaks before streaking of the inoculum.
3. Petri plate lid should never be lifted completely.
4. Plating of the medium should be done 24 h prior to performing the exercise.
4.9.3 Pour Plate Technique

zz Background
The pour plate technique was developed in the laboratory of Robert Koch. In this
technique, successive dilutions of the inoculums are added onto sterile Petri plates
to which melted and cooled agar media are poured and thoroughly mixed by rotat-
ing the plates; they are then allowed to solidify. After incubation, the plates were
observed for the presence of different individual colonies growing throughout the
medium.
zz Objective
To learn the pour plate technique
103 4
.. Fig. 4.2 Culture methods for
bacteria. a Streak plate method
and b liquid dilution method.
(Reproduced with permission
from Al-Dhabaan and Bakhali
2017)
zz Principle
A pour plate is a microbial isolation technique in which a small amount of inoculums
from broth culture is added by pipette to a sterilized petri plate follwed by pouring
a molten, cooled agar medium into the plate and thoroughly mixed the medium by
shaking the petri plate in a “8” shaped pattern to solidify the medium. Pour plates
allow microbes to grow both on the surface and within the medium. If the dilution
and volume of the inoculums, usually 1 cm3, are known, the viable count of the
sample per cubic centimeter can be determined. This method is very useful for grow-
ing microaerophiles that cannot tolerate exposure to oxygen as this method embeds
organisms in the medium.
zz Requirements
55 Mixed bacterial cultures
55 Bunsen burner
55 Beaker
55 Inoculation loop
55 Sterile Petri dishes

55 95% Ethyl alcohol
55 Glass marker
zz Procedure
1. Molten but cooled agar is taken in a conical flask.
2. A loopful of bacteria is taken and is thoroughly mixed with shaking the flask.
3. The contents of conical flasks are poured on to the sterile plates.
4 4. Then all plates were incubated in an inverted position at 37 °C for 24–48 h.
zz Result
1. The colonies of individual microorganism will be visible on agar surface in Petri
plates.
2. It will be observed that progressively poured plates will have lesser number of
colonies which will be distributed more or less sparsely in the plates; these colo-
nies may be transferred to other media or agar slants for further study.
zz Precaution
1. A fresh sterile pipette is used for each dilution.
2. The medium to be poured on the Petri plates should have a temperature of 45 °C.
3. While transferring a suspension from one tube to another it should be maintain
continuous shaking of the tubes for the uniform distribution of cells.
4. The plates should be incubated in an inverted position to prevent condensation
on the agar surface. Unless the surface is dry, it will be difficult to obtain discrete
surface colonies.
4.9.4 Spread Plate Technique

zz Background
The spread plate technique is used for the separation of a dilute, mixed population of
microorganisms so that individual colonies can be isolated. In this technique micro-
organisms are spread over the solidified agar medium with a sterile L-shaped glass
rod while the Petri dish is spun on a turntable.
zz Objective
To separate bacteria from a mixed culture using spread plate techniques
zz Principle
In spread-plate technique, inoculums are transferred to a solidified nutrient agar
medium in the Petri dish. The cells are spread over the surface of a solid agar
medium with the help of a sterile, L-shaped bent glass rod called spreader, while
the Petri dish is spun on a turntable (revolving plate form). Then the Petri plates
are incubated at 25 °C for 24–48 h. Once discrete, well-separated colonies develop
on the surface of a nutrient agar plate culture, and each may be picked up with a
sterile needle and transferred to a separate nutrient agar slants. Each of these new
slant cultures represents the growth of a single bacterial species and is designated as
a pure or stock culture.
4.10 · Staining Techniques
105 4
zz Requirements
55 Bunsen burner
55 Beaker of water
55 Inoculation loop
55 Sterile Petri dish
55 95% Ethyl alcohol, 500 mL beaker glass, marking pencil
zz Procedure
1. A bent glass rod is paced into a beaker and a sufficient amount of 95% ethyl alco-
hol is added to cover the lower bent portion.
2. With a sterile loop, a loop full of mixed culture is placed in the center of the
appropriately labeled nutrient agar plate that has been placed on the turntable.
3. The glass rod from the beaker is removed and is passed through the Bunsen
burner flame with the bent portion of the rod pointing downward to prevent the
burning alcohol from running down your arm. Allow the alcohol to burn off the
rod completely. Cool the rod for 10–15 s.
4. Remove the Petri dish cover and spin the turntable.
5. While the turntable is spinning, lightly touch the sterile bent rod to the surface
of the agar and move it back and forth. This will spread the culture over the agar
surface.
6. When the turntable comes to a stop, the cover is replaced.
7. The rod is immersed in alcohol and reflamed.
8. In the absence of a turntable, the Petri dish is turned manually and the culture is
spread with the sterile bent glass rod.
9. All plates are incubated for 24–48 h at 25 °C.
zz Results
After incubation, individual colonies may appear separately on the agar plate.
Colony morphology is observed and then individual isolated colonies are picked
up and purified through sub-culturing.
4.10 Staining Techniques

zz Background
The correct identification of microorganisms is of fundamental importance to any
microbial study. Visualization of microorganisms in their living state is quite difficult
not only because they are minute but also because they are transparent and prac-
tically colorless when suspended in an aqueous medium. Therefore, to study their
properties and to classify microorganisms into specific groups, biological stains and
staining procedures in conjunction with microscopy have become one of the major
tools in microbiology.
The staining process involves immersing the sample (before or after fixation and
mounting) in dye solution, followed by rinsing and observation. Many dyes, however,
require the use of a mordant, a chemical compound that reacts with the stain to
form an insoluble, colored precipitate. When excess dye solution is washed away, the
mordanted stain remains. Stains are classified as simple stain, differential stain, and
structural or special stains.
4.10.1 Simple Stains: Microbial Morphology

zz Background
4 Some staining techniques involve the application of only one dye to the sample which
is known as simple stain. Simple stain techniques can be performed with basic dyes
such as crystal violet or methylene blue, which are positively charged dyes that are
attracted to the negatively charged materials of the microbial cytoplasm. The most
commonly used basic stains are methylene blue, crystal violet, and carbolfuchsin.
zz Objectives
To perform simple staining procedure to visualize the morphological shape and
arrangement of bacteria cells
zz Principle
A simple stain will generally make all the organisms in a sample to appear in same
color, even if the sample contains more than one type of organisms. Simple stains
are of two types, basic and acidic. Basic dyes are called direct (positive) stain as they
immediately stain the microbial cell, while the acidic dyes are called (indirect) nega-
tive stain as they stain only the surrounding of the cell, leaving the cell unstained.
Simple stain is used to get information about cell shape, size, and arrangement. Basic
stains with a positively charged dye are employed for simple staining as many of the
cell components in microbes (for example, bacterial cell wall and nucleic acids) have
acidic constituents.
zz Requirements
55 Slant culture of 24-h grown microbes (e.g., Bacillus subtilis)
55 Methylene blue
55 Crystal violet
55 Carbolfuchsin
55 Bunsen burner
55 Inoculation loop
55 Staining tray
55 Microscope
55 Bibulous paper/blotting paper
55 Glass slide
zz Procedure
1. Prepare separate bacterial smears on glass slides using the supplied bacterial
culture.
2. Keep the slides on the staining tray and flood the smear with one of the basic
dyes for a specified period – methyl blue for 1–2 min/crystal violet for 20–60 s/
carbolfuchsin for 15–30 s. (Different dyes take different periods of time to react
with the cell.)
107 4
3. Wash the smear with tap water and remove the excess stain.
4. The slides are blot dried with blotting paper.
5. The slides are examined under oil immersion.
6. Describe the morphology of the microorganisms with reference to their, shapes
(bacilli, cocci, spirilli), and arrangements (chains, clusters, pairs).
zz Result
Microbes (bacteria) stain deep blue. If there are endospores within the cell, they
appear as clear areas.
4.10.2 Negative Staining (Indirect Staining)

zz Background
Negative staining uses a simple acidic dye (e.g., nigrosin and India ink). The nega-
tive stain does not stain the microbe (bacteria) but stains only the background. The
practical application of negative staining has two advantages: (i) cells will not shrink
or distort of bacteria because no heat fixing is necessary and (ii) some spirilli which
are difficult to stain can be observed by this method.
zz Objective
To perform negative staining
zz Principle
The negative charge of the stain is repelled by the negatively charged surface of bac-
teria. Since heat fixation is not done during this staining, the organism is not killed
during this process. Upon examination after staining, the microbes appear transpar-
ent in a dark background.
zz Requirements
55 Bacillus cereus or Aquaspirillumitersonii (24-h-old culture)
55 Nigrosin solution
55 Clear glass slides
55 Staining tray
55 Inoculation loop
55 Bunsen burner/spirit lamp
55 Lens paper
55 Microscope
zz Procedure
1. Place a drop of nigrosin stain at one end of the slide.
2. Transfer a loopful of the microbial culture into a drop of stain. Allow it to mix
thoroughly, stir it gently with the loop.
3. Take another clean glass slide and place its edge on the suspended microbe (in
stain) at an angle of 45° angle and prepare a thin smear on the slide.
4. Allow the smear to air-dry. Do not heat-fix the slide.
5. Examine the slides under oil immersion.
kObservation
The stained glass slide with the microbial smear can be observed under oil immersion
lens of the microscope.
zz Precaution
The smear should not form a thick layer. If it does, microscope light will not pass
through, or on dying the stain may crack. On the other hand, a too thin smear will
not allow sufficient contrast to observe the bacteria.
4
4.10.3 Differential Stains
zz Introduction
The differential stain technique distinguishes two kinds of organisms. This dif-
ferential technique separates bacteria into two groups, gram-positive bacteria and
gram-negative bacteria based on cell wall type, and this makes it an essential tool
for classification of bacteria. Another differential stain technique is the acid-fast
technique which differentiates species of Mycobacterium from other bacteria. An
acid-fast stain is used for bacteria with a high mycolic acid content in their cell wall,
making them resistant to the decolorizing step of the Gram stain.
4.10.3.1 Gram Staining

zz Background
Gram stain is one of the most widely applied differential stains in bacteriology. Gram
stain was first developed by Hans Christian Gram in 1884, a Danish physician. By
applying Gram staining, it is possible to classify all bacteria into either gram positive
or gram negative. The differential response of bacteria to Gram stain is related to the
chemical composition and architecture of the surface layers of the cell wall.
zz Objective
To perform Gram-staining technique
zz Principle
The Gram stain reaction is based on the difference in the chemical composition of
bacterial cell walls. In this staining procedure, the bacterial smear is subjected to a
stain (crystal violet), a mordant (dye fixative), and a decolorizing agent followed by
a counter stain (safranin). When the bacteria are stained with primary stain crystal
violet and fixed by the mordant, some of the bacteria are able to retain the primary
stain and some are decolorized by alcohol. The walls of gram-positive bacteria have
a thick layer of peptidoglycan and low lipid content. Decolorizing the cells causes
the thick walls to dehydrate and shrink, which closes the pores and prevents the
stain from exiting. So, the gram-positive bacteria appear blue or purple in color. In
case of gram-negative bacteria, it also takes up the crystal violet iodine complex but
as the cell walls of this type of bacteria are very thin, they become easily expose to
alcohol or decolorizer which dissolves their thin lipid layer and allows the crystal
violet iodine complex to leach out. Then, when again stained with safranin, they
take the stain and appear pink. The bacteria which retain the crystal violet stain
appear dark blue (or violet) and are called gram positive and those that stained red
109 4
or pink are called gram negative. The Gram’s stain requires four different solutions:
a basic or primary dye, a mordant, a decolorizing agent, and a counter stain.
zz Requirements
55 Bacterial culture
55 Gram staining reagents
–– Crystal violet
–– Gram’s iodine solution
–– Ethyl alcohol (95%)
–– Safranin
55 Staining tray, wash bottles, droppers
55 Distilled water
55 Glass marker
55 Inoculation loop
55 Spirit lamp/Bunsen burner
55 Bright field microscope
zz Procedure
1. Obtain clean and grease-free glass slides.
2. Prepare a smear by taking a loopful of bacterial culture.
3. The slide is air-dried and heat-fixed.
4. Crystal violet is applied to the smear for 30 s or 1 min.
5. Then the slide is rinsed off with water.
6. The slide is then flooded with Gram’s iodine for 1 min.
7. Then the slide is washed off with 95% alcohol first and then with water.
8. Safranin is added to it for about 1 min and is washed with water.
9. The slide is then air-dried and by applying immersion oil to the smear we can
observe it under microscope.
10. Prepare a bacterial smear and heat-fix on the slide using standard procedure.
Pour a few drops of crystal violet on the smear.
11. Wait for 1 min and wash with tap water. Then flood the smear with the Gram’s
iodine for 1 min and again wash with tap water.
12. Decolorize the stain with ethyl alcohol (95%) by dropping the reagent drop-wise
until crystal violet fails to wash from the smear.
13. Wash it with tap water and counterstain with safranin for 45 s and wash again
with water.
14. Blot dry the slides, using an adsorbent paper and air-dry.
15. Observe the slide under oil immersion objective of the microscope.
zz Results
Bacteria that appear violet (deep blue) are referred to as gram positive and those
appearing pink (slight red) are called gram negative.
Identify which smear has taken the violet stain and which has taken only the
counter stain.
zz Precautions
55 Don’t make the bacterial smears too thick.
55 Don’t expose the slides to 95% alcohol for longer period (. Fig. 4.3).
.. Fig. 4.3 Steps of Gram Gram positive Gram negative

staining. (Reproduced under a
Creative Commons Attribution
License 4.0 license from Parker Fixation
et al. 2016)
Crystal violet
4 Iodine treatment
Decolorization
Counter stain
safranin
4.10.4 Acid-Fast Staining

zz Background
Acid-fast staining is a widely used differential technique which was first developed by
Ziehl and later on modified by Neelsen. Therefore, this method is also called as Ziehl-
Neelsen (ZN staining) technique. The main aim of this staining is to differentiate
bacteria into acid-fast and non-acid-fast groups. Acid-fast organisms are those that
are capable of retaining the primary stain when treated with an acid (fast = holding
capacity). Members of the Actinomycetes under the genus Nocardia (N. brasilien-
sis and N. asteroides) are partially acid-fast. Oocysts of coccidian parasites, such as
Cryptosporidium and Isospora, also belong to acid-fast groups.
zz Objective
To perform acid-fast staining to differentiate acid-fast and non-acid-fast groups of
bacteria
zz Principle
Some bacteria resist colorization by both acid and alcohol and hence are referred
to as acid-fast microorganisms. Acid-fast procedure is used to distinguish acid-fast
bacteria (red in color) from non-acid-fast (blue color) bacteria. Acid fastness is due
to quite high lipid content of cell walls, particularly that of mycolic acid (a group
of branched chain hydroxyl lipid). When the smear is stained with carbolfuchsin,
it solubilizes the lipoidal material present in the mycobacterial cell wall, but by the
application of heat as a mordant, carbolfuchsin further penetrates through the lipoi-
dal wall and enters into the cytoplasm. Then the smear is decolorized by decolorizing
agent (3% HCl in 95% alcohol), but the acid-fast cells are resistant due to lipoidal
material in their cell wall which prevents the penetration of decolorizing solution.
The non-acid-fast organisms lack lipoidal material in their cell wall due to which they
are easily decolorized leaving the cells colorless. Then the smear is counterstained
with methylene blue. Only decolorized cells will absorb the counter stain methylene
blue and will appear blue, while non-acid-fast cells retain the red color.
111 4
zz Requirements
55 Mycobacterium culture
55 Concentrated carbolfuschin
55 Methyl blue
55 Decolorizing solvent: Ethyl alcohol (95%) 97 mL, concentrated HCl −3 mL
55 Loefflers’ alkaline methylene blue
55 Bunsen burner/spirit lamp
55 Inoculation loop
55 Glass slide
55 Staining tray, wash bottles, droppers
55 Microscope
55 Composition of carbolfuchsin stain
i Basic fuchsin 0.3 g

Ethanol (95%) 10 mL
Phenol (heat melted crystals) 5 mL
The basic fuchsin dissolved in ethanol, and then in phenol in water, is mixed and kept
for several days. Filter it before use.
55 Composition of decolorizing solvent
i Ethanol (95%) 97 mL
Hydrochloric acid (conc.) 3 mL
55 Composition of counter stain
i Methylene blue chloride 0.3 g

Distilled water 100 mL
zz Procedure
1. Prepare a smear of two bacteria on separate slides.
2. Allow the smears to dry in air and heat-fix the smears over the flame.
3. Heat the slides with a jet of steam coming from water bath for about 5–10 min.
4. Flood the smear with carbolfuchsin and steam for 3–5 min. Don’t boil it.
5. Then wash it with gentle steam of water until no color appears from effluent.
6. Decolorize with acid alcohol until the preparation appears faint pink or color-
less, i.e., for about 15–20 s.
7. Again wash with water as before.
8. Now counterstain with malachite green (1%) for 20–30 s and wash with water as
before. No heating is required.
9. Blot the slide dry with bibulous paper.
10. Then examine the slide under oil immersion objective using a microscope and
record the observations in the notebook.
zz Results
Acid-fast bacteria appear red and non-acid-fast bacteria appear blue.
4.10.5 Special Staining

4.10.5.1 Capsule Staining
zz Background
The purpose of the capsule stain is to reveal the presence of the bacterial capsule.
The water-soluble capsule of some bacterial cells is often difficult to see by standard
simple staining procedures or after the Gram stain. Capsule staining methods were
developed to visualize capsules and to yield consistent and reliable results. Capsule
4 may appear as clear halo when a fresh sample is stained by Gram or Leishman stain,
and by negative staining – – using India ink and nigrosin.
zz Objective
To stain and determine the presence (or absence) of a capsule and its shape and thickness
zz Principle
Certain bacteria and yeasts have a protective outer structure called a capsule. Since
the presence of a capsule is directly related to a microbe’s virulence (its ability to
cause disease), the ability to determine whether cells in a sample have capsules is
an important diagnostic tool. Capsules do not absorb most basic dyes; therefore, a
negative staining technique (staining around the cells) is typically used for capsule
staining. The dye stains the background but does not penetrate the capsules, which
appear like halos around the borders of the cell. The specimen does not need to be
heat-fixed prior to negative staining.
zz Requirements
55 Culture of Enterobacter e.g. Klebsiella, Clostridium, etc.
55 Congo red
55 Acid alcohol
55 Acid fuchsin
55 Staining tray
55 Inoculation loop
55 Glass slide
55 Blotting paper
55 Microscope
zz Procedure
1. A loop containing the bacterial innoculum is placed within India ink applied on
the slide.
2. A small portion of the culture is emulsified in a drop of ink.
3. A clean cover slip is placed over the preparation; prevent air bubbles. Press down
gently.
4. Then it is examined under dry objective.
4.10.5.2 Endospore Staining

zz Background
Chemically, the capsular material is a polysaccharide, a glycoprotein, or a poly-
peptide. Capsule staining is more difficult than other types of differential staining
113 4
procedures because the capsular materials are water soluble and may be dislodged
and removed with vigorous washing. Bacterial smears should not be heated because
the resulting cell shrinkage may create a clear zone around the organism, an artifact
that can be mistaken for a capsule.
zz Objective
To stain and observe the shape and size of the spore by differential staining technique
zz Principle
The capsule is non-ionic, so the dyes commonly used will not bind to it. Two dyes,
one acidic and one basic, are used to stain the background and the cell wall, respec-
tively. Negative staining methods contrast a translucent, darker-colored background
with stained cells but an unstained capsule. The background is formed with India
ink or nigrosin or Congo red. A positive capsule stain requires a mordant that pre-
cipitates the capsule. By counterstaining with dyes like crystal violet, methylene blue,
or carbolfuchsin, bacterial cell wall takes up the dye. Capsules appear colorless with
stained cells against dark background.
zz Procedure
1. Using sterile technique, add a loopful of bacterial culture to a tube with 1 mL
NaCl.
2. Add one drop of carbolfuchsin into the tube and mix gently.
3. Heat the mixture under flame for 1 min.
4. Place a drop of mixture onto the glass slide.
5. Place a drop of nigrosin onto the same glass slide next to the drop of mixture of
bacteria and dye.
6. Use another slide to drag the nigrosin-cell mixture into a thin film along the first
slide.
7. Allow to air-dry for 5–7 min (do not heat-fix).
8. Examine the smear microscopically (100×) for the presence of encapsulated cells
as indicated by clear zones surrounding the cells.
zz Results
55 Encapsulated bacteria: Clear halos (capsules) are observed around pink bacteria
against dark background
55 Bacteria without capsules: pink bacteria with no clear halos.
Examples of encapsulated and non-encapsulated bacteria:

55 Encapsulated bacteria: Bacillus anthracis, Klebsiella pneumoniae, Streptococcus
pneumoniae, Neisseria meningitidis, Clostridium perfringens
55 Non-encapsulated bacteria: Neisseria gonorrhoreae, etc.
4.10.6 Fungal Staining

zz Objective
To perform fungal mycelia staining by using lactophenol cotton blue (LPCB) stain
zz Principle
The fungal propagules either are hyaline (colorless) or of different colors. The hyaline
mycelia/spores/conidia, etc., and their cytoplasm can be stained by using lactophenol
and cotton blue. Cotton blue stains cytoplasm and results in light blue background,
whereas lactophenol acts as cleaning agent. The lactophenol cotton blue wet mount
preparation is the most widely used method for staining and observing fungi under
microscope. The preparation has three components: Lactophenol plus cotton blue,
Glycerol, and Lactic acid.
4
zz Requirements
55 Young fungal cultures (e.g., Aspergillus, Rhizopus, Penicillum sp.)
55 Mounting fluid: Lactophenol plus cotton blue
55 Glass slides
55 Cover slips
55 Needles or stiff inoculation wire
55 Ethanol (95%)
55 Bunsen burner/Spirit lamp
55 Euperol or nail polish
55 Microscope
zz Procedure
1. Young cultures (5–7 days old) of fungi growing on culture medium (PDA or Cza-
pek Dox) are prepared.
2. A drop of 70% alcohol is placed on a clean microscope slide.
3. A drop of mounting fluid is put in the center of a glass slide.
4. A portion of mycelia mat from fungal colony is transferred to a drop of mounting
fluid with the help of a flamed and cooled needle or stiff inoculation wire (inocu-
lation loop used for transferring bacteria or yeast is not suitable for transferring
fungal mycelia).
5. With the help of two needles, the fungal propagules are gently spread so that the
mycelia get separated and mixed with the stain.
6. The processes are repeated for all the fungal colonies.
7. Observe the slide(s) under low and higher magnification under the objective of
a microscope and study the types of conidia, conidiophore, and structure of
hyphae and their arrangement.
8. Identify the fungi following the mycological literature.
zz Result
Fungal mycelia will stain blue color forming a layer inside the unstained cell wall of
hyphae, conidiophores, phialides, and conidia, which are surrounded by a light blue
background on the slide.
zz Precautions
1. The preparation may be mounted in lactophenol only in case of dark color mold.
2. Air bubbles may also be removed by placing the slide in a vacuum chamber.
3. A gentle pressure to the cover glass may be applied to spread the fungal elements
to be examined, if not clearly visible.
4.11 · Microbial Growth Curve
115 4
4.11 Microbial Growth Curve
zz Background
Microbial growth is defined in terms of an increase in number of cell (population
number) which occurs by cell division. Bacteria normally reproduce by binary fis-
sion, thus doubling their number with each division. Estimation of bacterial growth
requires inoculation of a sterile broth medium and incubation of the culture under
optimum temperature and shaking condition.
zz Objective
To plot microbial growth curve for shake flask culture using turbidity method
zz Principle
Bacterial growth may be measured by a variety of techniques such as direct micro-
scopic cell count, standard plate count and, and indirect turbidometric method. The
turbidity measurement relates cell number to the turbidity (cloudiness) of a broth
culture. Turbidity or optical density (OD) is an indirect measure of bacterial num-
ber. The increase in turbidity indicates bacterial growth. OD is directly proportional
to the cell concentration. To measure the OD, spectrophotometer can also be used.
Preparation of bacterial growth curve requires to culture a bacteria for 24 h with
continuous shaking in flask and to measure OD of the culture broth at 600 nm wave-
length in a UV-VIS spectrophotometer at regular time intervals during the incuba-
tion period. When conditions for balanced growth are favorable, the bacterial growth
curve shows four distinct phases of growth such as lag phase, logarithmic phase,
stationary phase, and death phase (. Fig. 4.4).
zz Requirements
55 Sterile nutrient broth media (100 mL)
55 Inoculums
55 Laminar hood
55 Spirit lamp
55 Match box
.. Fig. 4.4 Sigmoid growth

curve of bacteria in culture
Stationary
No. of viable bacteria
phase
Log Death
phase phase
Lag
phase
Time period
55 Micro pipettes (1000 μL)

55 Sterile micro tips (1000 μL)
55 Centrifuge
55 Distilled water
55 Blotting paper
55 Tissue paper
55 Turbidometer
4
zz Procedure
1. 100 mL of nutrient broth is prepared in a 250 mL of conical flask and sterilized
using autoclave.
2. The sterile media was inoculated with the given inoculums of 2 mL aseptically in
the laminar hood and shaken well for uniform distribution of inoculums.
3. 1.5 mL of culture broth is aseptically removed from the shake flask using sterile
micropipette and added to a centrifuge tube.
4. The conical flask is incubated in the shaker incubator at 37 °C and 100 rpm.
5. The broth solution is centrifuged at 10,000 rpm for 10 min to get the pellet of
biomass.
6. Supernatant is decanted followed by addition of 1.5 mL of distilled water to the
pellet.
7. Place the centrifuge tubes in to the holes of vertex mixer platform and spin it
vigorously for 2 min to suspend the microbial pellet in the water.
8. The vertex mixer is switched off and tubes are taken out and observed for the
turbidity of the solution.
9. The OD of this turbid solution is taken at 600 nm using spectrophotometer and
recorded.
10. After 1 h, the orbital shaker is stopped and the shake flask is carefully taken out
and put it in the laminar hood.
11. Steps from 3 to 10 are repeated for 24 h of incubation time.
12. The growth curve is plotted taking time on X-axis and OD on Y-axis.
zz Result
A sigmoidal growth curve of the bacterium is obtained.
Observation table: measure of bacterial growth (OD) at 2-h intervals.
Incubation time (h) 0 2 4 6 8 10 12 14 16 18 20 22 24
OD
4.12 Isolation of Genomic DNA from Bacteria

zz Background
Genomic DNA (deoxyribose nucleic acid) constitutes the total genetic information
of an organism which is transferred from generation to generation. The genetic mate-
rials of almost all organisms are DNA, the only exceptions being some viruses that
have RNA (ribose nucleic acid). In prokaryotic organisms like bacteria, DNA is pres-
ent as a single, circular chromosome. In some bacteria, extra chromosomal DNA
(plasmid) is also present in addition to chromosomal DNA. Technique to obtain
4.12 · Isolation of Genomic DNA from Bacteria
117 4
enough quantity, purity, and integrity of nucleic acids is essential for molecular biol-
ogy procedures. It is an essential step in many downstream procedures such as clon-
ing, polymerase chain reaction (PCR), restriction enzyme digestions, microarray
analysis, and nucleotide sequencing.
Different procedures are adopted for the isolation of genomic DNA from dif-
ferent sources. It includes some basic steps like cell lysis, followed by deproteination
and recovery of DNA. Isolation of DNA usually begins with lysis or breakdown of
a tissue or cell and then with the separation of total DNA from RNA, protein, lipids,
polysaccharides, etc. Lysis process is essential for the destruction of protein structure
and release of nucleic acid from the nucleus. The DNA pellet can be resuspended in
either sterile distilled water or TE buffer (10 mM Tris: 1 mM EDTA) and stored at
4 °C for further use.
zz Role of Reagents Used in Genomic DNA Isolation

Tris–EDTA buffer
Tris acts as a common buffer to control pH. It helps in solubilizing the DNA and
protecting it from degradation. Tris interacts with the lipopolysaccharides present on
the outer membrane which helps permeabilize the membrane. This effect is enhanced
with the addition of EDTA. EDTA is a chelating agent that binds the metal ions like
the magnesium (Mg2+) required for DNase activity. Mg2+ is considered an essential
cofactor for action of most of the nucleases.
Sodium dodecyl sulfate (SDS)
Sodium dodecyl sulfate (SDS) is a strong anionic detergent that can solubilize the
membrane protein and lipids. This will help the cell membranes to break down and
expose the chromosomes to release DNA. Thus, SDS will disrupt all chromatin struc-
ture, transforming the compacted DNA into extended protein-free DNA molecules.
Sodium acetate
It is used in the precipitation of proteins present in chromosomal DNA.
NaCl solution
5 M NaCl solutions provide Na+ ions that block the negative charge of phosphate
in DNA. Negatively charged phosphate in DNA causes the phosphate molecules to
repel each other. The Na+ ions form an anionic bond with the negatively charged
phosphates, thereby neutralizing the negative charge and allowing the DNA mol-
ecules to come together.
RNase
It helps to remove RNA from a mixture of RNA and DNA and extract pure DNA
thus prevents any RNA contamination.
Proteinase-K
It degrades mostly protein impurities to get quality DNA product. It is also respon-
sible for inactivation of nucleases, thus preventing damage of isolated DNA.
Phenol: Chloroform: Isoamyl alcohol
Phenol: chloroform: isoamyl alcohol is used for extraction purpose. It separates a
mixture of molecules based on differential solubility of the individual molecules in
two different immiscible liquids. Chloroform in combination with phenol is more
efficient in denaturing proteins than when used alone. Chloroform isoamyl alcohol
binds to proteins and lipids of a cell membrane and dissolves them and thus forms
a precipitate. The liquid-protein complexes are present in organic phase and nucleic
acid in aqueous phase.
Phenol: chloroform: isoamyl alcohol aids in the separation of two phases and
decreases the process of foaming during extraction. Hence, this reagent is important
as it inhibit the formation of foam and lessen the chance of DNA trapping.
Precipitation of nucleic acids
Alcohol precipitation is the most commonly used method for nucleic acid precipita-
4 tion. Ethanol (2× volume) or isopropanol (2/3rd volume) is the standard alcohol
used for nucleic acid precipitation. The nucleic acid can be precipitated spontane-
ously and pelleted by centrifugation.
Ethanol/isopropanol
Ethanol/isopropanol is used for precipitation of genomic DNA. DNA is highly
insoluble in isopropanol. Isopropanol is a better alternative for ethanol due to its
greater potential for DNA precipitation in lower concentrations in lesser time.
TE Buffer
It protects the DNA from degradation. It acts as a buffer for the storage of DNA.
zz Objective
To isolate genomic DNA from bacterial sample by using phenol-chloroform extrac-
tion method.
zz Principle
DNA extraction starts by disrupting the cells and then by exploiting the differ-
ences in properties between DNA, protein, and other constituents. The sample is
first treated with extraction buffer containing EDTA, Tris/HCl, and CTAB. When
the cell membrane is exposed to the CTAB extraction buffer, the detergent captures
the lipids and proteins allowing the release of genomic DNA. In the extraction buf-
fer, EDTA works as a chelating component that binds with magnesium, while Tris/
HCl gives the solution a pH buffering capacity. In this extraction step, the proteins
and other cell lysates dissolved in the aqueous solution are separated from CTAB
nucleic acid complex. Under low salt concentration, the contaminants of the nucleic
acid complex do not precipitate and can be removed by extraction of the aqueous
solution with chloroform. The chloroform denatures the proteins and facilitates the
separation of the aqueous and organic phases. The nucleic acid is liberated from the
detergent by treating the aqueous solution with isopropyl alcohol, which precipitates
the nucleic acid. Under these conditions, the detergent which is more stable in alco-
hol than water can be washed out.
zz Requirements
55 Cooling centrifuge
55 Vortex
55 Water bath
55 Incubator
55 Freezer/deep freezer
4.12 · Isolation of Genomic DNA from Bacteria
119 4
zz Chemicals
Molecular biology grade chemicals were used for the study. Pyrogen-free, double-
distilled (glass), and deionized water should be used.
55 TE buffer (adjust the pH to 8.0.)
–– 10 mM Tris–HCl
–– 1 mM EDTA, pH 8.0.
55 SDS (10%)
–– Dissolve 10 g of SDS (sodium dodecyl sulfate) in 80 mL of distilled water and
adjust the volume to 100 mL with distilled water.
55 NaCl (5 M)
–– Dissolve 29.2 g of NaCl (MW 58.5) in 80 mL of distilled water. Adjust the
volume to 100 mL with distilled water.
55 CTAB/NaCl solution
–– 10% CTAB in 0.7 M NaCl in a beaker, 4.1 g NaCl was dissolved in 80 mL
water, and 10 g CTAB was slowly added while heating and stirring. If neces-
sary, heat to 60 ± 0.1 °C to dissolve. Final volume was adjusted to 100 mL.
55 Chloroform-isoamyl alcohol
–– 24:1 by volume. 5 mL
55 Isopropanol
55 Ethanol
–– 70% in water
55 Phenol-chloroform-isoamyl alcohol
–– 25:24:1 by volume. 5 mL
zz Procedure
1. Take 12-h-old bacterial cultures and centrifuge in a micro-centrifuge tube at
10,000 rpm for 10 min at 4 ± 0.1 °C.
2. Discard the supernatant and dissolve the pellet with 576 μL TE buffer by repeated
pipetting.
3. Add 30 μL of 10% SDS and incubate at 37 °C for 1 h. Then add 100 μL of 5 M
NaCl and mix the content thoroughly.
4. Again add 80 μL of CTAB/NaCl solution and mix the content thoroughly, incu-
bate for 10 min at 65 ± 0.1 °C.
5. Approximately equal amount of (0.7–0.8 mL) chloroform–isoamyl alcohol is added,
mixed thoroughly, and centrifuged at 10,000 rpm for 10 min in a micro-centrifuge.
6. Remove aqueous supernatant to a micro-centrifuge tube leaving the interface
behind.
7. Add equal volume of phenol chloroform, isoamyl alcohol, and extract thor-
oughly and spin in a micro-centrifuge for 10 min.
8. Transfer the supernatant to a fresh tube and add 0.6 volume of isopropyl alcohol
to precipitate the nucleic acid.
9. Shake the tubes back and forth until a strong white precipitate becomes clearly
visible.
10. Centrifuge the tubes at 10,000 rpm for 5 min and decant the supernatant.
11. Add 70% ethanol to the pellet to remove residual CTAB. Decant the ethanol
carefully and dry the pellet.
12. Resuspend the pellet in either sterile distilled water or TE buffer or store at 4 °C
till further use.
zz Result
The quantity and quality of DNA is measured by 0.8% agarose gel electrophoresis
and UV-visible spectrophotometer, respectively.
zz Precaution
1. DNase-free plastic wares and reagents must be used during the procedure.
2. Phenol is a strong acid that causes severe burns, whereas chloroform is a carcino-
gen. Therefore, they should be handled carefully.
4
4.13 NA Quantification and Quality Analysis by Agarose
D
Gel Electrophoresis
zz Background
The quantity and quality of isolated DNA are necessary to ascertain the approximate
quantity of DNA obtained and the suitability of DNA sample for further analysis.
This is important for many applications including digestion of DNA by restriction
enzymes or PCR amplification of target DNA. Two most commonly used techniques
for quantifying the amount of nucleic acid in a preparation are gel electrophore-
sis (qualitative) and spectrophotometric analysis (quantitative). Electrophoresis is a
technique used to separate and sometimes purify macromolecules, especially proteins
and nucleic acids that differ in size, charge, or conformation.
zz Objective
To visualize and quantify DNA by using agarose gel electrophoresis technique
zz Principle
Agarose gel electrophoresis is a procedure used to separate DNA fragments based
on their molecular weight and is an intrinsic part of almost all routine experiments
carried out in molecular biology. When charged molecules are placed in an elec-
tric field, they migrate toward either the positive or negative pole according to their
charge. In contrast to proteins, which can have either a net positive or net negative
charge, nucleic acids have a consistent negative charge imparted by their phosphate
backbone and, therefore, migrate toward the anode. DNA is electrophoresed within
a matrix or gel made up of agarose. The gel is immersed within an electrophoresis
buffer that provides ions to carry current and certain buffers to maintain the pH at
a relatively constant value. By varying the concentration of agarose, fragments of
DNA from about 200 to 50,000 bp can be separated by using standard electropho-
retic techniques. This method of quantification is based on ethidium bromide fluo-
rescent staining of DNA. Ethidium bromide is a fluorescent dye, which intercalates
between the stacked bases. DNA complex is much greater than the unbound dye. UV
irradiation at 254 nm is absorbed by the DNA and transmitted to the dye and the
bound dye itself absorbs the radiation at 302 and 366 nm. This energy is retransmit-
ted at 590 nm, the reddish orange region of the visible spectrum.
zz Requirements
55 Horizontal gel electrophoresis
55 UV transilluminator or gel documentation system
4.13 · DNA Quantification and Quality Analysis by Agarose Gel…
121 4
zz Chemical Reagents
55 50 × TAE (pH 8.5)
i Tris base 242 g

Glacial acetic acid 57.1 mL
EDTA 37.2 g
Water 1000 mL
55 Ethidium bromide solution
i EtBr 10 mg
Distilled water 1 mL
EtBr is dissolved in 1 mL of sterile distilled water by continuous stirring.
55 Agarose solution
–– Agarose – 0.8 g
–– TAE buffer – 100 mL
55 Agarose is dissolved in TAE buffer and boiled till agarose dissolves completely
and a clear solution is formed.
–– Gel loading dye (6×)
–– Bromophenol blue – 0.25%
–– Xylene cyanol – 0.25%
–– Sucrose – 40%
–– Marker
zz Procedure
1. Dissolve 0.8 g of agarose in 100 mL of 1× TAE buffer and boil till agarose com-
pletely dissolves and forms a clear solution.
2. Place the comb at the edge of the gel-casting tray and pour the boiled agarose into
the gel-casting tray after adding 10 μL of ethidium bromide and allow to solidify.
3. After solidification, gently remove the comb and place the tray into the gel tank.
4. Pour 1× TAE buffer till the gel is submerged.
5. Mix 5 μL of DNA with 1 μL of gel-loading dye and load into the wells. Addi-
tion of dyeallows us to note the extent to which the samples might have migrated
during electrophoresis.
6. Load at least 1 or 2 wells with uncut, good-quality DNA (50 and 100 ng) as
molecular standards.
7. Run the electrophoretic gel at 70 V till the dye has migrated one-third of the
distance in the gel.
zz Result and Observation

Visualize the DNA using a UV transilluminator and quantify in comparison with the
fluorescent yield of the standards (. Fig. 4.5).
zz Precaution
Ethidium bromide should not be added to the agarose gel in very hot condition as the
vapors of EtBr are reported to be carcinogenic.
.. Fig. 4.5 Genomic DNA in

agarose gel. (Reproduced with
permission from Kuhn et al.
2018)
4.14 DNA Quantification by Spectrophotometric Method

zz Background
Analysis of UV absorption by the nucleotides provides a simple and accurate esti-
mation of the concentration of nucleic acid in a sample. This method is however
limited by the factors such as the quantity of DNA and the purity of the prepara-
tion. Accurate analysis of the DNA preparation may be impeded by the presence of
impurities in the sample or if the amount of DNA is too little.
zz Objective
To quantify DNA by using spectrophotometric method
zz Principle
The absorption properties of DNA can be used for detection, quantification, and
assessment of purity. Purines and pyrimidines in nucleic acid show absorption max-
ima around 260 nm (e.g., dATP: 259 nm, dCTP: 272 nm, dTTP: 247 nm), whereas
the proteins typically absorb at 280 nm. If the DNA sample is pure and without sig-
nificant contamination from protein and organic solvents, the ratio of OD260/OD280
can be determined to assess the purity of the sample. Lower A260/280 values may
indicate protein contamination. Lower A260/230 values indicate contamination with
salts or some solvents (e.g., phenol). A ratio near 2 indicates a highly pure nucleic
acid sample (Luebbehusen 2010). Although λmax is constant, the extinction coeffi-
cient of nucleic acids depends on their environment. The presence of RNA, sheared
DNA, etc., could interfere with accurate estimation of total genomic DNA.
zz Requirements
55 TE buffer
55 Sample DNA
4.15 · PCR Amplification
123 4
zz Procedure
1. Take 1 mL of TE in a cuvette and calibrate the spectrophotometer at 260 nm as
well as 280 nm.
2. Add 10 μL of each DNA sample to 990 μL of TE buffer and mix well.
3. Use TE buffer as a blank in other cuvette of the spectrophotometer.
4. Note the OD260/OD280 values on spectrophotometer.
5. Calculate the OD260/OD280 ratio.
zz Result
1. A ratio of 1.8:2.0 denotes that the absorption in the UV range is due to nucleic
acids.
2. A ratio lower than 1.8 indicates the presence of proteins and/or other UV
absorbers.
3. A ratio higher than 2.0 indicates that the sample may be contaminated with chlo-
roform or phenol. In either case (<1.8 or >2.0), it is advisable to reprecipitate the
DNA.
zz Estimation
The amount of DNA can be quantified using the following formula:
OD 260 × dilution factor × no. of oligonucleotide

DNA concentration ( µg/mL ) =
1000
Spectrophotometric conversions for nucleic acids
55 1A 260 of ds DNA = 50 μg/mL
55 1A 260 of 260 of ss oligonucleotides = 33 μg/mL
55 1A 260 of ss RNA = 40 μg/mL
4.15 PCR Amplification

zz Background
The polymerase chain reaction (PCR) is a technique used for in vitro amplification of
specific DNA sequence to millions of copies within a short time developed by Kary
Mullis in 1983 (Mullis and Faloona 1987). Most PCR methods typically amplify
DNA fragments between 0.1 and 10 kilo base pairs (kb) in size, although some tech-
niques allow for amplification of fragments up to 40 kb in size. The amount of ampli-
fied product is determined by the available substrates in the reaction, which become
limiting as the reaction progresses.
zz Objective
To study the 16S rRNA gene amplification by polymerase chain reaction
zz Principle
The method relies on thermal cycling, consisting of cycles of repeated heating and
cooling for enzymatic replication of the DNA. As PCR progresses, the DNA gener-
ated is itself used as a template for replication, setting in motion a chain reaction in
which the DNA template is exponentially amplified. There are three major stages of
PCR which are repeated for 30–40 cycles. These stages include (i) denaturation, (ii)
annealing, and (iii) extension. In the first cycle, the double-stranded template DNA
is denatured by heating to above 90 °C for about 15 s. The temperature is then cooled
to 40–60 °C. Cooling of the mixture in this range allows the primers to hybridize
with the appropriate flanking regions. The primers are oriented on the template, so
their 3′ ends are directed toward each other. The precise temperature is critical and
each PCR system has to be defined and optimized. After the annealing step is over,
the temperature is raised to 70 °C. At this temperature, complementary hybrids are
4 obtained with high specificity.
zz Requirements
55 Thermal cycler
55 PCR tube (0.2 mL)
55 Micropipette
55 Vortex
55 Spinner
55 Freezer/deep freezer
zz Chemicals
Molecular biology-grade chemicals are used for the study. Pyrogen-free, double-
distilled (glass), deionized water is used.
(a) Nuclease-free water
(b) 10× Taq buffer A
(c) 10 mM dNTPs mix
(d) Primer (universal primer)
(i) 27F (5′-AGAGTTTGATCCTGGCTCAG-3′)
(ii) 1492R (5′-GGTTACCTTGTTACGACTT-3′)
(e) Taq polymerase
(f) Genomic DNA
zz Procedure
1. Isolate the bacterial genomic DNA by following the genomic DNA isolation pro-
tocol of bacteria described earlier.
2. Prepare 25 μL of reaction mixture containing 10 pmol of both the primers (27 F
(5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTAC-
GACTT-3′)) in a thermal cycler, 100–150 ng DNA, 5 mM dNTPs, 1× Taq poly-
merase buffer containing 15 mM MgCl2, and 0.5 μL of Taq DNA polymerase
(5U μL−1).
3. Mix the reaction mixture well by vortexing.
4. Settle down the reaction mixture in the PCR tube.
5. Set the PCR program: initial denaturation at 94 °C for 5 min, 30 cycles of 94 °C
for 1 min, 50 °C for 1 min, 72 °C for 2 min, and final elongation at 72 °C for
10 min.
6. Separate the amplified product on 1% agarose gel.
7. After gel electrophoresis, perform ethidium bromide staining.
8. After staining when viewed under a UV transilluminator, the DNA appears pink.
Other strains such as Sybr Gold or Sybr Green II may also be used.
4.16 · Sequence Analysis
125 4
zz Precautions
55 Add all the chemicals carefully.
55 Wear and change gloves often.
55 Always change to fresh pair of gloves when leaving and entering PCR reagents
mixing areas. Change gloves whenever contamination between samples is possible.
4.16 Sequence Analysis

zz Background
In recent years, identification of unknown bacterial isolates has been enhanced via
PCR amplification of 16S rRNA gene sequencing and subsequent sequence analysis.
Portions of the 16S rRNA gene sequences within bacteria are identical in all known
bacteria. Conversely, between these homologous regions, there are unique sequences
of DNA associated with specific bacteria. The so-called “universal” primers have
been designed that anneal to the conserved rRNA gene sequences, allowing amplifi-
cation of the unique sequences within the conserved regions.
zz Principle
Aliquots of amplified gene that result from PCR with these universal primers can be
sequenced inexpensively in commercial laboratories, or in many cases at university
facilities. Once the sequence of the amplified rRNA is known, it can be used to iden-
tify the original bacterial source of RNA at the genus or species level. Computerized
sequence databases have been compiled on large numbers of bacterial species. This
allows for a comparison of an unknown rRNA gene sequence product with the known
bacterial sequences that exist in the data base. Several computer software sequence
analysis programs are available to aid in sequence searches. One such data base is
the Basic Local Alignment Search Tool (BLAST), which is provided by the National
Center for Biotechnology Information (NCBI) and is available on the Internet. This
program allows researchers to perform sequence comparisons between known rRNA
sequences and the amplified rRNA sequence of interest. For example, a sequence
from an unknown soil isolate can be compared to all known sequences in the database,
allowing for the isolate to be identified, or at least determine what organism it is most
closely related to. MEGA software can be used to determine the phylogeny relation-
ship of the microorganism with its related neighbor using the 16S rRNA sequence.
zz Procedure
1. Download your unknown 16S rRNA gene sequence from the BlackBoard Web-
site to your computer. If you did not get a good sequence, there would be some
extra files on the BlackBoard that you may download instead of yours.
2. Open your browser (Netscape, Internet Explorer, etc.): 7 http://www.ncbi.nlm.
nih.gov/BLAST
3. Under “Basic BLAST,” click on “nucleotide blast.” A new page will open up.
4. This page has several components. These components and the parameters that we
choose are as follows:
55 Enter Query Sequence: Either paste your sequence directly in the box given
or press the “Browse” button and find your file on your computer to be used
in the search. You can also give a name to your search in the “Job Title” box.
55 Choose Search Set: Since our search does not involve human or mouse data-
bases, click on the “Others (nr etc.)” button. Click on the drop-down menu
and choose “16S ribosomal RNA sequences (Bacteria and Archaea).”
55 Next in the “Exclude” option, place a checkmark in the box for “Uncul-
tured/environmental sample sequences.” Leave the optional “Organism” and
“Enterez Query” as they are.
55 Program Selection: Click on the radio button next to “Highly similar sequences
(megablast).”
4 55 Finally press the big blue BLAST button on the left panel to get to the results.
However, if you find that you need to find more similar species, first press
“Algorithm parameters” located underneath the BLAST button and increase
the number of results from the default 100 to 250 or even 500, then press the
BLAST button.
5. After pressing the BLAST, a new page appears that may get refreshed a few times.
Finally a final page will appear that will give you an ID # (Query ID), the number
of nucleotides present in your sequence (Query Length), and few other infor-
mation. Underneath you will see red bars, each of which represents a matching
species. Scrolling down further, you can see the species names that match your
unknown. The best match is at the top and the matching becomes less accurate
as you go down. Pressing on the “Accession” brings up a rather comprehensive
description of the matching species, while pressing the “Max Score” shows the
actual matching of nucleotides between your sample and the matching species.
The species with the highest Max Score is usually the species that should be taken
as your unknown result. However, sometimes a few species have the same Max
Score, and then you may need to read about the differences among them by con-
sulting the Bergey’s manual and checking the described characteristics against
your unknown.
6. Make a printout of the first two to four pages of these results and include it in
your report.
4.17 Sampling of Air Microflora

zz Background
Microbial flora of air are those organisms that are found temporarily suspended
in air or carried about on dust particles or droplets (Stetzenbach et al. 2004). A
collection of airborne biological particles is called a bioaerosol. Bioaerosols are
generated by a wide variety of natural and human-made processes including cough-
ing, sneezing, wave action, splashes, wind, cooling towers, ventilation systems, etc.
Temperature and relative humidity are the two most important factors affecting the
survival of microorganisms in the airborne state. Sampling microorganisms from the
air requires simultaneous processing of collected sample. Microbial numbers in air
are generally quite low, and it is impractical to collect large volumes of air for later
processing. Collection of viable airborne microorganisms requires approaches that
minimize sampling stress and capture the particle size of bioaerosol droplets in which
airborne microorganisms typically occur. There are three principal methods used to
quantify microorganisms in the air: (1) impingement (trapping of airborne practices
4.17 · Sampling of Air Microflora
127 4
in a liquid matrix), (2) impaction (forced deposition of airborne particles on a solid
surface), and (3) filtration (trapping of airborne particles by size exclusion).
zz Objective
To perform sampling of air microflora and determination of microbial density
zz Principle
One of the simplest methods of isolating and detecting microorganisms in the air
is the use of gravity (or settling) plates (Geller et al. 2007). Gravity plates are Petri
plates filled with sterile media which are left open with the agar surface exposed.
Microorganisms carried on by dust or droplets simply “settle” on the agar surface
and, following incubation, reproduce over and over to eventually form individual
colonies. The nutrient medium we will be using in our Petri dishes for this exercise is
called Plate Count Agar (or PCA).
zz Requirements
55 500 mL conical flask
55 Cotton
55 Alcohol
55 Bunsen burner
55 Nutrient agar
55 Sabouraud dextrose agar
55 Blood agar
55 Petri plates
55 Autoclave
55 Biological incubator
zz Procedure
1. Settle plate method using three open 8.5 cm diameter Petri dishes (60.0525 cm2
areas) containing different cultures are used. This method allows the bacteria- or
fungi-carrying particles to settle on culture media.
2. Bacteria are collected on nutrient agar (NA) and blood agar (BA) to which an
antifungal agent (griseofulvin) has been incorporated to inhibit the growth of
fungi, while fungi are collected on Sabouraud dextrose agar (SDA) plates to
which an antibacterial agent (chloramphenicol) has been incorporated to inhibit
the growth of bacteria.
3. After the solidification of the plates, the plates are exposed to air for about 30 min
for fungus and 5–15 min for bacteria.
4. Then the plates are incubated for 24 h at 37 °C for bacteria and for 5–7 days at
25 °C for fungi.
5. To avoid self-contamination of agar plates during air sampling, sterile gloves,
mouth masks, and protective gown are worn, and before using the agar plates,
they are also checked visually for any microbial growth.
zz Result
1. The sampling is performed to obtain information on the concentration of all
viable particles in the air. Subsequently, the bacterial and fungal cultures can be
identified by microscopic characteristics and biochemical reactions.
2. Determination of microbial density

Following incubation of culture plates, bacterial and fungal colony forming
units (CFU) were enumerated. Afterward, the mean colony forming units per
cubic meter (CFU/m3) of air of the plates collected in triplicates were determined
using the following equation as described.
−1
N = 5a × 104 ( bt )
Where N is the microbial CFU/m3 of indoor air; a is the number of colonies per
4 Petri dish; b is the Petri plate surface area (cm2); and t is the exposure time of the
petri dish (minutes).
3. After the microbial density of the resultant colonies on each plate has been deter-
mined, the colonial morphology of the different colonies formed was noted and
identical colonies were sub-cultured into nutrient Agar (NA) or Sabouraud dex-
trose agar (SDA) plates, incubated appropriately, and stored for further identifi-
cation and characterization.
4.18 Antibiotic Assay

zz Background
Antibiotics are commonly used for the treatment of infectious diseases. Once the
causative organism is identified for specific disease and is isolated, it is important to
test the sensitivity of the organism to the effective antibiotics. In the clinical labo-
ratories, antibiotic-impregnated discs are commonly used to identify the antibiotic
sensitivity of the causative organism. The effectiveness of the antibiotic in this test
is based on the size of the zone of inhibition. The zone of inhibition also depends
on the diffusibility of antibiotic, the size of inoculums, type of medium, and other
factors. The concentration of antibiotic at the edge of zone of inhibition represents
the minimal inhibitory concentration (MIC) of the antibiotic. Alternatively, the anti-
biotic disc with different concentration of antibiotic could be employed in the test.
The minimal inhibitory concentration is the lowest concentration of antibiotic that
exhibits the zone of inhibition on the assay plate.
zz Objective
To study the antibiotic sensitivity of bacterial strains and to determine the minimal
inhibitory concentration (MIC) of antibiotics to bacterial strain
zz Principle
Because of convenience, efficiency, and cost, the disk diffusion method is probably
the most widely used method for determining antimicrobial resistance in clinical set-
ting. The zone around an antibiotic disk that has no growth is referred to as the zone
of inhibition. This zone is then measured in millimeter and compared to a standard
interpretation chart used to categorize the isolate as susceptible, intermediately sus-
ceptible, or resistant.
zz Requirements
55 Antibiotic discs
55 Mueller–Hinton agar
4.18 · Antibiotic Assay
129 4
55 Bacterial cultures (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus
aureus)
55 Cotton swab
55 Petri plates
zz Procedure
(A) Antibiotic Sensitivity Test
1. Select and label the cultures that are to be used for the antibiotic sensitivity assay
(E. coli, Pseudomonas aeruginosa, Staphylococcus aureus).
2. Prepare the Mueller–Hinton agar plates (at least three plates).
3. Aseptically take a swab of the test culture and inoculate the surface of the Muel-
ler–Hinton agar plate completely so as to make a lawn.
4. Allow at least 5 min for the agar surface to dry before applying disc.
5. Take forceps and sterilize the tip by dipping in alcohol and then by flaming. Allow
to cool.
6. Carefully take the antibiotic disc and place over the agar plate at least 15 mm
from the edge of the plate. Gently press the disc to give a better contact with the
agar.
7. Place at least six different antibiotic discs at the same distance apart from each
other in the agar plates.
8. Incubate the plates in inverted position for 16–18 h at 37 °C.
zz Result
1. Observe the zone of inhibition around the antibiotic discs.
2. Indicate whether test organism is resistant (no zone of inhibition) or sensitive
(clear zone of inhibition) to the antibiotic.
(B) Determination of Minimal Inhibitory Concentration of the Antibiotic

1. Prepare four nutrient agar plates.
2. Select a test organism and an antibiotic from the previous experiment that showed
good inhibition zone.
3. Inoculate the nutrient agar plate with the selected test organisms as lawn culture.
4. Allow the plates to dry for 5 min.
5. Place a disc impregnated with different concentrations of antibiotic (10–500 μg/
mL).
6. Incubate the plates for 12–18 h.
7. Measure the zone of inhibition on the plate by a ruler.
zz Result
1. Tabulate the obtained concentration of antibiotic versus the diameter of the zone
of inhibition.
2. Plot a graph taking the concentration of antibiotic on X-axis and the square of
the diameter of the zone of inhibition along Y-axis.
3. The straight line that intercepts the X-axis is the minimal concentration of the
antibiotic required for inhibiting the growth of the test organism.
4.19 Coliform MPN Test for Bacteriological Examination of Water

zz Background
The presence of coliform bacteria in water is an indication that disease-causing bac-
teria also may be present and that the water is unsafe to drink. Coliform bacteria (of
which Escherichia coli is a member) in food or water has been accepted as indicative
of recent fecal contamination and the possible presence of frank pathogens. Coliform
bacteria occur normally in the intestines of humans and other warm-blooded ani-
4 mals and are discharged in great numbers in human and animal wastes. In polluted
water, coliform bacteria are found in densities roughly proportional to the degree of
fecal pollution. When members of the coliform group are present, other kinds of
microorganisms capable of causing disease also may be present. MPN test and the
membrane filter test have been the methods most commonly used for the detection of
coliforms in water. Presence of coliform bacteria is the most common indicator used
in microbial examination of water. Total coliforms are grouped into two categories:
(1) fecal coliform (thermo-tolerant coliform) and (2) non-fecal coliform.
zz Principle
E. coli could be easily detected by its ability to ferment glucose (later changed to lactose).
The MPN test for coliforms consists of three steps: a presumptive test, a confirmation
test, and a completed test. The first step is the presumptive test. A set of tubes of lauryl
sulfate tryptose (LST) lactose broth is inoculated with samples of water and incubated.
Lauryl sulfate is a surface-active detergent which inhibits the growth of gram-positive
organisms while encouraging the growth of coliforms. Coliforms use any oxygen present
in the broth and then ferment the lactose producing acid and gas under anaerobic condi-
tions. Gas formation in 24 or 48 h is a positive test. Once it has been established that gas-
producing lactose fermenters are absent in the water, it is presumed to be safe. However,
gas formation may also be caused by non-coliform bacteria. Some of these, such as
Clostridium perfringens, are gram-positive. To confirm the presence of gram-negative
lactose fermenters, the next step is to inoculate media such as Levine’s eosin methylene
blue (EMB) agar, Endo agar, or brilliant green lactose bile (BGLB) broth from positive
presumptive tubes in what is called the confirmed test. In some cases, the organisms must
be isolated and stained to provide the competed test. Positive BGLB tubes are streaked
on eosin–methylene blue (EMB) agar. The two dyes, eosin and methylene blue, also
inhibit the growth of gram-positive organisms. Typical colonies are isolated on nutrient
agar slants and inoculated into LST broth. If gas is now formed by 24 or 48 h, a Gram
stain is made from the growth on the slant. If the cells are gram negative after examina-
tion under oil and there is no indication of spores, the completed test is considered to be
positive. Further biochemical studies (IMViC) may be performed on isolated cultures.
In practice the competed test is seldom performed. All three tests are necessary to prove
that an organism in a water sample is in truth a coliform. In actual practice, when it has
been shown that the presumptive and confirmed tests give essentially the same results,
then the completed step is generally not done because of the time it takes.
zz Requirements
55 Water sample
55 Three test tubes containing Durham tubes and double-strength LST lactose
broth (DSLB)
55 Six test tubes containing Durham tubes and single-strength LST lactose broth (SSLB)
4.19 · Coliform MPN Test for Bacteriological Examination of Water
131 4
55 10 mL pipette
55 1 mL pipette
55 Pipette bulb
55 Incubator at 35 °C
zz Procedure
First Period
zz Presumptive Test
1. Set up three DSLB and six SSLB tubes as illustrated in Step 1. Label each tube
according to the amount of water that is to be dispensed to it: 10 mL, 1.0 mL, and
0.1 mL, respectively.
2. Mix the bottle of water to be tested by shaking 25 times.
3. With a 10 mL pipette, transfer 10 mL of water to the DSLB tubes.
4. With a 1.0 mL pipette, transfer 1 mL of water to each of the middle set of SSLB
tubes and 0.1 mL to each of the last three SSLB tubes.
5. Incubate the tubes at 35 °C for 48 h.
zz Results
1. Examine the tubes and record the number of tubes in each set that have a gas
bubble(s) in the inverted Durham tube.
2. Record this data for your report.
Second Period
zz Materials
55 Incubated tubes from the previous week
55 Petri plates of Levine’s EMB agar
55 Petri plates of Endo agar
55 Inoculation loop
55 Gas burner
zz Confirmed Test
1. Select one positive lactose broth tube from the presumptive test and streak one
plate of each of Levine’s EMB agar and Endo agar.
2. Use a streak method which will produce good isolation of colonies such as
described. If all your tubes are negative, borrow a positive tube from another
student.
3. Incubate the plate for 24 h at 35 °C.
Third Period
zz Materials
Incubated plates from the previous week
zz Results
The media are checked for the presence of coliform colonies.
Results are recorded. If no coliform colonies are present, the water is considered
safe to drink.
Take-Home Messages
55 In natural environment, microorganisms usually exist in a mixed population.
However, pure cultures are required for several microbiological experiments. Thus,
pure cultures are prepared in the laboratory by serial dilution and pour plating
methods and their cultures are maintained at 4 °C.
55 Sterilization is essential to make any object free from contamination. It can be
achieved by exposure to lethal, physical, or chemical agents and, in special case
such as heat-labile solution, by filtration method.
4 55 Steam heat used for sterilization of aqueous solution and dry heat are principally
used to sterilize the glassware. Chemical sterilizing agents are also useful in the
sterilization of volatile substances.
55 Bacterial growth takes place very fast when inoculated in a suitable medium. The
term “growth of microorganism” usually refers to the total population rather than
the size or mass of an individual microorganism. Growth of microorganism can
be determined by spectrophotometrically measuring bacterial growth in liquid
medium. pH changes in the medium have a profound influence on growth activi-
ties and survival of microorganisms.
55 Most of the microorganisms are colorless and therefore are invisible when sus-
pended in an aqueous medium. Therefore, staining procedures are performed to
make microorganisms visible under microscopes. Staining can be achieved either
by simple staining (using single dye) or by differential staining (using more than
one dye) to distinguish different strains or different structures within a cell.
55 Culture media vary in form and composition depending upon the requirement of
the species to be cultivated. Thus, several media are available for use in microbiol-
ogy which offers advantage for isolation, maintenance, and characterization of
certain group of microorganism.
55 Multiple tube fermentation test or most probable number test is used to determine
the presence of coliform bacteria in the water sample which has a wide spread use
to detect any contamination due to fecal material.
55 Antibiotic sensitivity test will be performed by the commonly used agar diffu-
sion method which is designed to determine the smallest amount of the antibiotic
needed to inhibit the growth of microorganism.
55 Air sampling is the process of capturing contaminants in a known volume of air.
In active monitoring, a microbiological air sampler physically draws a known vol-
ume of air through particle collection device, which can be liquid or a solid culture
media or a nitrocellulose membrane, and the quantity of microorganisms present
is measured in CFU/m3 of air. Microflora suspended in air or carried with dust
particles or droplets are known as air air-borne microflora. Air microflora can be
quantified by the following methods: (1) impingement (trapping of airborne prac-
tices in a liquid matrix), (2) impaction (forced deposition of airborne particles on
a solid surface), and (3) filtration (trapping of airborne particles by size exclusion).
55 DNA isolation, purification, and amplification are routine procedures in many molec-
ular biology analyses. Besides sequence analysis of DNA, RNA, and peptide is essen-
tial to understand the structure, function, and evolution of the microorganism. In this
chapter, you will learn the methods of isolation of genomic DNA from bacteria, PCR
amplification of bacterial DNA as well as the bioinformatics approaches like BLAST
homology searching and phylogeny tree construction for sequence analysis.
Bibliography
133 4
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135 5
Plant Tissue Culture

Techniques and Nutrient
Analysis
Contents
5.1 Plant Propagation and Tissue Culture – 136

5.1.1 Preparation of Culture Media – 136
5.1.2 Callus Culture – 138
5.1.3 Cell Suspension Culture – 141
5.1.4 Protoplast Culture – 143
5.1.5 Artificial Seeds/Synthetic Seeds – 146
5.2 DNA Isolation and Analysis – 148

5.2.1 Isolation of DNA from Plant Sample – 148
5.2.2 Agarose Gel Electrophoresis – 151
5.2.3 Spectrophotometric Quantification of DNA – 153
5.3 Plant Nutrient Analysis – 154

5.3.1 Estimation of Protein Content in Plant Samples – 154
5.3.2 Estimation of Fatty Acid – 155
5.3.3 Estimation of Ammonia Nitrogen – 156
5.3.4 Estimation of Phosphorus in Plant Sample – 157
5.3.5 Estimation of Potassium in Plant Sample – 158
5.3.6 Estimation of Sulfur – 159
5.3.7 Estimation of Boron in Plant Sample – 160
5.3.8 Estimation of Zn, Cu, Mn, and Fe in Plant Sample – 162
Bibliography – 163

136 Chapter 5 · Plant Tissue Culture Techniques and Nutrient Analysis

You will learn compositions of different media required for plant tissue culture and differ-
ent culturing techniques employed for plant tissues. Further, you will learn the techniques
and principles behind the isolation of DNA from plant tissues along with its quantifica-
tion. Finally, you will learn different elements required for plant growth and development.
You will become skilled at the principles and techniques behind the analysis of different
nutrients required for plant growth.
5.1 Plant Propagation and Tissue Culture

5
5.1.1 Preparation of Culture Media
zz Background
The basal medium is formulated so that it provides all the compounds needed for
plant growth and development, including certain compounds that can be made
by an intact plant, but not by an isolated piece of plant tissue. Macronutrients are
required in millimolar (mM) quantities, while micronutrients are needed in much
lower (micromolar, μM) concentrations. Vitamins are organic substances that are
parts of enzymes or cofactors for essential metabolic functions. Sugar is essential for
in vitro growth and development. Plant growth regulators (PGRs) at a very low con-
centration (0.1–100 μM) regulate the initiation and development of shoots and roots
on explants on semisolid or in liquid medium cultures. Auxin and cytokinins are the
two most important classes of PGRs used in tissue culture.
zz Objective
Preparation of MS (Murashige and Skoog 1962) basal medium for plant tissue
culture
zz Principle
A defined nutrient medium is composed of inorganic salts, a carbon source, nitrogen
source, vitamins, amino acids, and growth regulators. A gelling agent is also used in
the static medium. There are different types of media that are used for plant tissue
culture purpose out of which Murashige-Skoog (Murashige and Skoog 1962), Whites
(White 1963), Eriksson (Eriksson 1965), B5 (Gamborg et al. 1968), N6 medium (Chu
et al. 1975), Nitsch’s medium (Nitsch and Nitsch 1969) are quite popular medium
and are widely used in plant tissue culture. Among these, MS medium (Murashige
and Skoog 1962) is commonly regarded as the most suitable basic tissue culture
medium for plant regeneration. The numbers in MS are used to indicate the sucrose
concentration in the medium, for example, MS0 contains no sucrose and MS20 con-
tains 20 g/l sucrose.
zz Requirements
55 Amber bottles
55 Plastic beakers (100 ml, 500 ml, and 1000 ml)
55 Measuring cylinders (500 ml)
55 Glass beakers (50 ml)
5.1 · Plant Propagation and Tissue Culture
137 5
55 Disposable syringes (5 ml)
55 Disposable syringe filter (0.22 μm)
55 Autoclaved centrifuge tubes (2 ml)
55 Stand
55 Autoclave
55 pH meter
55 Benzylaminopurine
55 Naphthalene acetic acid
zz Preparation of Stock Solutions

55 The concentrated solutions of the desired composition of a medium are pre-
pared, which are known as stock solutions. These stock solutions are stored prop-
erly and at the time of use diluted adequately to get the desired concentration.
Usually, the stock solutions are prepared in higher concentrations: 10× or 100×
concentrations.
55 For the preparation of stock solutions, chemicals are dissolved either in double
distilled water or in demineralized water. While dissolving the nutrients in water,
one compound is added at a time to avoid precipitation. For macronutrients stock
solution, calcium chloride is dissolved separately in water and then added to the
rest of the solution to avoid precipitation.
55 Preparation of growth regulators: Growth regulators are not soluble in water.
The compounds should be dissolved in few milliliter of solvents (e.g., ethanol).
Similarly, cytokinin is dissolved in 1N NaOH. Following the dissolution, water is
added slowly to make up to the requisite volume (. Table 5.1).
zz Procedure
Preparation of MS medium using the stock solution
1. About 400 ml of distilled water is taken in a 2 l conical flask.
2. Each of 50 ml/l solution of stocks A and B are poured into the conical flask.
3. Then 5 ml/l each of other stock solutions are added to it.
4. Further, 3% (w/v) of sucrose per liter is added to the medium.
5. Then the solution is mixed thoroughly.
6. The pH of the medium is adjusted to 5.6–5.8 by adding few drops of 1N HCl or
1N NaOH.
7. Following that about 6 g/l of agar is added to the medium and mixed thoroughly
with intermittent stirring.
8. Then the volume is made up to 1 liter and heated until the agar is fully mixed.
9. Then the medium is immediately poured into culture flasks or tubes and closed
with non-absorbent cotton or polypropylene caps.
10. The culture flasks and tubes are then autoclaved at 121 °C at 15 lbs pressure for
15 minutes.
11. Following autoclaving, the medium is cooled in laminar air flow to 50–60 °C.
12. Hormones which are prepared earlier by filtering through 0.22 mm membrane
filter are dispensed into sterile cooled medium.
13. Then the culture flasks or tubes are cooled further and stored at 4 °C.
.. Table 5.1 Composition of revised MS medium
A. Mineral sails
Macronutrients Minor elements
Salts ml/l mM Salts mg/1 μM
NH4NO3 1650 N 41.2 H3BO3 6.2 100
KNO3 1900 18.8 MnSO4·4H2O 22.3 100
CaCl2·2H2O 410 3.0 ZnSO4·4H2O 8.6 30

5 MgSO4·7H2O 370 1.5 KI 0.83 5.0
KH2PO4 170 1.25 Na2MoO4·2H2O 0.25 1.0
Na2-EDTA 37.3a Na 0.20 CuSO4·5H2O 0.025 0.1
FeSO4·7H2O 27.8a Fe 0.10 CoCl2·6H2O 0.025 0.1
B. Organic constituents
Sucrose 30 g/l Agar 10 g/1
Edamin (optional) 1 g/l myo-Inositol 100 mg/l
Glycine 2.0 mg/l Nicotinic acid 0.5 mg/l
Indole Acetic acid 1–30 mg/l Pyridoxin·HCI 0.5 mg/l
Kinetin 0.04–10 mg/l Thiamin·HCI 0.1 mg/l
Reproduced with permission from Murashige and Skoog (1962)

a5ml/l of a stock solution containing 5.57 g FeSO4·7H2O and 7.45 g Na2-EDTA per liter of H2O
zz Result
The prepared medium is stored for 3–4 days and checked for any contamination. If
sterilization is not properly done, then the medium gets contaminated. If the medium
is contaminated, it should be discarded.
Preparation of other plant tissue culture media
Few other important media for plant tissue culture can be prepared using the
composition given below following the above-discussed method (. Table 5.2).
5.1.2 Callus Culture

zz Objective
To induce callus from the supplied plant part.
zz Principle
Callus, a mass of undifferentiated plant cells, has the capacity to regenerate into a
whole plant. Explants from stems, leaves, roots, flowers, or seeds of the plant can be
induced to form callus. It is usually carried out on solidified gel medium in the pres-
ence of growth regulators and initiated by inoculation of small explants or sections
139 5
.. Table 5.2 Composition of media frequently used for Plant tissue culture (Saad and
Elshahed 2012)
Medium Components MS Gs W LM VW Km M NN
(mg/l)
Macronutrients
Ca3(PO4)2 200.0
NH4NO3 1650.0 400.0 720.0
KNO3 1900.0 2500.0 80.0 525.0 180.0 180.0 950.0
CaCl2·2H2O 440.0 150.0 96.0 166.0
MgSO4·7H2O 370.0 250.0 720.0 370.0 250.0 250.0 250.0 185.0
KH2PO4 170.0 170.0 250.0 150.0 150.0 68.0
(NH4)2SO4 134.0 500.0 100.0 100.0
NaH2PO4·H2O 150.0 16.5
CaNO3·4H2O 300.0 556.0 200.0 200.0
Na2SO4 200.0
KCl 65.0
K2SO4 990.0
Micronutrients
KI 0.83 0.75 0.75 80.0 0.03
H3BO3 6.20 3.0 1.5 6.2 6.2 0.6 10.0
MnSO4·4H2O 22.30 7.0 0.75 0.075 25.0
MnSO4·H2O 10.0 29.43
ZnSO4·7H2O 8.6 2.0 2.6 8.6 0.05 10.0
Na2MoO4·2H2O 0.25 0.25 0.25 0.25 0.05 0.25
CuSO4·5H2O 0.025 0.025 0.25 0.025 0.025
CoCl2·6H2O 0.025 0.025 0.025
Co(NO3)2·6H2O 0.05
Na2EDTA 37.3 37.3 37.3 74.6 37.3 37.3
FeSO4·7H2O 27.8 27.8 27.8 25.0 27.8 27.8
MnCl2 3.9 0.4
Fe(C4H4O6)3·2H2O 28.0
Vitamins and other supplements
Inositol 100.0 100.0 100.0 100.0
Glycine 2.0 2.0 3.0 2.0 2.0

(continued)
.. Table 5.2 (continued)
Thiamine HCl 0.1 10.0 0.1 1.0 0.3 0.3 0.5
Pyridoxine HCl 0.5 0.1 0.5 0.3 0.3 0.5
Nicotinic acid 0.5 0.5 0.5 1.25 5.0
Ca-panthothenate 1.0
Cysteine HCl 1.0
Riboflavin 0.3 0.05
5 Biotin 0.05 0.05
Folic acid 0.3 0.5
MS Murashige and Skoog, Gs Gamborg et al., W White, LM Lloyd and McCown, VW Vacin and
Went, Km Kudson modified, M Mitra et al., NN Nitsch and Nisch media
from established organ or other cultures (the inocula). The initiation of callus forma-
tion from explants is referred to as callus induction or primary callus. Secondary cal-
lus culture is initiated from primary callus. The callus culture cannot be maintained
at the same medium for longer period, as it would lead to cell loss, thereby reducing
cell division and secondary metabolite production. Hence callus should be subcul-
tured regularly once in 4–5 weeks. Auxin, at a moderate to high concentration, is
the primary hormone used to produce callus. In some species, a high concentration
of auxin and a low concentration of cytokinin in the medium promote abundant
cell proliferation with the formation of callus. Callus culturing is performed in the
dark, while light can encourage the differentiation of the callus. Due to their size and
nature, callus cultures have an inherent degree of heterogeneity. Callus can be very
compact or friable.
zz Requirements
55 Fresh carrot roots
55 Beakers
55 Conical flasks
55 Cotton plugs
55 Agar agar
55 Sterilized blades
55 Blade holder
55 Micropipettes
55 Sterilized microtips
55 Cork borers
55 Petri dishes
55 5% Teepol
55 70% v/v ethanol
55 0.8% Sodium hypochlorite solution
55 MS medium with 0.5 mg/2,4-dichlorophenoxyacetic acid.
141 5
zz Procedure
1. A fresh healthy tap root of carrot is taken and washed thoroughly under running
tap water to remove surface dirt.
2. The tap root is then dipped in 5% “Teepol” for 10 minutes following which it is
washed.
3. The surface of the tap root is then sterilized by immersing in 70% (v/v) ethanol
for 1 minute, followed by 20–25 minutes in 0.8% sodium hypochlorite solution.
4. Then the root is washed thrice in sterilized distilled water to remove hypochlorite
solution completely followed by blotting it dry using sterile tissue paper.
5. Then it is transferred to a sterilized Petri dish containing a filter paper.
6. Then a series of transverse slices of root 1 mm in thickness are cut using sharp scal-
pel. (Each piece contains a whitish circular ring of cambium around the pith. Cut an
area of 4 mm2 across the cambium from each piece so that each piece contains part
of phloem. Cambium and xylem size and thickness of explant should be uniform.)
7. Then each piece is transferred to a sterile Petri dish and the lid of Petri dish is
properly placed.
8. The cap of culture tube is removed and the explants are transferred by holding
the tube at an angle of 45° to the nutrient medium – Gamborg’s B5 or MS basal
medium supplemented with 0.5 mg/2,4-dichlorophenoxyacetic acid.
9. The cap is placed immediately on the open mouth of each tube.
10. Then after inoculation the culture tubes are incubated in dark at 25 °C for
4–6 weeks in the culture room.
11. The culture tubes are observed carefully and discarded if any culture shows signs
of contamination.
12. Usually, after 4 weeks, the explants are supplemented with 2, 4-D. After substan-
tial callus formation, the best callus explant is selected.
13. Then small pieces from callus region are excised into two or three pieces and each
piece of callus tissue is transferred to a tube containing fresh medium to produce
large amount of calluses.
zz Results
The calluses will proliferate at the cutting edges of the explants. Then white and frag-
ile undifferentiated mass of calluses appear in the MS basal medium supplemented
with growth regulators and other additives (. Fig. 5.1).
zz Precautions
Throughout the manipulation process, keep the forceps and scalpels in 95% ethanol
and heat thoroughly before use.
5.1.3 Cell Suspension Culture

zz Objective
To study the suspension culture
zz Principle
Tissues and cells cultured in a liquid medium produce a suspension of single cell and
clumps ranging from few to many cells, which are called suspension cultures. For
5
.. Fig. 5.1 Callus formation from carrot root. (Reproduced with permission from Oleszkiewicz et al.
(2018))
s uspension culture, cells can be isolated from in vitro plant materials either by mechan-
ical means or by enzymatic digestion and from callus induced from any explants. Cell
suspension culture can be obtained from virtually any part of the plant, just as callus
cultures. Suspension cultures when maintained under controlled conditions of light,
temperature, and aeration follow a predictable pattern of growth curve. Suspension
cultures should be sub-cultured frequently and regularly to maintain viability.
zz Requirements
55 Seeds of Nicotiana tobaccum
55 Cotton plugs
55 Agar agar
55 Forceps
55 Sterilized blades and blade holder
55 Cork borers
55 Petri dishes
55 5% Teepol
55 HgCl2
55 70% ethanol
55 Sodium hypochlorite solution
55 MS containing 2,4-dichlorophenoxyacetic acid (Conc. 1–2 mg/l)
55 Shaker
55 Centrifuge
55 Microscope
zz Procedure
1. The seeds are submerged in water with a few drops of detergent in beaker follow-
ing which seeds are cleaned with the help of cheese cloth.
2. The seeds are then submerged in 70% alcohol for 30–60 seconds following which
the alcohol is decanted.
3. The seeds are then transferred to a flask or beaker containing 20–40% sodium
hypochlorite for 15–20 minutes following which the seeds are rinsed 5 times with
sterile water.
143 5
4. Then 2–3 seeds are placed per culture vessel on the surface of MS agar medium
without growth regulators.
5. The cultures are then incubated at 25 °C for 16 hours photoperiods under
1000 lux intensity for 1–2 weeks and the germination of the seedlings is checked.
6. The germinated seedlings are then collected when the cotyledons are fully
expanded and the epicotyl is beginning to emerge.
7. The shoot apex is excised with or without cotyledons and stem base is inserted
into the medium and incubated for at 25 °C in dark condition.
8. The development of callus is noted in the culture medium at 3–4 weeks.
9. Then small pieces of callus of 0.5 g weight are subcultured on the same fresh
medium for proliferation.
10. Then a piece of callus is transferred to a sterile Petri dish.
11. Then the callus is broken into 20–30 small pieces of 2 cm diameter with the help
of forceps and small pieces of callus are transferred to the liquid media using
forceps incubated on a gyratory shaker set at 125 rpm placed in a temperature-
controlled room.
12. Then it is subcultured every week. For the first few subcultures, a portion of the
spent medium is removed and replaced with fresh medium with the help of a
large bore-sized pipette.
13. The establishment of suspension culture is confirmed once small cluster and
aggregates of cells are found to be dispersed.
zz Results
The cell suspension formed is examined under a microscope by taking an aliquot
from the flask on a glass slide under sterile condition.
5.1.4 Protoplast Culture

zz Background
Protoplast is a spherical, osmotically sensitive cell that refers to the cellular content
excluding cell wall, but it possesses plasma membrane. The protoplasts can regen-
erate cell wall, dedifferentiate to form callus, redifferentiate into shoots, roots, or
embryos, and produce a complete plantlet. Protoplasts can undergo fusion and take
up genes which offer great prospects in genetic engineering and crop improvement
programs. Generally, mesophyll tissues from leaves are preferred for isolation of pro-
toplast. Alternatively protoplasts can be prepared from the cultured cell materials.
zz Principle
Plant cell wall is composed of different types of polysaccharides such as cellulose,
hemicelluloses, and pectin. Cellulose microfibrils are cross-linked by highly branched
hemicelluloses, which are composed primarily of xyloglucans and embedded in a
matrix of pectic polysaccharides. Therefore, success of protoplast isolation pri-
marily lies with yield of large population of uniform and highly viable protoplasts.
Enzymatic dissolution of different components of the plant cell wall is essential for
isolation of protoplasts which depends upon use of several enzymes.
Protoplast can be isolated from cells by mechanical method and/or enzymatic
method.
In mechanical method, the cells are plasmolysed, causing the protoplast to shrink
away from the cell wall. The plasmolysed cells are cut with sharp blade to release
the protoplasts. The enzymatic method involves sequential enzymatic hydrolysis
or mixed enzymatic hydrolysis. The cells are separated by using pectin hydrolyzing
enzyme such as macerozyme or pectolyase. Once the cells are separated, they are
washed in CPW solution free of enzymes but containing plasmolyticum by gentle
centrifugation. The pellet is retained and resuspended in second enzymes, like, cellu-
lases and hemicellulases, used to hydrolyze the remaining cell wall components. Once
the protoplasts are released, they are washed with CPW to remove the debris. Thus
sequential isolation takes place in two steps.
5 5.1.4.1 Protocol-1
zz Requirements
55 Fresh and healthy leaves
55 Forceps
55 13% w/v Mannitol solution.
55 Digesting enzymes (0.4% w/v, cellulase and 0.15% w/v pectinase and sometimes
hemicellulase)
55 Phosphate buffer pH – 7
Buffer solution: – Tris–MES 20 mM (pH 5.5)

Mannitol 0.9 M
Washing buffer: – Tris–MES 20 mM (pH 7.5)
Mannitol 0.9 M
55 Digestion solutions
55 Solution 1: Macerozyme (0.1%) and cellulase (1%) in buffer solution
55 Solution 2: Pectolyase (0.1%) and caylase (1%) in buffer solution
zz Procedure
1. Mature and fresh leaves are collected from healthy plants and washed in tap water
to remove adhering soil particles followed by sterilization with sodium hypochlo-
rite solution for 10 minutes.
2. The leaves are then washed with sterile distilled water to remove the traces of
sodium hypochlorite.
3. Then buffer is added to maintain osmotic pressure.
4. The lower epidermis is then removed with forceps carefully.
5. The stripped surface of leaf is then kept in mannitol solution (13% w/v) for
3 hours to allow plasmolysis of cells.
6. About 1 g of leaves is peeled off and transferred into enzyme m ixture and incu-
bated for 12–18 hours at 25 °C.
7. Then leaf debris are removed with the help of forceps.
8. The enzyme solution containing protoplasts is filtered with a nylon mesh (45 mm)
and the filtrate is centrifuged at 75 g for 5 minutes.
9. The supernatant is decanted and 13% mannitol is added and centrifuged fol-
lowed by decantation.
10. Step 9 is repeated thrice and finally specific concentration of protoplast suspen-
sion is prepared (. Fig. 5.2).
145 5
.. Fig. 5.2 Pictorial representation of isolation of protoplast. (Reproduced with permission from Tor-
res (1989))
5.1.4.2 Protocol 2
zz Requirements
55 Fresh and healthy leaves
55 Forceps
Buffer solution: – Tris–MES 20 mM (pH 5.5)

Mannitol 0.9 M
Washing buffer: – Tris–MES 20 mM (pH 7.5)
Mannitol 0.9 M
55 Digestion solutions
55 Solution 1: Macerozyme (0.1%) and Cellulase (1%) in buffer solution
zz Procedure
1. Mature and fresh leaves are collected from healthy plants and washed in tap water
to remove adhering soil particles followed by sterilization with sodium hypochlo-
5 rite solution for 10 minutes.
2. The leaves are then washed with sterile distilled water to remove traces of sodium
hypochlorite.
3. The leaves are then cut into pieces and transferred to a Petri dish containing
15 ml of digestion solution 1 and incubated at 26 °C in the dark and under gentle
shaking (50 rpm).
4. After 10, 20, and 30 minutes of incubation, 20 μL of the sample is transferred
onto a slide and observed under microscope.
5. Finally, after 30 minutes of incubation, the digestion solution is removed using a
pipette and filtered through a nylon mesh or other filter (75–150 μm) with a funnel.
6. The Petri dishes are rinsed by 5 ml of washing solution.
7. The liquid is collected in a conical tube and centrifuged for 10 minutes at 500 rpm.
8. The protoplasts are isolated and are in the form of pellet.
9. The supernatant is removed with a pipette and discarded.
10. The pellet is washed with 5 ml of washing buffer and gently resuspended in the
solution with a Pasteur pipette.
11. Then it is centrifuged for 10 minutes at 500 rpm.
12. Step 11 is repeated twice and after the third centrifugation, the pellet is resus-
pended in 300 μL of washing solution.

The protoplast suspension can be checked under microscope for viability and quality.
zz Precaution
Never let the pellet without liquid.
5.1.5 Artificial Seeds/Synthetic Seeds

zz Background
Artificial or synthetic seeds refer to encapsulated explants such as shoot tips, axil-
lary buds, and somatic embryos in cryoprotectant material like hydrogel, alginate
gel, ethylene glycol, dimethyl sulfoxide, and others that can be developed into a
plant. Artificial seeds/synthetic seeds are most commonly described as encapsu-
lated somatic embryos. The idea of developing synthetic seeds or artificial seeds was
first conceived by Murashige in 1977. Initially, the development of synthetic seeds
had been restricted to encapsulation of somatic embryos in a protective jelly. Large
147 5
numbers of somatic embryos are produced in vitro as suitable planting materials.
Technology is developed to encapsulate them to produce artificial or synthetic seeds.
zz Objective
To develop artificial seed/synthetic seeds
zz Principle
The artificial seed structure mimics that of the conventional seed. It consists of both
explant material, which imitates the zygotic embryo in the conventional seed, and
the capsule (gel agent and additional materials such as nutrients, growth regulators,
anti-pathogens, bio-controllers, and bio fertilizers), which emulates the endosperm
in the conventional seed. The principle for alginate encapsulation formation depends
on the exchange of ions between Na+ in sodium alginate with Ca2+ in CaCl2·2H2O,
which happens when sodium alginate droplets involving the artificial embryos or any
other plant propagule is dropped into the CaCl2·2H2O solution, producing stable
explant beads. The solidity and rigidity of the capsule depend on two major factors:
concentration of gelling agents and mixing duration.
zz Requirements
55 Carrot seeds
55 Scalpel
55 Forceps
55 Dissecting needle
55 Petri dishes
55 Pipettes
55 Culture tubes
55 MS media with 2,4-dichlorophenoxyacetic acid (2.0 mg/l) and 3% sucrose for cal-
lus induction
55 Agar agar (6–0.8%)
55 2.4% Sodium alginate solution
55 75–100 mM autoclaved CaCl2 solution
zz Procedure
1. The carrot seeds are germinated in vitro. After 2 weeks, collect aseptically germi-
nated seedling with fully expanded cotyledons.
2. Then each seedling is placed on a sterile Petri dish, one at a time to prepare explants.
3. Then excise hypocotyls from each seedling and cut them transversely into two
equal halves.
4. Then the hypocotyl sections are inoculated on to a semisolid agar, and culture ves-
sels are sealed with parafilm and incubated in culture room condition for 4–6 weeks.
5. The callus is induced using aseptically germinated carrot seedling in MS medium
supplemented with 2,4-dichlorophenoxyacetic acid (2.0 mg/l) and 3%(w/v)
sucrose for callus induction.
6. Then small pieces of callus are excised and subcultured in fresh medium with the
same composition every month in order to maintain callus stocks.
7. A piece of carrot callus is transferred to a sterile Petri dish and split into small
pieces of carrot callus with forceps.
8. Then it is incubated for 1 week on a gyratory shaker set at 125 rpm.
.. Fig. 5.3 Artificial synthetic

seed. (Reproduced with permis-
sion from Saiprasad (2001))
9. A small sample is aseptically removed and transferred into a Petri dish.

10. Then formation of single cells and cell clusters and embryogenic cells is observed.
11. The cell suspension cultures are then transferred to a flask of liquid MS medium
without plant growth regulators.
12. Then it is incubated in the gyratory shaker.
13. The cultures are observed weekly for signs of somatic embryogenesis, and differ-
ent stages of somatic embryos will be visible after 1–2 weeks of transfer.
14. The suspension culture is filtered through 250 μ sieve to obtain separated somatic
embryos. The somatic embryos are transferred to liquid MS medium without any
plant growth regulators.
15. Then autoclaved 2.4% (w/v) sodium alginate solution is added.
16. A flask containing 75–100 mM autoclaved calcium chloride solution is taken.
17. Then sodium alginate solution with somatic embryos are taken in wide mouth
droppers and dropped into a flask containing 75–100 mM calcium chloride solu-
tion (30–40 drops per minute).
18. The resultant beads are allowed to remain in the calcium chloride solution for
30–40 minutes.
19. The beads are then collected by filtering through a nylon mesh.
20. The calcium chloride solution is recycled for making more beads but fresh solu-
tion is better (. Fig. 5.3).
5.2 DNA Isolation and Analysis
5.2.1 Isolation of DNA from Plant Sample
zz Background
The extraction of genomic DNA from plant material requires cell lysis, inactiva-
tion of cellular nucleases, and separation of the desired genomic DNA from cellular
debris. Ideal lysis procedure is rigorous enough to disrupt the complex starting mate-
5.2 · DNA Isolation and Analysis
149 5
rial (plant tissue), yet gentle enough to preserve the target nucleic acid. One of the
most widely followed extraction procedures is the protocol developed by Murray
and Thompson in 1980 for the extraction and purification of DNA from plants and
plant-derived foodstuff.
zz Objective
To isolate genomic DNA from supplied plant tissue
zz Principle
Cetyltrimethylammonium bromide (CTAB) captures lipids and proteins and other
cell lysates of the cell membrane allowing the release of the genomic DNA. However,
under low salt (NaCl) concentration, CTAB forms an insoluble complex with the
nucleic acids while the other cell components of the plant cell, viz., polysaccha-
rides, phenolic compounds, and contaminants remain in the supernatant and can be
washed away. The DNA is then released from DNA-CTAB complex by precipitat-
ing it by using sodium acetate-containing solution. The DNA is then purified with
ethanol or isopropanol. The EDTA binds with Mg, thereby decreasing the DNase
activity.
zz Requirements
55 Plant sample (leaf or any other part)
55 Mortar and pestle (sterile)
55 Spatula (sterile)
55 1.5 ml Centrifuge tube (sterile)
55 Liquid nitrogen
55 CTAB (2×)
–– CTAB – 10 g
–– 5 M NaCl – 140 ml
–– 2 M Tris–Cl, pH 8.0 – 25 ml
–– 0.5 M EDTA – 20 ml
–– Total volume is 200 ml
55 Chloroform and isoamyl alcohol solution in the ratio of 24:1
55 3 M Sodium acetate (pH 5.2):
–– Sodium acetate: 408.1 g
–– Sterile H2O: 800 ml
–– The pH of the solution is adjusted to 5.2 with glacial acetic acid and the volume
is made up to 1000 ml.
55 DNase-free RNase A
–– RNase A (10 mg/ml) in 10 mM Tris (pH 7.5) containing 15 mM NaCl
The solution is heated to 100 °C for 15–20 minutes to make it DNase free and then
it is cooled slowly to room temperature. Then small aliquots are made and stored at
−20 °C.
55 TE buffer (10 mM Tris, 1 mM EDTA), pH 8.0
–– Tris – 1.211 g
–– EDTA – 0.372 g
–– Sterile H2O ~800 ml
–– The pH is adjusted to 8.0 and the volume is made up to 1000 ml with sterile H2O.
zz Procedure
1. About 100 mg of plant sample is taken in a mortar and pestle and homogenized
to powder in the presence of liquid nitrogen and then transferred to a sterile
1.5 ml centrifuge tube.
2. Then about 1.5 ml of preheated extraction buffer (at 65 °C) is added to the
homogenate and mixed vigorously and incubated in water bath at 65 °C for
1 hour.
3. The temperature of the mixture is then brought down to room temperature and
then 667 μl chloroform: isoamyl alcohol (24:1) is added followed by mixing gen-
tly by inverting for a period of 15–20 minutes.
5 4. Then the mixture is centrifuged at 10,000 rpm at 4 °C for 10 minutes and then the
supernatant is collected followed by addition of 700 μl of cold isopropanol.
5. The mixture is then precipitated and then the nucleic acid is centrifuged for
5–10 minutes at 2000 rpm for 5 minutes at RT.
6. Then 500 μl of 70% ethanol is poured into the mixture to wash the pellet and the
supernatant is decanted and pellet is dried at RT.
7. Then the pellet is dissolved in 50 μl of 0.1 × TE+RNase (100 μg/ml) and incu-
bated for 1 hour at 37 °C.
8. The volume of DNA sample is increased to 250 μl with 0.1 × TE.
9. Then equal volume (250 μl) of chloroform: phenol: isoamyl alcohol (25:24:1) mix
is added to it and kept for 5 minutes and spun for 15 minutes at 12,000 rpm.
10. The upper aqueous layer is carefully separated and equal volume of chloroform
is added to the mixture (to remove phenol).
11. Then the mixture is centrifuged for 15 minutes at RT. Then the top layer is trans-
ferred to fresh tubes and the volume is measured.
12. Then about 1 ml of chilled absolute ethanol and 1/10th volume of 3M sodium
acetate (pH 5.2) are added and kept at −20 °C for 1 hour.
13. Following this, the mixture is centrifuged for 10 minutes.
14. Then the pellet is washed with 70% ethanol and centrifuged for 10 minutes.
15. The pellet is dried and dissolved in sterile water (. Fig. 5.4).
.. Fig. 5.4 Schematic presentation of DNA isolation from plants

151 5
The purity or quality of the extracted DNA is calculated by either spectrophotomet-
ric test or agarose gel electrophoresis.
Spectrophotometric Test
The optical density (OD) of the prepared DNA sample is checked at 260 and
280 nm. The pure DNA has an A260/A280 ratio of 1.8–2.0 in 10 mM Tris–Cl,
pH 8.5. However, any change in A260/A280 ratio indicates the presence of con-
taminants such as proteins or phenols or other salts.
Agarose Gel Electrophoresis
The electrophoresis of DNA samples on 0.8% agarose gel with DNA markers and
their staining with ethidium bromide are carried out to check the quality of the
extracted DNA.
About 2–4 μl of DNA sample is used to run in 0.8% agarose gel.
zz Precaution
1. Fresh plant sample should be used.
2. All the chemicals should be prepared freshly and stored accordingly.
5.2.2 Agarose Gel Electrophoresis

zz Background
Agarose gel electrophoresis is a widely used method for separation, identification,
and preparation of nucleic acids based on their molecular weight and is an intrin-
sic part of almost all routine experiments carried out in molecular biology. Agarose
is a linear polysaccharide having repeated basic unit agarobiose (galactose and
3,6-anhydrogalactobiose). Molten agarose forms a gel by hydrogen bonding and the gel
pore size depends on agarose concentration. The lower the concentration of agarose,
the higher the pore size. Agarose gels have a large range of separation, but relatively
low resolving power. By varying the concentration of agarose, fragments of DNA from
about 200 to 50,000 bp can be separated using standard electrophoretic techniques. This
method of quantification is based on ethidium bromide fluorescent staining of DNA.
zz Objective
To separate the extracted genomic DNA from plant sample by agarose gel electro-
phoresis
zz Principle
The DNA molecules are separated by electrophoresis based on their size, shape, and
magnitude of net charge on the molecules. Low concentration of agarose gel with
large pore permits separation of high molecular weight molecules and vice versa.
However, the relationship between fragment size and rate of migration is nonlin-
ear, since larger DNA fragments have greater frictional drag and are less efficient
in migrating through the agarose polymer. Ethidium bromide is a fluorescent dye,
which intercalates between the stacked bases of DNA. UV irradiation at 254 nm is
absorbed by the dye-stuffed DNA and the bound dye itself absorbs the radiation and
retransmits at 590 nm in the reddish-orange region of the visible spectrum.
zz Requirements
1. Extracted DNA sample
2. Agarose (0.8% in 1× TAE)
3. TAE buffer (50×)
Tris base 242 g
Acetic acid 57.1 ml
EDTA 37.2 g
Double distilled H2O to make up to 1000 ml
(1× TAE buffer is used for tank buffer and for preparation of gel)
4. Gel loading dye (5×)
55 Bromophenol blue 0.25%
5 55 Xylene cyanol 0.25%
55 Sucrose 40%
55 Marker
5. Ethidium bromide
10 mg in 1 ml 1× TAE buffer
zz Procedure
1. The casting chamber is sealed and placed on a flat surface
2. 0.8 % agarose solution is prepared by using 1× TAE buffer. The agarose is boiled
in TAE buffer to dissolve the agarose.
3. Then the agarose solution is cooled to 50 °C. At this stage, ethidium bromide is
added so that the final concentration is about 0.5μg/ ml.
4. Then the agarose solution is poured into the gel casting tray and the comb is set
in the gel casting tray.
5. Then the casting tray is left for some time at room temperature for the gel to
solidify.
6. Then the comb is removed and the gel is placed in the running tank so that the
gel is submerged in the running buffer (1× TAE buffer).
7. Then about 2 μl of loading dye is mixed with 10 μl of the extracted DNA sample
and loaded into the well.
8. The electrophoresis unit is allowed to run at 50 V until the dye migrates to bot-
tom of the gel or positive electrode.
9. Then the power supply is stopped and the gel is removed.
10. The gel is observed under UV transilluminator.

The ethidium bromide that intercalates DNA shows orange light which can be
observed (. Fig. 5.5).
zz Precaution
55 While placing the comb, ensure that there is enough space between the bottom
of comb.
55 Allow proper formation of wells and avoid sample leakage.
55 Before loading samples, remove any air bubbles from the wells.
55 Avoid using high voltage as it can cause smearing of DNA.
55 Stock solution of ethidium bromide (10 mg/ml) should be stored in dark bottles.
55 Wear suitable eye and face protection while working with UV light.
153 5
.. Fig. 5.5 Figure showing the quality of extracted DNA. The upper lane is for samples extracted by
the standardized method after modification; the lower lane is for the same samples extracted by the
conventional method. M is the molecular marker (100 bp). (Reproduced with permission from Aboul-
Maaty and Oraby (2019))
5.2.3 Spectrophotometric Quantification of DNA

zz Objective
To quantify the amount of DNA present in the given sample
zz Principle
Under extreme acid conditions, DNA is initially depurinated quantitatively followed
by the dehydration of sugar to ῳ-hydroxylevulinylaldehyde. This aldehyde condenses,
in acidic medium, with diphenylamine to produce a deep-blue colored condensation
products with absorption maximum at 600 nm. This method is commonly applied for
DNA samples that range from 50 to 500 mg.
zz Requirements
55 DNA standard (0.5 mg/ml)
55 Isolated DNA sample
55 Saline citrate solution (0.15 M NaCl, 0.015 M Na3 citrate)
55 Diphenylamine reagent
–– Diphenylamine: 5 g
–– Glacial acetic acid: 500 ml
–– Conc. H2SO4: 13.75 ml
55 It can remain stable for 6 months at 2 °C. This should be warmed to room tem-
perature and swirled to remix before use.
zz Procedure
1. Test tubes containing 1, 2, and 3 ml aliquots of the isolated DNA sample dis-
solved in standard saline citrate and similar aliquots of DNA standard are taken.
2. Then the volume is adjusted to 3 ml with H2O.
3. Following this, about 6 ml of diphenylamine reagent is added to each tube, and
after mixing, the tubes are heated in a boiling water bath for 10 minutes.
4. Then the tubes are cooled to room temperature.
5. The absorbance of the solution is read at 600 nm against the blank.
6. A standard graph is constructed taking A600 (ordinate) vs. quantity of DNA
(abscissa).
5 7. Then the concentration of DNA dissolved in the saline citrate s olution is calcu-
lated from the standard curve.
5.3 Plant Nutrient Analysis

zz Background
Elements considered essential for plants include carbon (C), oxygen (O), and hydro-
gen (H) available from carbon dioxide (CO2) in the air, water (H2O) obtained from
either rainfall or from the soil; mineral elements such as nitrogen (N), phospho-
rus (P), potassium (K), calcium (Ca), magnesium (Mg), and sulfur (S), known as
macronutrients; and boron (B), chlorine (Cl), copper (Cu), iron (Fe), manganese
(Mn), molybdenum (Mo), and zinc (Zn), known as the micronutrients. All these ele-
ments are available from the soil and/or are applied as soil amendments, such as lime
and fertilizers, which are then taken up through the plant roots. The concentration
of the essential elements in plants is expressed on a dry-matter basis in either percent
or grams per kilogram (g/kg) for macronutrients, and either parts per million (ppm)
or milligrams per kilogram (mg/kg) for micronutrients.
5.3.1 Estimation of Protein Content in Plant Samples

zz Objective
To quantitate the protein content of the given plant sample
zz Background
The Kjeldahl nitrogen method is one the most frequently used procedures for mea-
suring nitrogen and in turn the protein content in biological materials.
zz Principle
The amino nitrogen (–NH2) is oxidized by sulfuric acid in the presence of catalyst
(NH4)2SO4. The ammonium ion is converted to NH3 by NaOH and collected by
distillation. The NH3 is then quantitatively titrated against standard acid (HCl or
H2SO4) of known strength and nitrogen in the sample is computed.
zz Requirements
55 Plant sample
55 H2O2
5.3 · Plant Nutrient Analysis
155 5
55 Conc. H2SO4
55 Catalyst mixture
55 CuSO4 and selenium in a ratio of 20:1
55 Digestion catalyst mixture
It is a mixture of K2SO4 and catalyst mixture in a ratio of 10:1. The catalyst
mixture increases the boiling point of digestion mixture and shortens the digestion
time. The ideal temperature is 360–400 °C.
55 NaOH – 40%
55 Standard H2SO4 – 0.01N
55 Standard NaOH – 0.01N
55 Methyl red indicator
zz Procedure
1. About 5 g of plant sample grounded and powdered is transferred to Kjeldhal’s
digestion flask.
2. Then about 10 ml of H2SO4 is added followed by 5 ml of H2O2 in small lots. H2O2
is added to oxidize the organic material and to minimize foaming and frothing.
3. Then 1 g of digestion mixture is added and heated slowly.
4. The completion of digestion is indicated by absence of mixture followed which
the tubes are taken out & cooled to room temperature.
5. Then the digested material is carefully transferred to the macro distillation flask
with repeated washings.
6. Then about 10 ml of 0.1 N H2SO4 is taken in a 250 ml conical flask and 2–3 drops
of methyl red indicator is added.
7. The end of the condenser is dipped into the mixture.
8. Then 25 ml of NaOH (40%) is added and distillation is started.
9. The distillation is continued for 5 minutes.
10. Then the excess standard acid is titrated against standard alkali taken in the burette.

55 The color change is from pink to yellow.
55 Calculations
55 Volume of acid taken (V1) = 40 ml
55 Normality of acid (N1) = 0.1 N
55 Volume of alkali (V2) = V2
55 Normality of alkali (N2) = 0.1 N
( V1 N1 - V2 N 2 ) ´ 0.014 ´100
55 % of N2 =
5
5.3.2 Estimation of Fatty Acid

zz Objective
To determine the fatty acid content in the supplied sample
zz Principle
Free fatty acids are extracted from lipids of feed sample by using Dolls extraction
mixture 40:10:1 (heptane:isopropanol:acetic acid). Then the heptane phase is taken
and evaporated. The free fatty acids form a complex with cupric ions when mixed with
copper reagent. The colored complex formed with copper is soluble in chloroform
and diethyldithiocarbamate is used as a color-developing agent.
zz Requirements
55 Copper reagent
–– 1 volume of 1 M triethanolamine
–– 1 volume of 1 N acetic acid
–– 10 volumes of 6.45% Cu(NO3).23H2O
55 Diethyldithiocarbamate reagent
5 –– 0.1% (w/v) solution of sodium diethyldithiocarbamate in redistilled secondary
butanol. (Both the reagents are stored in the refrigerator and used within 7 days.)
55 Standard fatty acids solution
–– 10–100 mM myristic, palmitic, stearic, and linoleic acids in chloroform
zz Procedure
1. 5 mL of the chloroform solution of fatty acids of feed sample is taken in a centri-
fuge tube.
2. Then 2.5 ml of copper reagent is added to it.
3. The tubes are stoppered and shaken vigorously for 1 minute and centrifuged for
a few minutes.
4. The supernatant aqueous phase is removed by suction with hypodermic needles.
5. A portion (2.5 ml) of chloroform layer is taken into a clean dry tube.
6. Then 0.5 ml of diethyldithiocarbamate reagent is added to the chloroform solu-
tion and mixed.
7. The absorbance is read at 440 nm.
8. All measurements are also made against a blank solution that had been subjected
to the same procedure.
9. A standard curve is made with various concentrations ranging from 10 to 100 mM
of myristic, palmitic, stearic, and linoleic acids.

The sample reading is matched with the curve and the amount of free fatty acid is
calculated.
5.3.3 Estimation of Ammonia Nitrogen

zz Objective
To quantify the protein content of the given plant sample
zz Requirements
55 Micro-Kjeldahl distillation unit
55 Burette
55 Conical flask
55 Volumetric flask
55 Funnel and funnel stand
55 Tashiro’s indicator
157 5
55 N/7 H2SO4
55 Carbonate-free magnesium oxide
55 40% Sodium hydroxide
55 Plant sample
zz Procedure
1. 2–4 g of plant sample is ground and mixed in water and then filtered.
2. The residue is then washed with water and the required volume is made up with
water.
3. About 5 ml of filtrate is taken and mixed with magnesium oxide at a concentra-
tion of 25 mg/ml.
4. A conical flask containing 5 ml of Tashiro’s indicator is placed at the end of
the condenser of micro-Kjeldahl distillation apparatus so that the tip of the
condenser is completely dipped inside the indicator to avoid escape of released
ammonia during distillation.
5. Then 5 ml of filtrate sample is taken in the distillation unit.
6. About 10 ml of 40% NaOH is added sufficient to make the contents alkaline (till
the contents turn blue or black) and then the receiving end is immediately closed
with a pinchcock.
7. The funnel is sealed with a little amount of distilled water to avoid escape of
ammonia.
8. The contents are steam distilled by boiling the water in a round-bottom flask
connected to the distillation unit.
9. Then about 30 ml of distillate is collected to ensure that the nitrogen in the form
of ammonia is distilled.
10. The distillation is stopped when red color turns to green.
11. The distillate is repeated without taking the filtrate.
zz Result
55 Amount of ammonia nitrogen present in the sample is calculated as follows:
100 ´ Y ( B - B1 ) ´ 0.002
55 % of Nitrogen in Ammonia =
X´W
where
Y = volume (mL) of filtrate
X = volume (mL) of aliquot taken for distillation
B = volume (mL) of N/7 H2SO4 consumed for the titration of test sample
B1 = volume (mL) of N/7 H2SO4 consumed for the titration of blank distillate
W = weight (g) of oven-dried sample taken for filtration 0.002 g = 1 mL of N/7 H2SO4
5.3.4 Estimation of Phosphorus in Plant Sample

zz Objective
To estimate the phosphorous content in the supplied plant sample
zz Principle
The reaction is based on the formation of a complex between vanadate, molybdate,
and orthophosphate radicals to give a yellow color heteropoly compound in the pres-
ence of HNO3 medium. The yellow color formation is due to the substitution of
oxyvanadium/oxymolybdate radical by orthophosphate radical to give a heteropoly
compound. The intensity of color can be read at 470 nm.
zz Requirements
55 Plant sample
55 Barton’s reagent
55 Solution A: 25 g of ammonium molybdate dissolved in 400 ml distilled water.
55 Solution B: 1.25 g of ammonium metavanadate dissolved in 300 ml boiling
water.
5 55 The solution B is cooled down and 250 ml of conc. HNO3 is added. The solution
is again cooled down to room temperature and then solution A is poured into
solution B and the mixture is diluted to 1000 ml.
55 Standard P solution
55 0.2165 g of potassium dihydrogen orthophosphate is dissolved in 1000 ml dis-
tilled water to prepare a 50 ppm P solution. Then from the stock solution, 1, 2,
3, 4, and 5 ppm solutions are prepared by taking 1 ml, 2 ml, 3 ml, 4 ml, and 5 ml
solution.
zz Procedure
About 1 g of plant sample is digested with triacid mixture (HNO3: HClO4: H2SO4
in 9:3:1 ratio).
1. 1 g of ground plant sample is taken in a 300 ml Kjeldhal distillation flask. 10 ml
of acid mixture (HNO3:HClO4 in 3:1 ratio) is added.
2. The temperature is raised until the production of NO2 ceases.
3. The heating is continued till the volume is reduced to about 3–5 ml.
4. The completion of digestion is confirmed when the liquid becomes clear.
5. After cooling, distilled water is added and the contents are transferred to a 50 or
100 ml volumetric flask through Whatman’s no.1 filter paper and the volume is
made up.
6. About 5 ml of the digested sample is taken in a 50 ml volumetric flask.
7. Then about 10 ml of vanadomolybdate reagent (Barton’s reagent) is added and
the solution is diluted to 50 ml with distilled water.
8. The color developed is read after 10 minutes in UV-Visible spectrophotometer at
470 nm and the absorbance is noted.
9. A standard curve for “P” is prepared by using different concentration (1, 2, 3, 4,
and 5 ppm of “P”) and the concentration of P is calculated from the curve.

“P” in the given plant sample = reading from the standard graph × dilution factor
5.3.5 Estimation of Potassium in Plant Sample

zz Background
Potassium (K) is an important nutrient in plants and aids in the production of
starches, controls root growth, and regulates the opening and closing of stomatal
pores. It helps plants to resist drought and the effects of excessive temperatures.
159 5
zz Objective
To estimate the potassium content in the supplied plant sample
zz Principle
Potassium (K) is usually estimated by flame spectrophotometer technique. The atoms
take energy from flame and get excited to higher energy orbits. With the recapture
of lost electrons, they release energy of specific wavelength characteristic of each
element; therefore, this technique can be used for identifying the element. Further,
the intensity of the light is directly proportional to the concentration of the atoms
of these elements.
zz Requirements
55 Plant sample
55 Flame emission spectrophotometer, wavelength settings at 548 nm
55 Potassium (K) Primary Standard (1000 mg/l)
–– About 1.9067 g oven-dried and desiccated potassium chloride (KC1) is trans-
ferred to a 1000 ml volumetric flask and the volume is made up to 1000 ml with
deionized water.
55 Working standards
–– A series of working standards ranging between 0 and 100 mg/l is prepared from
primary standard solution for flame emission spectrophotometer.
zz Procedure
1. About 1 g of plant sample is digested with triacid mixture (HNO3:HClO4:H2SO4
in 9:3:1 ratio).
2. Then 5 ml of digested sample is pipetted out into a 50 ml volumetric flask and the
volume is made up to 50 ml with deionized water.
3. Then this solution is fed to the flame photometer and absorbance is read using
filter for K at 548 nm.
4. A standard curve is prepared by plotting the readings of known standard against
their concentration.
5. From the standard curve, the concentration of K in the extract is calculated.

“K” in the given plant sample = reading from the standard graph × dilution factor
5.3.6 Estimation of Sulfur

zz Objective
To determine the sulfur content in the supplied sample
zz Principle
Requirements
55 Gum acacia – acetic acid solution
–– 5 g pure gum acacia in powder is dissolved in 500 ml of hot water and filtered in
hot condition through No. 42 filter paper. It is cooled and diluted to 1 liter with
dilute acetic acid.
55 Barium chloride
55 Standard stock solution (2000 mg S/liter)
–– 10.89 g of oven-dried potassium sulfate is dissolved in 1 liter of water.
Standard working solution (10 mg S/liter)

55 2.5 ml of the stock solution is diluted to 500 ml.
zz Procedure
1. 1 g of plant material is taken in a digestion flask.
2. About 10–15 ml of nitric acid:perchloric acid in 3:1 ratio is added and mixed
5 together.
3. The flask is placed on a hot plate till digestion is over.
4. The solution is filtered once digestion is over into a 100 ml conical flask.
5. The solution is made up to 100 ml with distilled water and stored in air tight
container.
6. 10 ml of aliquot from the digested extract is taken.
7. 1 g of BaCl2 is added and shaken for 1 minute.
8. Then 1 ml of gum acacia acetic–acid solution is added and the volume is made
up to the mark and shaken for a minute.
9. The blank is run in the same manner.
10. The turbidity is measured after 25–30 minutes of precipitation at 440 nm.
11. The S content in the sample is read from the standard curve against the similar
absorbance as noted for the sample.
zz Result and observation

S content (μg) in 1 g of sample = concentration of S (μg/ml) as read from the stan-
dard curve × dilution factor
5.3.7 Estimation of Boron in Plant Sample

zz Background
A number of techniques are used to determine the boron content in plant tissues,
such as titrimetric, spectrometric, and colorimetric methods.
zz Objective
To determine the boron (B) content in plant sample
zz Principle
This method is based on the principle of formation of colored complex between
azomethine and H3BO3 in aqueous media. The complex is stable over a range of
0.5–10 μg of B/ml at pH 5.1. The maximum absorbance for the colored complex is
420 nm.
zz Requirements
55 Plant tissue
55 Deionized type 1 water
161 5
55 Hydrochloric acid (2N HC1)
55 Azomethine-H (0.5% w/v):
55 0.5 g azomethine-H is dissolved in 100 ml of 1% L-ascorbic acid by gentle heating
at 30 °C and the volume is made up to 100 ml with deionized water.
zz Buffer Reagent
250 g of ammonium acetate (NH4C2H3O2) and 15 g of EDTA are dissolved in 400 ml
of distilled water. Then 125 ml of glacial acetic acid is slowly added.
zz Calcium Hydroxide Suspension

0.4 g of Ca(OH)2 is added to 100 ml of distilled water.
zz Wash Solution (0.4N HC1)

33.3 mL conc. HC1 is diluted to 1 L with deionized water
zz Calcium Chloride 0.01M

1.11 g of Ca(Cl)2 is added to 900 ml distilled water and the volume is made up to
1000 ml.
zz Standard Boron Solution

Stock solution (1000 mg B/l)
5.715 g boric acid (H3BO3) is taken in a 1000 ml volumetric flask and the volume
is made up with deionized water
zz Working Standards
A series of working standards, 0.5–6 mg B/l, by appropriate dilutions of the
1000 mg B/l stock standard.
zz Procedure
1. About 1.25 g dried plant material is ground and sieved into a silica crucible.
2. The crucibles are placed in a cool furnace and ash at 500°C for 4 hours.
3. The crucible is removed from the muffle furnace and cooled down and then 2.5 ml
2N HCl is added.
4. 10 ml deionized type 1 water is added after 15 minutes to give a total volume of
12.5 ml.
5. The solution is filtered using a Whatman No. 540 filter paper into a plastic vial.
6. 1 ml of aliquot blank and diluted boron standards are taken in a 10 ml tube and
2 ml buffer is added.
7. Then 2 ml of azomethine H reagent is added.
8. Absorbance of the solution is taken at 420 nm.
9. A standard curve is prepared by taking standard B solutions.

B content (μg) in sample = concentration of B (μg/ml) as read from the standard
curve × dilution factor
5.3.8 Estimation of Zn, Cu, Mn, and Fe in Plant Sample

zz Objective
To determine the Zn, Cu, Mn, and Fe in plant sample
zz Principle
The concentration of Zn, Cu, Mn, and Fe elements in plant sample is done by atomic
absorption spectrometry technique. The different components of an atomic absorp-
tion spectrophotometer (AAS) include a sample introduction system, an excitation
source containing a hollow cation lamp, a nebulizer, a flame burner, a chopper, and
5 a detector. An atom of an element is capable of absorbing light energy characteristic
of that element. The analyte containing the element(s) of interest is atomized into
the flame. So photons are generated from a hollow cathode lamp whose cathode is
made of the element for determination. Therefore, when photons are passed through
the flame containing atoms of the element of interest, the photons get absorbed.
The degree of absorption is proportional to the concentration of the element in the
flame. The measured difference between the light intensity passing around the flame
and that passing through the flame determine the concentration of the element in the
atomized solution.
zz Requirements
55 Plant tissue
55 Extracting solution
–– Diethylenetriamine penta-acetic acid (DTPA) 1.9679 g
–– Triethanol amine (TEA) 13.3 ml
–– CaCl2·2H2O – 1.47 g in 200 ml distilled water
–– All the above are mixed and diluted to 900 ml and the pH is adjusted to 7.3 with
6N HCl and the final volume is made up to 1000 ml.
55 Stock standard solution
–– The stock solutions of the different elements are prepared by dissolving their
wires. About 1 g of wire is dissolved in nitric acid in 1:1 ratio and diluted to
1000 ml with distilled water to obtain a final concentration of 1000 μg/ml.
–– For Zn: 4.398 g/l of ZnSO4·7H2O
–– For Cu: 3.929 g/l of CuSO4·5H2O
–– For Fe: 4.977 g/l FeSO4·7H2O
–– For Mn: 3.598 g/l MnSO4·H2O
–– Then a series of standard solutions of 0.5, 1, 1.5, 2, and 2.5 ppm are prepared for
each metal.
zz Procedure
1. 0.5 g of plant material is taken in a digestion flask.
2. About 10 ml of nitric acid: perchloric acid in 4:1 ratio is added and mixed together.
3. The flask is placed on a hot plate till digestion is over.
4. The solution is filtered once digestion is over into a 100 ml conical flask.
5. The solution is made up to 50 ml with distilled water and stored in air tight con-
tainer.
6. The sample is then fed to AAS and the micronutrient content is calculated.
Bibliography
163 5
The micronutrient content in sample (mg/kg) = concentration read on AAS × 100
Take-Home Messages
55 Plant cells are totipotent, i.e., whole plant can be regenerated from single non-
sexual cells. As a necessary precursor to most plant transformation systems, there
must be methods established to manipulate plant tissues and cells in sterile tissue
culture media.
55 Plant tissue culture refers to growing of plant cells, tissues, organs, seeds, or other
plant parts in a germ-free environment in a culture medium. The culture media
are composed of complex mixture of salts, organic supplement consisting of vita-
mins and amino acids, carbon source, gelling agent, plant growth regulators, and
antibiotics.
55 The majority of a plant’s DNA is found within the nucleus of each cell. Specific
segments of the nuclear DNA, called genes, contain all the information required
for the cell to make proteins that are responsible for traits.
55 Plant analysis is critical to improving crop nutrition and yield. From the nutri-
tional standpoint, plant analysis is based on the principle that the concentration of
a nutrient within the plant is an integral value of all the factors that have interacted
to affect it.
55 Use of plant analyses knowledge is an integral part of most agronomic research
and considered as a useful tool for crop consultants to monitor production fields.
Bibliography
Aboul-Maaty NAF, Oraby HAS. Extraction of high-quality genomic DNA from different plant orders
applying a modified CTAB-based method. Bull Natl Res Cent. 2019;43:25. https://doi.org/10.1186/
s42269-019-0066-1.
Chu CC, Wang CC, Sun CS, Hsu C, Yin KC, Chu CY, Bi FY. Establishment of an efficient medium for
anther culture of rice through comparative experiments on the nitrogen. Sci Scinica. 1975;18:659–68.
Eriksson T. Studies on the growth requirements and growth measurements of cell cultures of
Haplopappus gracilis. Physiol Plant. 1965;18:976–93.
Gamborg OL, Miller RA, Ojima K. Nutrient requirements of suspension cultures of soybean root cells.
Exp Cell Res. 1968;50:151–8.
Gul F, Hussain A, Jan G, Hamyun M. Genomic DNA extraction for molecular identification of endo-
phytic fungi: an easy and efficient protocol. Biosci Biotech Res Asia. 2017;14(2):667–71.
Jackson ML. Boron determination for soil and plant tissues. In: Soil chemical analysis. 1958, p. 370–87
Kalra PK. Handbook of reference methods for plant analysis. Washington: CRC Press; 1998.
Katoch R. Analytical techniques in biochemistry and molecular biology. New York: Springer; 2011.
Kowalenko CG, Van Laerhoven CJ. Total sulfur determination in plant tissue. In: Handbook of refer-
ence methods for plant analysis; 1998. p. 93.
Lindsay WL, Norvell WA. Development of a DTPA test for Zn, Fe, Mn and Cu. Soil Sci Soc Am J.
1978;42:421–8.
Mathur J, Koncz C. Protoplast isolation, culture, and regeneration. In: Martinez-Zapater JM, Salinas J,
editors. Arabidopsis protocols. Methods in molecular biology, vol. 82. Totowa: Humana Press;
1998.
Motsara MR, Roy RN. Food and agriculture organization of the United Nations. Rome: FQA; 2008.
Murashige T, Skoog F. A revised medium for rapid growth and bio assays with tobacco tissue cultures.
Physiol Plant. 1962;15(3):473–97.
Nitsch JP, Nitsch C. Haploid plants from pollen grains. Science. 1969;163:85–7.
Oleszkiewicz T, Klimek-Chodacka M, Milewska-Hendel A, Zubko M, Stroz D, Kurczynska E, Boba A,
Szopa J, Baranski R. Unique chromoplast organisation and carotenoid gene expression in
carotenoid-rich carrot callus. Planta. 2018;248:1455–71.
Saad AIM, Elshahed AM. In Leva A, Rinaldi LMR, editors. Plant tissue culture media, recent advances
in plant in vitro culture. IntechOpen; 2012 https://doi.org/10.5772/50569. Available from: https://
www.intechopen.com/books/recent-advances-in-plant-in-vitro-culture/plant-tissue-culture-media.
Saiprasad GVS. Artificial seeds and their applications. Resonance. 2001;6(5):39–47.
Torres KC. Overview of protoplast isolation and culture. In: Tissue culture techniques for horticultural
crops. Boston: Springer; 1989.
White PR. The cultivation of animal and plant cells. 2nd ed. New York: Ronald Press; 1963.
5
165
Supplementary
Information
Index – 167

https://doi.org/10.1007/978-981-15-6252-5
167 A–C
Index
A Atomic absorption spectrophotometer

(AAS) 162
Acid-fast staining 108, 110 Autoclaving 87
–– principles 110 Auxin 136, 140
–– procedure 111 Azomethine-H 161
–– requirements 111
–– results 111
Agarose gel electrophoresis 151 B
–– DNA quantification and quality
analysis 120, 122 Barium chloride 54
Barton’s reagent 158
–– precaution 121
Basic BLAST 125
–– principles 120
Beef extract 93
–– procedure 121
Bioaerosols 126
–– requirements 120, 121
Biochemical oxygen demand (BOD),
–– result and observation 121
definition 46
–– precaution 152
Biochemical oxygen demand (BOD) in water
samples 45
–– calculation 49
–– quality of extracted DNA 153
–– determination of DO 49
–– dilution of samples 49
–– procedure 47, 48
Air microflora 126–128
Alcohol precipitation 118
–– alkaline iodine 47
Alkaline iodine 44, 47
–– allylthiourea solution 47
Alkalinity of waste water
–– BOD-free water 47
–– calcium carbonate 43
–– calcium chloride solution 47
–– ferric chloride solution 47
–– complete setup for titration process 41
–– Manganese sulfate (MnSO4) 47
–– materials requirement 41
–– phosphate buffer solution 47
–– Methyl orange indicator 41
–– sodium hydroxide solution 47
–– phenolphthalein indicator 41
–– sodium thiosulfate 47
–– sulphuric acid 41
–– starch solution 47
–– phenolphthalein alkalinity 42
–– sulphuric acid 47
–– procedure 42
–– setup for 48
–– tabulation 42
–– theory 46, 47
–– theory 40, 41
Biological hazards 7
–– total alkalinity 42
Biosafety 85
Allylthiourea solution 47
Biotechnology laboratory, safety signs in 9–11
Ammonia nitrogen estimation 156
BlackBoard Website 125
Blood agar (BA) 127
Boric acid 56, 75
–– results 157
Boron content estimation 160
Ammonium acetate solution 77
Ammonium molybdate solution 79
Antibiotic assay 128, 129
Antibiotic sensitivity test 129
Artificial/synthetic seeds 148
Brilliant green lactose bile (BGLB) broth 130
–– definition 146
–– encapsulated somatic embryos 146
–– procedure 147, 148
C
–– requirements 147 Calcium chloride solution 47
Assay media 94 Calcium hydroxide suspension 161
168 Index
Callus culture 138 –– complex media 93

–– callus formation from carrot root 142 –– differential media 94
–– precautions 141 –– enrichment media 94
–– principles 138, 140 –– natural media 93
–– procedure 141 –– reducing media 94
–– requirements 140 –– selective media 94
Callus formation from carrot root 142 –– semi-synthetic media 93
Callus induction 140 –– simple media 93
Capsule staining 112 –– synthetic media 93
–– principles 112 –– nutrient agar (NA) slants and plates
–– procedure 112 –– precedure 95
–– requirements 112 –– preparation 95, 96
Carbonate removal 68 –– principles 94
Cell suspension culture –– requirements 95
–– principle 141, 142 –– results 96
–– procedure 142, 143 –– potato dextrose agar medium (PDA) 96
–– requirements 142 –– principles 96
–– results 143 –– procedure 97
Cellulose microfibrils 143 –– requirements 96
Cetyltrimethylammonium bromide (CTAB) –– results 97
captures 149 Culture methods for bacteria 103
Chemical oxygen demand (COD) in water Cyanobacteria isolation 98
samples 51 –– principles 98
–– calculation 51 –– procedure 99
–– COD reflux unit 50 –– requirements 98
–– procedure 51 –– results 99
–– requirements 50, 51 Cytokinins 136, 137
–– theory 49, 50
Chemical oxygen demand (COD),
definition of 49 D
Chloride content in soil samples 73 Diethyldithiocarbamate reagent 156
–– calculation 73 Differential media 94
–– procedure 73 Differential stains, gram stain 108
–– requirements 72, 73 –– precautions 109
–– theory 72 –– principles 108, 109
Chloride content in water samples 54 –– procedure 109
–– calculation 54 –– requirements 109
–– procedure 53, 54 –– results 109
–– requirements 53 –– steps of 110
–– theory 53 Digestion catalyst mixture 155
Chloroform 117 Digestion reagent 56, 75
Chloroform isoamyl alcohol 117 Diphenyl amine indicator 70
Chromic acid solution preparation 87 Dissolved oxygen (DO) content in water
Coliform MPN test for bacteriological samples
examination of water 130, 131 –– calculation 46
Colony forming units (CFU) 128 –– principles 43
Complex media 93 –– procedure 45, 46
Congo red 113 –– reactions 44
Containment, definition of 85 –– requirements
Copper reagent 156 –– alkaline iodine 44
Cu element estimation –– Manganese sulfate (MnSO4) 45
–– principles 162 –– sodium thiosulfate 45
–– procedure 162 –– starch solution 45
–– requirements 162 –– setup for 44
–– result and observation 163 –– tabulation 46
Culture media preparation 92 –– theory 43
–– media types Distillation process 57, 76
–– assay media 94 DNA isolation and analysis
169 C–G
Index
–– agarose gel electrophoresis 151 Flame spectrophotometer technique 159

–– precaution 152 Free fatty acids 155
–– principles 151 Fungal staining 113
–– procedure 152 –– precautions 114
–– quality of extracted DNA 153 –– principles 114
–– requirements 152 –– procedure 114
–– result and observation 152 –– requirements 114
–– from plant sample 148, 150 –– results 114
–– agarose gel electrophoresis 151
–– precaution 151 G
Gas safety in laboratory 5
General laboratory safety guidelines
–– biotechnology laboratory, safety signs in 9–11
–– spectrophotometric test 151
–– chemical/laboratory safety education,
–– spectrophotometric quantification of DNA
products of 5
–– fire safety 3, 8
–– gas safety 5
–– general calculations used in laboratory
DNase 120
experiments 11
Dry heat sterilization 89
–– complex multistep dilution and
concentration 14
E –– concentration expression terms 11–12
–– concentration of common commercial
Electrical conductivity strength acids 14
–– definition of 38 –– parallel dilutions 13
–– of waste water –– sequential dilutions 13
–– calculation 40 –– serial dilution 12
–– materials requirements 38 –– volumetric flask 15
–– results 40 –– volumetric pipette 15
–– sample conductivity meter setup 39 –– glassware used in environmental lab 16–19,
–– theory 38 21–23, 26
Endospore staining 112, 113 –– instruments used in environmental lab 27, 28,
–– principles 113 30–34
–– procedure 113 –– issues for proper maintenance of laboratory
–– results 113 safety 2
Enrichment media 94 –– laboratory safety clothes and accessories 6
Environmental Microbiology Laboratory 85 –– rules of good laboratory practice 3
Eosin methylene blue (EMB) agar 130 –– test facilities and several levels of
Ethanol/isopropanol 118 management 4
Extraction solution 79 –– types of hazards 7
Genomic DNA (deoxyribiose nucleic acid) from
bacteria 117
F –– chemical requirements 119
Fatty acid estimation 155 –– precaution 120
–– principles 155, 156 –– principles 118
–– procedure 156 –– procedure 119
–– requirements 156 –– reagents
–– result and observation 156 –– ethanol/isopropanol 118
Fe element estimation –– NaCl solutions 117
–– principles 162 –– nucleic acids precipitation 118
–– procedure 162 –– phenol
–– requirements 162 –– chloroform
Ferric chloride solution 47 –– isoamyl alcohol 117
Ferroin indicator solution 50 –– Proteinase-K 117
Ferrous ammonium sulfate 51, 70 –– RNase 117
Filtration 89 –– sodium acetate 117
Fire safety in laboratory 3, 8 –– sodium dodecyl sulfate (SDS) 117
170 Index
Genomic DNA (deoxyribiose nucleic acid) from –– procedure 116

bacteria (cont.) –– requirements 115
–– TE Buffer 118 –– results 116
–– Tris–EDTA buffer 117 –– sigmoid growth curve of bacteria 115
–– requirements 118 Microbial growth, definition of 115
–– results 120 Microbial techniques
Glasswares cleaning –– agarose gel electrophoresis (See Agarose gel
–– autoclaving 87 electrophoresis)
–– precautions 88 –– air microflora 126–128
–– procedure 87, 88 –– antibiotic assay 128, 129
–– requirements, chromic acid solution –– cleaning glasswares 87
preparation 87 –– autoclaving 87
–– sterilization 88 –– chromic acid solution preparation 87
Good microbiological technique (GMT) 86 –– precautions 88
Gram negative 108, 109 –– procedure 87, 88
Gram-positive bacteria 108 –– sterilization 88
Gram stain 108 –– coliform MPN test for bacteriological
–– precautions 109 examination of water 130, 131
–– principles 108, 109 –– culture media preparation 92
–– procedure 109 –– assay media 94
–– requirements 109 –– complex media 93
–– results 109 –– differential media 94
–– steps of 110 –– enrichment media 94
Growth regulators preparations 137 –– natural medium 93
–– nutrient agar (NA) slants and plates
H (See Nutrient agar (NA) slants and
plates)
Heat sterilization 89 –– potato dextrose agar medium (See Potato
Horizontal gel electrophoresis 120 dextrose agar medium (PDA))
–– reducing media 94
I –– selective media 94
–– semi-synthetic medium 93
India ink 113 –– simple media 93
Indirect staining, See Negative staining (indirect –– synthetic medium 93
staining) –– Cyanobacteria isolation (See Cyanobacteria
isolation)
K –– genomic DNA from bacteria (See Genomic
DNA (deoxyribiose nucleic acid) from
Kjeldahl method 75, 76
bacteria)
Kjeldahl nitrogen method 154
–– laboratory etiquette 86, 87
–– laboratory safety 85, 86
L –– microbial growth curve (See Microbial growth
Lactophenol cotton blue (LPCB) stain 113, 114 curve)
Lauryl sulfate tryptose (LST) lactose broth 130 –– pH determination 90
Leishman stain 112 –– precaution 91
–– procedure 90
M –– requirements 90
Manganese sulfate (MnSO4) 45, 47 –– pH effect on growth of microbes 91, 92
Material safety data sheet (MSDS) 85 –– calibration of pH meter 92
Max Score 126 –– precaution 92
MEGA software 125 –– principles 91
Methyl orange indicator 41 –– requirements 91
Microbial density 128 –– results 92
Microbial growth curve –– sample preparation and measurement 92
–– measure of bacterial growth 116 –– polymerase chain reaction (PCR)
–– principles 115 amplification 123–125
171 G–P
Index
–– pure cultures isolation (See Pure cultures –– calculation 57

isolation techniques) –– procedure
–– sequence analysis 125, 126 –– digestion process 57
–– spectrophotometric method, DNA –– distillation process 57
quantification by 122, 123 –– reagents 56
–– staining techniques (See Staining techniques) –– requirements 56
–– sterilization –– theory 56
–– definition of 89 NoChromix® cleaning solution 88
–– dry heat 89 Nucleic acids precipitation 118
–– filtration 89 Nutrient agar (NA) 127
–– moist heat 89 Nutrient agar (NA) slants and plates
–– of other materials 90 –– preparation 95, 96
–– radiation (UV radiation) 89 –– principles 94
Micronutrients 154 –– procedure 95
Minimal inhibitory concentration (MIC) 128, 129 –– requirements 95
Minimum growth pH 91 –– results 96
Mn element estimation Nutrient medium, definition of 136
–– requirements 162 O
Modified Winkler test 43
Optical density (OD) 115, 151
Moist heat sterilization 89
Organic matter and organic carbon content
Moisture content in soil
in soil samples 72
–– definition 62
–– procedure 71
–– procedure 62
–– setup for titration experiment 71
–– theory 62
–– theory 70
Molten agarose 151
Organic matter oxidation 68
Mueller–Hinton agar plates 129
Oxygen scavenging media 94
Murashige and Skoog medium (MS
medium) 136, 141
–– compositions 138 P
–– using stock solutions 137
Parallel dilutions 13
Particle size in soil samples
N –– automated particle-measurement
NaCl solutions 117 instruments 65
Natural medium 93 –– calculations 69, 70
Negative staining (indirect staining) 107 –– carbonate removal 68
–– observation 108 –– dispersion 69
–– precaution 108 –– fractions separations 69
–– principles 107 –– organic matter oxidation 68
–– procedure 107 –– particulate characterization 66
Nigrosin 113 –– ranges of particle size 64
Nitrogen content in soil samples –– requirements
–– Kjeldahl method 76 –– calcium chloride solution 65
–– procedure –– dispersing agent 65
–– digestion 75 –– hydrogen peroxide 65
–– distillation 76 –– instruments and glassware 65
–– volumetric analysis 76 –– sand determination 69
–– reagents 75 –– sieving machine in 68
–– requirement 75 –– silt and clay determination 69
–– theory 74, 75 –– theory 64
Nitrogen content in water samples Perchloric acid 79
172 Index
pH determination 90 –– requirements 154, 155

–– precaution 91 –– result and observation 155
–– principles 90 –– sulphur estimation 159
–– procedure 90 –– principles 159
pH of soil –– requirements 159, 160
–– classification 63 –– result and observation 160
–– digital pH meter 63 –– Zn, Cu, Mn, Fe elements estimation
–– procedure 63 –– principles 162
–– theory 62, 63 –– procedure 162
Phenol 117 –– requirements 162
Phenolphthalein indicator 41, 56, 74, 75, 79 –– result and observation 163
Phenolpthanlein indicator 42 Plant tissue culture techniques
Phosphorous content estimation –– artificial/synthetic seeds 148
–– principles 157, 158 –– definition 146
–– procedure 158 –– encapsulated somatic embryos 146
–– requirements 158 –– principles 147
–– result and observation 158 –– procedure 147, 148
Phosphorus content in soil samples –– requirements 147
–– procedure 79, 80 –– callus culture 138
–– requirements 79 –– callus formation from carrot root 142
–– standard curve plot 80 –– principles 138, 140
–– theory 79 –– procedure 141
Physical hazards 7 –– requirements 140
Plant growth regulators (PGRs) 136 –– cell suspension culture
Plant nutrient analysis –– principle 141, 142
–– ammonia nitrogen estimation 156 –– procedure 143
–– procedure 157 –– requirements 142
–– requirements 156, 157 –– results 143
–– results 157 –– culture media preparation 136
–– boron content estimation 160 –– basal medium 136
–– principles 160 –– macronutrients 136
–– procedure 161 –– MS medium preparations using stock
–– requirements 160, 161 solutions 137
–– result and observation 161 –– others plant tissue culture media,
–– Boron content estimation procedure 161 preparation of 138
–– elements 154 –– plant growth regulators (PGRs) 136
–– fatty acid estimation 155 –– plant tissue culture media, composition
–– principles 155, 156 of 139–140
–– procedure 156 –– principle 136
–– requirements 156 –– requirements 136, 137
–– result and observation 156 –– stock solutions preparations 137
–– micronutrients 154 –– DNA isolation and analysis (See DNA
–– phosphorous content estimation isolation and analysis)
–– principles 157 –– plant propagation and tissue culture 136
–– procedure 158 –– protoplast culture 143
–– requirements 158 –– precaution 146
–– result and observation 158 –– principles 143, 144
–– potassium (K) estimation 158, 159 –– procedure 144, 146
–– principles 159 –– protoplast isolation 145
–– procedure 159 –– requirements 144–146
–– requirements 159 –– results 146
–– result and observation 159 Plant tissue culture techniques nutrient analysis
–– protein content estimation 154 –– artificial/synthetic seeds, procedure 148
–– Kjeldahl nitrogen method 154 –– callus culture
–– principles 154 –– precautions 141
–– procedure 155 –– procedure 141
173 P–S
Index
–– cell suspension culture, procedure 142, 143 –– requirements 103

–– protoplast culture –– results 104
–– principles 143, 144 –– serial dilution method 99, 101
–– procedure 144 –– principle 100
Plasmolysed cells 144 –– procedure 100
Polymerase chain reaction (PCR) –– requirements 100
amplification 123–125 –– spread plate technique 104
Potassium (K) estimation 158 –– principles 104
–– principles 159 –– procedure 105
–– result and observation 159 –– streak plate method 100, 103
Potassium chromate indicator 53, 72 –– precautions 102
Potassium content in soil samples –– principles 101
–– flame photometer 78 –– procedure 102
–– standard curve plot 78
–– theory 77 R
Potassium dichromate solution 50, 70
Potato dextrose agar medium (PDA) 96 Radiation (UV radiation) 89
–– principles 96 Reducing media 94
–– procedure 97 RNase 117
–– results 97
Potato extract 97 S
Pour plate technique 102 Sabouraud dextrose agar (SDA) 127, 128
–– precaution 104 Secondary callus culture 140
–– principles 103 Selective media 94
–– procedure 104 Semi-synthetic medium 93
–– requirements 103 Sequence analysis 125, 126
–– results 104 Sequential dilutions 13
P-propiolactone/formalin 86 Serial dilution agar plate method 100
Primary callus 140 Serial dilution method 12, 99, 101
Protein content estimation 154 –– principle 100
–– Kjeldahl nitrogen method 154 –– procedure 100
–– principles 154 –– requirements 100
–– procedure 155 Silver nitrate solution 53, 72
–– requirements 154, 155 Simple media 93
–– result and observation 155 Simple stains, microbial morphology 106
Proteinase-K 117 –– principles 106
Protoplast culture 143 –– procedure 106, 107
–– precaution 146 –– requirements 106
–– principles 143, 144 –– results 107
–– protocol-1 Sodium acetate 117
–– procedure 144 Sodium chloride 53, 72
–– protoplast isolation 145 Sodium dodecyl sulfate(SDS) 117
–– requirements 144 Sodium hydroxide 47, 56, 79
–– protocol-2 Sodium hydroxide–sodium thiosulfate
–– procedure 146 reagent 56, 75
–– requirements 145, 146 Sodium thiosulfate 45, 47
–– results 146 Soil quality analysis
Pure cultures isolation techniques 99 –– moisture content in soil
–– pour plate technique 102 –– calculation 62
–– precaution 104 –– procedure 62
–– principles 103 –– requirements 62
–– procedure 104 –– theory 62
174 Index
Soil quality analysis (cont.) –– principles 114

–– particle size in soil samples –– procedure 114
–– automated particle-measurement –– requirements 114
instruments 65 –– results 114
–– calcium chloride solution 65 –– gram stain 108–110
–– calculations 69, 70 –– negative staining (indirect staining) 107
–– carbonate removal 68 –– observation 108
–– dispersing agent 65 –– precaution 108
–– dispersion 69 –– principles 107
–– fractions separations 69 –– procedure 107
–– hydrogen peroxide 65 –– requirements 107
–– instruments and glassware 65 –– simple stains, microbial morphology 106
–– organic matter oxidation 68 –– principles 106
–– particulate characterization 66 –– procedure 106, 107
–– ranges of particle size 64 –– results 107
–– sand determination 69 Stannous chloride solution 79
–– sieving machine in 68 Starch solution 47
–– silt and clay determination 69 Sterilization 88
–– theory 64 –– definition of 89
–– pH of soil –– dry heat 89
–– classification 63 –– filtration 89
–– digital pH meter 63 –– moist heat 89
–– procedure 63 –– of other materials 90
–– theory 62, 63 –– radiation (UV radiation) 89
Spectrophotometric analysis (quantitative) 120 Stock solutions 77, 161
Spectrophotometric quantification of DNA –– definition of 137
122, 123 –– MS medium preparations using 137
–– principles 153 –– preparations 137
–– procedure 154 Stock standard solution 162
–– requirements 153 Streak plate method 100, 103
Spectrophotometric test 151 –– precautions 102
Spread plate technique 104 –– principles 101
–– results 105 Sulfate content in soil samples
16S rRNA gene sequence 125 –– calculation 74
Staining techniques 105 –– procedure 74
–– acid-fast staining 110 –– requirements 73
–– principles 110 –– theory 73
–– procedure 111 Sulfuric acid 87
–– requirements 111 Sulphate content in water samples
–– results 111 –– determination 55
–– capsule staining 112 –– procedure 55
–– principles 112 –– requirements 54, 55
–– procedure 112 –– sample standard calibration curve 55
–– requirements 112 –– theory 54
–– differential stains 108 Sulphur estimation 159
–– endospore staining 112, 113 –– principles 159
–– procedure 113 –– requirements 159, 160
–– results 113 –– result and observation 160
–– fungal staining 113 Sulphuric acid 41, 47
–– precautions 114 Synthetic medium 93
175 S–Z
Index
T V
Total dissolved solids (TDS) Vitamins 136
–– calculation 53 Volumetric analysis 76
–– evaporating disc for 52
–– procedure 52
–– requirements 52 W
–– theory 52
Total organic carbon (TOC) 70 Whatman filter paper 77, 79
Total Suspended Solids (Tss) 52–53 Winkler’s titration method 43
Toxic hazards 7
Tris –EDTA buffer 117
Turbidimetric method for sulphate content Z
–– procedure 55 Ziehl-Neelsen (ZN staining) technique 110
–– requirements 54, 55 Zn element estimation
Turbidometric method 115 –– principles 162
U –– requirements 162
Universal primers 125

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Learning Materials in Biosciences

Jayanta Kumar Patra

More information about this series at http://www.­springer.­com/series/15430

Gitishree Das Hrudayanath Thatoi

ISSN 2509-6125 ISSN 2509-6133 (electronic)

© Springer Nature Singapore Pte Ltd. 2020

Jayanta Kumar Patra

Swagat Kumar Das

1  eneral Guidelines of Laboratory Safety, Calculations

2 Water Quality Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

3 Soil Quality Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

4 Isolation, Culture, and Biochemical Characterization of Microbes . . . . . . . . 83

5 Plant Tissue Culture Techniques and Nutrient Analysis . . . . . . . . . . . . . . . . . . . . 135

5.3.5 Estimation of Potassium in Plant Sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158

Jayanta Kumar Patra, M.Sc., Ph.D.,

Gitishree Das, M.Sc., Ph.D.,

Swagat Kumar Das, B.Pharm., M.Tech., Ph.D.,

Hrudayanath Thatoi, M.Sc., M.Phil, Ph.D.,

1.1 General Guidelines of Laboratory Safety – 2

1.2 Fire Safety in a Laboratory – 3

1.3 Gas Safety in a Laboratory – 5

1.4 Safety Signs in Biotechnology Laboratory – 9

1.5  eneral Calculations Used in Laboratory

1.6  asic Laboratory Glassware and Instruments Used in an

© Springer Nature Singapore Pte Ltd. 2020

What You Will Learn in This Chapter

1.1 General Guidelines of Laboratory Safety

Maintaining safety inside a laboratory is of utmost importance for scientific person-

Good Laboratory Practice (GLP):

IF IN DOUBT, ASK - DON’T PUT SAFETY AT RISK !

1.2 Fire Safety in a Laboratory

Management Management Management Corporate quality

TFM chronic TFM repro- TFM TFM

Knowledge Caring for

1.3 Gas Safety in a Laboratory

.. Fig. 1.4 Laboratory safety

.. Table 1.1 Different types of hazards in a laboratory

Physical hazards Runaway reactions

High pressure/low pressure

Gases and gas cylinders

Toxic hazards Target organ toxicants

Biological hazards Human specimens/some animal specimens

Reproduced with permission from Hill Jr. (2019)

.. Fig. 1.5 Fire safety alarm

Harmful or Irritant Flammable Explosives Oxidizer

Corrosive Environmental Respiratory Gloves Required

Eye or Face Protective Clothing Protective Footwear Eye Protection

Ear Protection Fire Extinguisher Fire Hose Safety Flammable Gas

Fire Blanket Nonflammable Gas International Reactive Material

Prohibition Non-potable Water Do Not Touch No Open Flames

Do Not Eat or Do Not Enter Carcinogen Hazard Low Temperature

Hot Surface Magnetic Field Optical Radiation Laser Warning

Compressed Gas Non-ionizing Generic Warning Ionizing Radiation

Remote Control Biohazard Sign High Voltage Warning Toxic Chemical

Toxic Substance Irritant Emergency Call Escape Route Sign

Fire Triangle First Aid Safety Shower Eye Wash

1.5 General Calculations Used in Laboratory Experiments

There are different types of calculations such as molarity, normality, percent by

.. Table 1.2 Concentration expression terms

More information about this series at http://www.springer.com/series/15430

1 eneral Guidelines of Laboratory Safety, Calculations

2 Water Quality Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

3 Soil Quality Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

4 Isolation, Culture, and Biochemical Characterization of Microbes . . . . . . . . 83

5 Plant Tissue Culture Techniques and Nutrient Analysis . . . . . . . . . . . . . . . . . . . . 135

5.3.5 Estimation of Potassium in Plant Sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158

1.1 General Guidelines of Laboratory Safety – 2

1.2 Fire Safety in a Laboratory – 3

1.3 Gas Safety in a Laboratory – 5

1.4 Safety Signs in Biotechnology Laboratory – 9

1.5 eneral Calculations Used in Laboratory

1.6 asic Laboratory Glassware and Instruments Used in an

1.1 General Guidelines of Laboratory Safety

1.2 Fire Safety in a Laboratory

1.3 Gas Safety in a Laboratory

1.5 General Calculations Used in Laboratory Experiments

1.6 asic Laboratory Glassware and Instruments Used

Round- These types of flask They are used