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Laboratory ManualFor

Biotechnology

Laboratory ManualFor

Biotechnology

ASHISH S. VERMA Ph.D. Professor

Amity Institute of BiotechnologyAmity University Uttar Pradesh

NOIDA (UP), India

SURAJIT DAS Ph.D. Assistant Professor

Department of Life Science National Institute of Technology

Rourkela (Odisha), India

ANCHAL SINGH Ph.D. Assistant Professor

Amity Institute of BiotechnologyAmity University Uttar Pradesh

NOIDA (UP), India

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Visakhapatnam-530013,Ph-2782609(M)09440100555,visakhapatnam@schandgroup.com(Marketing Office)© 2014, Ashish S. Verma, Surajit Das and Anchal SinghAll rights reserved. No part of this publication may be reproduced or copied in any material form (including photocopying or storing it in any medium in form of graphics, electronic or mechanical means and whether or not transient or incidental to some other use of this publication) without written permission of the copyright owner. Any breach of this will entail legal action and prosecution without further notice.Jurisdiction : All disputes with respect to this publication shall be subject to the jurisdiction of the Courts, Tribunals and Forums of New Delhi, India only.

First Edition 2014

ISBN : 978-93-83746-22-4 Code : 03 425

printed in india

By Rajendra Ravindra Printers Pvt. Ltd., 7361, Ram Nagar, New Delhi -110 055 and published by S. Chand & Company Pvt. Ltd., 7361, Ram Nagar, New Delhi -110 055.

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This manual is dedicated to

Master Aviral Singh

Awesome, Naughty and Sweet

“Robbin, The Great ”Wish, we all could be a little more like him.

Anchal Singh Surajit Das Ashish S. Verma

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In the last century, biological sciences have seen an unprecedented development and growth. In particular, during the last quarter of the preceding century the growth of biological sciences was so prolific that it was difficult to keep a tab on its pace for the development. Last century has given few really important discoveries like structure of DNA as proposed by Watson and Crick, for which they were awarded with the Nobel Prize. Before Watson and Crick, few famous scientists have extensively worked in the area of bacterial genetics. Since the structure of DNA was elucidated, biological sciences took a quantum jump. The discoveries which used to be in the realm of scientists and were confined only within the boundaries of research laboratories, received unprecedented importance due to their applications towards the improvement in our life. Therefore, it became necessary to bring these discoveries and methods to the public domain. Introduction of this vast knowledge, to public domain can only be achieved by imparting this education to the students. If students are getting this knowledge they will surely imply their perception and speculation along with the information provided to them by their parents and grandparents. So by just brining this knowledge to the text books, we have introduced this information at least for three generations i.e., students, their parents and grandparents. This was done by developing various courses in Biotechnology. In the present scenario, it is very difficult to pinpoint that academician or administrator who decided to bring biotechnology in the students curriculum, certainly they have done great services to the society and humanity.

We find that biotechnology education has received tremendous acceptance from society as a part of educational curriculum. That is the reason different universities and higher education institutions are offering various courses in Biotechnology. Some of such courses are B.Sc., B.Tech., M.Tech., M.Sc. Even MBA in Biotechnology has been offered by some universities. Academicians started realizing that we have to provide more rigorous training in this subject, which has lead to the development of new courses like Dual Degrees, Integrated M.Sc., Integrated M.Tech., etc. Some institutions realized that the real potential of this subject can only be tapped with advanced training to the students, and as a result, some academic institutions are offering Integrated Ph.D. program. Once a student has been awarded doctorate he/she could be of real benefit to the society with their advance knowledge and training. Since these students are trained in such a manner that their ideas can be easily translated to achieve the goal via research and development.

The genesis of this Lab Manual itself is a journey for each of us individually, later on this journey became for us. It is true that everyone has to start his/her journey alone and with time one finds like-minded companions. During the course of journey, each and every one of us start relying on experiences, wisdom and intellectual decisions of each other. I (Dr. Ashish) too have many travelers and companions during this journey. I will reveal their contributions one-by-one along with the narration in this text.

I used to be a researcher, and teaching was a part of my profession. Since March 2008, I opted to be full time teacher by accepting an offer as Professor of Biotechnology at Amity University Uttar Pradesh (AUUP), NOIDA, India. I started teaching Immunology, Immunotechnology, Immunodiagnostics as well as Animal Biotechnology to the Undergraduate and Postgraduate students of Biotechnology. Here, I have numerous opportunities to interact with students as well as faculty members on daily basis. Most of the faculty members face the problem of unavailability of literature and information about the protocols to be demonstrated to their students during their Lab Courses. Some instructors are lucky to have sufficient enough information, if they are dealing with some of the well-established disciplines of biological -sciences like Biochemistry, Immunology, etc. because they can find protocols easily in standard laboratory books. Since these established disciplines are in educational curriculum for decades, therefore literature and textbooks for these subjects have been evolved and optimized to cater to the need of its end users. However for other subjects like bioprocess technology, animal biotechnology, plant biotechnology, etc. we are really at the dead end for the books and laboratory manuals. I am not trying to say that this information is not available. This information is available in different lab manuals, books and off course, on world wide web (www) i.e., internet. The main issue that I came across is that this information is either not available in cohesive manner or if available it is of higher standard. Therefore, it is not possible for students to correlate these experiments and some times students are unable to grasp the design and significance of an experiment. Some of these major limitations started getting strength, when I visited different biotechnology departments to deliver lecture or as examiner. My thoughts were resonated by my counterparts in academic arena. Almost all academicians, who are in this profession face the same problem. This made me think, that what can I do something to alleviate this void from the scenario of Biotechnology education. Somehow, this thought was never out of mind rather it got strengthened as I have more and more opportunities of teaching and working as instructor to lab courses.

While mentally, I started formulating this plan to develop a Laboratory Manual for the Biotechnology students. Does my plan sound good and make sense was my major concern until and unless I could receive some feedback. At this

Preface

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juncture, one of my colleagues Dr. Anchal Singh, Assistant Professor, Amity Institute of Biotechnology, AUUP, NOIDA became a companion traveler along this journey. We both were working together since a long time on various academic projects, so my long interaction had built up a trust on her academic acumen and intellectual excellence. One day, I was fiddling with papers, and she saw some anxiety on my face, a natural question from a colleague was ‘Ashish! What are you up to?” My immediate reply to her was that I am trying to develop a Laboratory Manual for Biotechnology students. Her instantaneous reply was “Ashish! That’s a great idea.” This will be very helpful to the students of Biotechnology. Her words at the time of need were a great solace to my perplexed mind. I could not resist myself from asking her to join me in this journey. Then, we started working together on this project. The first step was to develop a theme for the Lab Manual, we both worked on it and kept discussing to what to have and what not to have in it? No doubt, inputs of Dr. Anchal Singh did help to develop this project a cake walk.

Now the next hurdle was how to get published and who will publish it? Being a professor, I have privilege that representatives of different publishers in India visit my office with references to different aspects of published work. One time, Mr. M.S. Bisht had visited to my office, we floated the idea of this Lab Manual to him. He instantly agreed to publish it. As per the requirement, we submitted the proposal for the review and it was approved by their review committee. So, now the ball started rolling and we started working on this project. Various times me and Dr. Anchal came across with some limitations, as we were not experts of all the sections, we needed some one else to share his/her expertise. We discussed so many names during our discussion but were unable to come up with any suitable name.

One day, I suggested the name of Dr. Surajit Das to Dr. Anchal. She agreed that it is the best choice. Me and Dr. Anchal both know Dr. Das very well, as he was our colleague and we had numerous interactions with him. Now the question which had to be answered was “Would he be ready to join us?” Then I talked to Dr. Das, who is at present working as Assistant Professor at National Institute of Technology, Rourkela. He did give ears to our proposal and agreed to work with us on this project. He was working on this project with full zeal and effort even when he was awarded with funds to carry out his research work abroad. We are happy to know that during this journey Dr. Das has been blessed with a beautiful daughter who keeps him busy a lot. Even then we admire his professionalism and commitment towards the work. This way the my journey which started alone but I feel blessed that now I have two admirable companion travelers, who were always with me, and it is the joint effort of all of us that we are able to bring this project to this level.

There is another wonderful person who has a major role during this project, his name is Master Aviral Singh, son of Dr. Anchal Singh. He has become an integral part of this project because of his inquisitiveness about what his Mumma (Mother) is doing all the time. Dr. Anchal has to bear the brunt of his anger and irritation some time, but most of the time Aviral has become a source of enthusiasm by providing us lighter moments to relieve the stress of the work. We are dedicating this book to Master Aviral Singh as we felt that Master Aviral Singh is an inseparable part of this venture.

At the end, we would like to express our view that we tried our best to bring this manual to the best possible form and shape. Being academicians, we understand that “No Book or Manual is Perfect”. We worked on this philosophy to do our best; we hope you may still find it really useful. We will always wait for your comments, either positive or negative. We promise you that we won’t shy off hearing your comments or criticisms. It is the criticism which can be a primer for the improvement and to bring excellence to our efforts. Please feel free to share your thoughts and suggestion as how to improve this manual for the next editions. We would like to hear from you that what other sections/experiments need to be added, what is missing, and what is wrong? What are your expectations and where we failed to come up to those expectations?

Off course, biotechnology courses are offered throughout the country, the objective of all these courses is the same to train young students to be Future Biotechnologists. Nevertheless every course has minor differences from each other that would also be reflected in their laboratory courses and subjects along with the experiments to be performed as laboratory exercises. It is not impossible to include the syllabi of all the courses in one book. So, our efforts were focused towards the development of a manual which could be used by majority of students and faculty members all across the board rather than focusing on one institution or university. We hope that those experiments can also be adopted easily along with this lab manual.

With these lines, we hope to find your support.

Ashish S. VermaSurajit Das

Anchal Singh

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This book “Laboratory Manual for Biotechnology Students” is a comprehensive protocol version of the latest in protocols, basic background, and potential implications of biotechnology in clinic, animal and plant industries. To define the term, biotechnology is a technology based on biology - biotechnology harnesses cellular and biomolecular processes to develop technologies and products that help improve our lives and the health. The biological processes of microorganisms have been used for more than 6,000 years to make useful food products, such as bread and cheese, and to preserve dairy products; and implications of biotechnologies in medical and environmental sciences are coming up. For students who would like to learn about biotechnology, there is a need of a manual where they can find easy-to-follow protocols, relevant figures, and logical thinking before starting an experiment. This manual serves above purposes very well. The manual has 12 sections and each section has about 20 experiments. The sections are on the following topics: Before We Start; Animal Biotechnology; Plant Biotechnology; Biochemistry; Immunology; Microbiology; Enzymology and Enzyme Technology; Hematology and Serology; Bioprocess Technology and Fermentation Technology; Bioanalytical Techniques; Molecular Biology and Recombinant DNA Technology; Cell Biology and Cytogenetics; Appendix (9 in numbers). To make the book for general use, both animal and plant biotechnologies have been covered.

Colored illustrations and brief outlines (flowcharts) of the protocols are the major strength of the book as they help in following the protocol with minimum efforts. Most of the sections suggest additional experiments which provide students motivation to understand science better. Hematology and serology sections will benefit those students who would pursue health-related careers. Precautions while handling viral infected blood samples are also mentioned in different sections of the book.

I strongly encouraged students to read this book and follow protocols as described. I am sure they will gain the knowledge in the field and learn how to conduct biotechnology experiments properly. The book should be useful for university researchers, biotechnology industry personnel and diagnostic companies. This book is a welcome and needed orientation to the many issues that are particularly relevant to the impact that biotechnology has and will have on day-to-day life.

Mukesh Verma

Foreword

MUKESH VERMA, Ph.D.

Chief, Methods and Technologies Branch,Program Director, Epidemiology and Genomics

Research Program Division of Cancer Control and Population Sciences,

National Cancer Institute (NCI), National Institutes of Health (NIH),

Suite# 4E102, 9609 Medical Center Drive,

Rockville, MD 20850. USA

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At the outset, we would like to express our gratitude to ‘The GOD’. It is because of The God Almighty that we exist in this world. God has gifted us (as Human Beings) a brain to hypothesize, analyze, courage to dream and motivation to achieve our dreams.

Dr. Anchal would like to thank her dad Mr. Kanhaiya Ji Singh, mom Ms. Mohini Singh, brother Mr. Abhisar, his wife Mrs. Meenakshi and their son Aarav for their support, love and help. Anchal’s nine years old son Aviral deserves a special gratitude as his unstoppable questions and witty answers which never made her to realize the stress and work pressure of book writing and editing.

I (Dr. Ashish) would like to express my indebtedness to my mother Ms. Sushma Saxena, because I do exist in the present state due to her great efforts to raise and to groom me. She has always been the person in my life whom I can bank upon for anything, anytime. I wish my father Dr. Har Swarup Verma should be with us to see this book. My brother Mr. Saumya Swarup, his wife Ms. Nimisha Swarup, their kids Utkarsh and Shreeparna have also supported me, as and when I needed them. Similarly, my sisters and their family members have always encouraged me to remain focused towards the completion of this book. It is not that only family members who have touched me during the development of this book, but Master Aviral (son of Dr. Anchal) with his inquisitiveness and unending innocent queries worked as refresher for me as well as for Dr. Anchal too.

I (Dr. Surajit) thank my beloved parents for their everlasting encouragement and whole-hearted blessings. My wife Mrs. Sutapa stood with me at every endeavor of mine with her support, encouragement and love. My three years old daughter Soumili (Brishti) is really a breeze of fresh air when needed after a long work day. Her naughtiness and never-ending queries are nothing but the feeling of joy and happiness. Last but not the least, financial support for research and development project from funding agencies, viz. Department of Biotechnology, Ministry of Science and Technology, Government of India and National Fund for Basic, Strategic and Frontier Application Research in Agriculture (NFBSFARA), Indian Council of Agricultural Research (ICAR) are duly acknowledged. The members of the Laboratory of Environmental Microbiology and Ecology (LEnME) at the Department of Life Science, National Institute of Technology, Rourkela have been supportive during the writing of this book and deserve appreciations.

We would like to acknowledge the unconditional support that we have received from Dr. A.K. Chauhan, Founder President, Amity University Uttar Pradesh, NOIDA. Our special thanks go to Prof. Ajit Varma who was always with us as and when we needed his support and advice on various aspects of this book. We will not do justice, if we do not acknowledge the role of Prof. Soom Nath Raina, who is one of our colleagues. For us, Prof. Raina is more than a colleague, his affection, care and concerns made him a part of our extended family. Prof. Raina has always encouraged us to go for it and was always been helpful to get us focused, whenever we where getting out of track. We would like to acknowledge the support provided by our colleagues Drs. Girish Sharma, Roumi Deo, Shivani Sharda, Bhawna Rathi, Aarti Sharma Neeraj Kumar, Neeraj Khare and Amit Kaushik.

Our students Priyadarshini Mallick, Shruti Rastogi, Sneha Saran, Shishir Agrahari, Deepak Kushwaha, Faisal Adeeb, Shankar Chanchal, Mohammed Faheemuddin and Ajay Yadav contributed directly or indirectly towards this book. We remain thankful for the support of our students. Some of them offered help and support to organize us better, some of them offered their viewpoints, and some of them did not forget to criticize us, but whatever our students’ have done towards this book is admired by us. We have observed that the biggest motivation for teachers is always their students and students’ need. We would like to convey our thanks to Mr. Udai Pratap Singh, who was initially involved with this project and was extremely helpful for stimulating discussions and formulation of project. Somehow, Mr. Singh decided not to continue to be associated with this project, therefore we have no other choice except to honor his request.

We are really fortunate to have Mr. Dinesh Kumar as a team member for this project. Mr. Dinesh Kumar is working with us since we have joined this organization. For us, it is impossible to express our indebtedness for his dedication towards the completion of this book. We were just amazed to see his interest in this project and his alertness about the work. Many a time, we found that we missed something and Mr. Kumar was able to bring those errors to our attention so that those mistakes could be corrected and omissions could be incorporated. It will be unfair for us, if we fail to acknowledge the help and support offered to us by Mr. Yogendra Singh. Mr. Singh joined our group soon after we joined this organization. Somehow while working with me, Mr. Singh was able to recognize my addiction for coffee, and he was always there to fulfill my required doses and need for caffeine by filling our coffee cups with freshly brewed coffee every now and then. His support has made us to work long hours and relieved boredom of consistent writing, discussing and search for required material.

Acknowledgements

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We are thankful to the management and the staff of S. Chand & Company Pvt. Ltd., New Delhi for help and support in publication of this manual.

At the end we would like to thank all those people who have helped us directly or indirectly towards completion of this manual. There is no doubt that we gained so many insights, ideas, suggestions, as well as comments from our colleagues and laboratory support staff of Amity Institute of Biotechnology, Amity University Uttar Pradesh, NOIDA for which we will always remain indebted to them.

Ashish S. VermaSurajit Das

Anchal Singh

Disclaimer : While the authors of this book have made every effort to avoid any mistakes or omissions and have used their skill, expertise and knowledge to the best of their capacity to provide accurate and updated information, the authors and the publisher do not give any representation or warranty with respect to the accuracy or completeness of the contents of this publication and are selling this publication on the condition and understanding that they shall not be made liable in any manner whatsoever. The publisher and the authors expressly disclaim all and any liability/responsibility to any person, whether a purchaser or reader of this publication or not, in respect of anything and everything forming part of the contents of this publication. The publisher shall not be responsible for any errors, omissions or damages arising out of the use of the information contained in this publication.Further, the appearance of the personal name, location, place and incidence, if any; in the illustrations used herein is purely coincidental and work of imagination. Thus the same should in no manner be termed as defamatory to any individual.

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How to Refer This Manual?

This manual is developed and designed for the students and instructors of Biotechnology. Biotechnology as such contains so many subjects which make the course versatile but complicated. Every university has its own syllabus, therefore these subjects are offered in different semesters. To simplify location of contents, this manual is divided in 12 sections. Every subject has been assigned a two Alphabet code, which will be helpful to locate the section easily in the manual.

Subjects Code

Animal Biotechnology AB

Before We start BS

Bioanalytical Techniques BT

Biochemistry BI

Bioprocess Engineering and Fermentation Technology BP

Cell Biology and Cytogenetics CB

Enzymology and Enzyme Technology ET

Hematology and Serology HS

Immunology IM

Microbiology MI

Molecular Biology and Recombinant DNA Technology MB

Plant Biotechnology PB

SUFFIXES AND PREFIXESApart from subject codes, prefixes and suffixes are included which will help you to understand and locate the desired content easily in this manual. Some suffixes are in numeral, while some are alphabetical. We are explaining all those giving appropriate examples, so that they can be easily followed.

SUFFIXES“E”: Capital “E” means Experiments at Cross-roads. This is at the end of each section. This contains experiments

mentioned into another section which can be tried into this section. e.g., AB-E: mean experiments at cross-road.“G”: Capital “G” means general information about the section. It contains some of the general information related

with the section. e.g., AB-G: It is the additional information related with Animal Biotechnology section. “I”: Capital “I” means Introduction of section. Introduction is at the beginning of the section, and contains the

introduction related to the section. e.g., AB-I: Is the introduction of section Animal Biotechnology. “S”: Capital “S” refers to supplementary experiments related to the section. e.g., CB-3S: It is a supplementary

experiment for CB-3. “V”: Capital “V” means Viva of the section. This is at the end of each section. This contains the question for viva-voice

related to the section. e.g., AB-V: It is the Viva for section Animal Biotechnology.“1”: Numerals like 1, 2…. Are also used as suffixes. They represent the experiment number for the respective sections.

e.g., AB-1: Is the 1st experiment of Animal Biotechnology section.

PREFIXESFig: It is the abbreviated form for Figure. A numerical and alphabetical code after fig. identifies with the experiment

number. e.g., Fig-AB-1: This means this figure is included in experiment number 1 (AB-1).OT: It is the prefix for the table in that experiment. This contains a numerical code to identify with the experiment

number. e.g., OT-AB-1: This means it is the observation table for experiment number 1 (AB-1).

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1. Before We Start (BS) 1–57 BS-I: Introduction 3 BS-1: Biotechnology Laboratory:

Safety Guidelines 5 BS-2: Biosafety, Biohazard and Biosafety

Management 7 BS-3: Material Safety Data Sheet (MSDS) 13 BS-4: Solutions for Dilutions 29 BS-5: pH and Buffers 33 BS-6: Graphical Representation: A Know How 40 BS-7: Common Instruments in Daily Usage 42 BS-8: Good Laboratory Practices (GLP) 47 BS-9: Rationale for Laboratory

Experimentation 50 BS-10: Data Upkeep in Laboratory Note Book 54 BS-V: Viva 57

2. Bioanalytical Techniques (BT) 59–131

BT-I: Introduction 61 BT-1: pH Adjustment 63 BT-2: Homogenization 65 BT-3: Sonication 69 BT-4: Centrifugation 71 BT-5: Testing of Laminar Flow Hood 74 BT-6: To Check CO2 Incubator 78 BT-7: Beer Lambert’s Law 80 BT-8: Preparation of Absorbance Curve 82 BT-9: Adsorption Column Chromatography 84 BT-10: Paper Electrophoresis of Amino Acids 87 BT-11: Paper Chromatography 89 BT-12: Thin Layer Chromatography (TLC) 93 BT-13: Cellulose Acetate Electrophoresis 96 BT-14: Void Volume and Total Volume 98 BT-15: Determination of Molecular Weight 101 BT-16: Affinity Column Chromatography 103 BT-17: Agarose Gel Electrophoresis for DNA 106 BT-18: SDS-PAGE Electrophoresis 109 BT-19: Coomassie Blue Staining 114 BT-20: Western Blotting 116 BT-21: Ponceau S Staining 118 BT-22: Gel Drying 120 BT-23: Gel Documentation 122

Contents

BT-G: General Information 124 BT-E: Experiments at Cross Roads 130 BT-V: Viva 131

3. Cell Biology and Cytogenetics (CB)133–200

CB-I: Introduction 135 CB-1: Study of Ultrastructure of Cell Organelles 137 CB-2: PTC Testing 141 CB-3: Isolation of Chloroplast 143 CB-3S: Hill Reaction 145 CB-4: Cell Fractionation 147 CB-4S: Mitochondrial Marker Enzyme Assay 150 CB-5: Vital Staining of Mitochondrion 152 CB-6: Pinocytosis 154 CB-6S: Amoeba Culture 156 CB-7: Phagocytosis 158 CB-7S: Yeast Culture 160 CB-8: Differential Staining of DNA and RNA 162 CB-9: Feulgen Staining 164 CB-10: Glycogen Staining 167 CB-11: Apoptosis in Mouse Thymus/Thymocytes 170 CB-12: Mitosis in Onion Root Tip 173 CB-13: Effect of Colchicine on Mitosis 176 CB-14: Mitosis in Blood Cells 179 CB-15: C-Banding of Metaphase Chromosome 182 CB-16: G-Banding of Metaphase Chromosome 184 CB-17: Identification of Male and Female

Drosophila 186 CB-18: Polytene Chromosome 188 CB-19: Effect of Heat Shock on Polytene

Chromosome 191 CB-20: Meiosis in Grass Hopper’s Testis 193 CB-G: Drosophila Culture 195 CB-E: Experiments at Cross Roads 199 CB-V: Viva 200

4. Microbiology (MI) 201–259 MI-I: Introduction 203 MI-1: Sterility Testing of Media 205

MI-2: Disposal of Microbial Waste 207 MI-3: Antiseptic Property of Alcohol 209 MI-4: Phenol Coefficient 211 MI-5: Bacterial Growth Curve 213 MI-6: Bacterial Colony Counting 215 MI-7: Gram’s Staining 217 MI-8: Isolation of Bacteria 219 MI-9: Antibiogram 223 MI-10: Storage of Microorganisms 225 MI-11: Mounting of Fungi 227 MI-12: Warcup Method 229 MI-13: Enumeration of Bacteriophages 232 MI-14: Resazurin Test 234 MI-15: Methylene Blue Reductase 236 MI-16: IMViC Test 238 MI-17: Test for Water Potability 242 MI-18: Isolation of Bacteriophages from Sewage 245 MI-19: Streptomycin Resistant Mutant 247 MI-20: Penicillin Activity in Penicillinase Positive and Negative Bacteria 250 MI-21: Enzyme Induction 252 MI-22: Bacterial Conjugation 254 MI-23: Effect of Osmotic Pressure 256 MI-E: Experiments at Cross Roads 258 MI-V: Viva 259

5. Biochemistry (BI) 261–326 BI-I: Introduction 263 BI-1: Qualitative Tests for Carbohydrate 265 BI-2: Anthrone Method for Carbohydrate Estimation 276 BI-3: DNS Method for Reducing Sugar 278 BI-4: Estimation of Blood Glucose 280 BI-5: Isolation and Quantitation of Starch 282 BI-6: Isolation and Quantitation of Glycogen 284 BI-7: Analysis of Complex Carbohydrate Mixture 287 BI-8: Solubility Test for Lipid 288 BI-9: Qualitative Test for Fatty Acids Or Titrimetric Method for Fatty Acids 290 BI-10: Test for the Unsaturation of Fatty Acids 292 BI-11: Acrolein Test for Glycerol 294 BI-12: Iodine Value of Fat 296 BI-13: Acid Value of Fat 298 BI-14: Estimation of Blood Cholesterol Or Liebermann-Burchard Test 300 BI-15: Extraction of Lipids 302 BI-16: Quantitative Test for Amino Acids 305

BI-17: Estimation of Protein 308 BI-18: Isolation of Protein 314 BI-19: Estimation of Nucleic Acid 316 BI-20: Determination of Inorganic Phosphorus 321 BI-21: Determination of pKa Value of Weak Acid. 323 BI-E: Experiments at Cross Roads 325 BP-V: Viva 326

6. Molecular Biology and Recombinant DNA Technology (MB) 327–381

MB-I: Introduction 329 MB-1: DEPC Water Preparation 331 MB-2: Isolation of Genomic DNA from Bacteria 333 MB-3: Isolation of Genomic DNA from Plants 335 MB-4: Isolation of Genomic DNA from Animal Cells 338 MB-5: Plasmid DNA Isolation 341 MB-6: Concentrating DNA 344 MB-7: RNA Isolation 346 MB-8: Polymerase Chain Reaction (PCR) 348 MB-9: Reverse Transcription (RT) Or c-DNA Synthesis 351 MB-10: Two-Step RT-PCR 353 MB-11: One-Step RT-PCR 356 MB-12: Purification of PCR Product 359 MB-13: DNA Extraction from Agarose Gel 361 MB-14: Purification of Modified Oligonucleotide 364 MB-15: DNA Ligation 366 MB-16: Restriction Digestion 368 MB-17: Competent Cell Preparation 370 MB-18: Bacterial Conjugation 372 MB-19: Bacterial Transduction 375 MB-20: Bacterial Transformation 378 MB-E: Experiments at Cross Roads 380 MB-V: Viva 381

7. Enzymology and Enzyme Technology (ET) 383–436

ET-I: Introduction 385 ET-1: Enzymatic Method for Determination of

Blood Glucose 387 ET-2: Enzyme Activity: Effect of Enzyme

Concentration 390 ET-3: Enzyme Activity: Effect of Incubation Time 393

(xiii)

ET-4: Enzyme Activity: Effect of Temperature 395

ET-5: Enzyme Activity: Effect of pH 397 ET-6: Enzyme Activity: Effect of Substrate

Concentration 399 ET-7: Enzyme Extraction: Amylase from Bacteria 401 ET-8: Enzyme Extraction: b-amylase from

Barley 403 ET-9: Enzyme Extraction: Catalase from Plant 405 ET-10: Enzyme Extraction: Alkaline Phosphatase from Liver 407 ET-11: Zymography 409 ET-12: Trypsinization of Adherent Cells 412 ET-13: Enzymatic Digestion of Tissue 414 ET-14: Enzyme Immobilization: Agarose

Beads 416 ET-15: Enzyme Immobilization/Entrapment in

Alginate 419 ET-16: Enzyme Immobilization: Magnetic Beads 421 ET-17: Conjugation of Enzyme with Antibody 424 ET-18: Protein Digestion with Immobilized

Enzyme 427 ET-19: IgG Digestion with Immobilized Pepsin 429 ET-20: Isolation and Purification of Enzyme 432 ET-E: Experiments at Cross Roads 435 ET-V: Viva 436

8. Animal Biotechnology (AB) 437–497 AB-I: Introduction 439 AB-1: Preparation and Sterilization of Incomplete Medium 441 AB-2: Preparation and Sterilization of Complete Medium 444 AB-3: Heat Inactivation of Serum 446 AB-4: Cell Counting 447 AB-5: To Test Cell Viability 450 AB-6: Thawing of Cells 452 AB-7: Freezing of Cells 454 AB-8: Culture and Sub-culture of Suspension Cell Line 456 AB-9: Culture and Sub-culture of Adherent Cell Line 459 AB-10: MTT Assay 462 AB-11: Transfection of Mammalian Cells 465 AB-12: Culture of Primary Suspension Cells 468 AB-13: Culture of Primary Adherent Cells 471 AB-14: Transformation of Lymphocytes 474

AB-15: Qualitative Test for Apoptosis 477 AB-16: Quantitative Test for Apoptosis 480 AB-17: Cell Cycle Synchronization at G0

Stage by Serum Starvation 482 AB-18: BFU-E from Mouse Bone Marrow 484 AB-19: CFU-GM from Mouse Bone Marrow 487 AB-20: Chorioallantoic Membrane (CAM)

Assay 490 AB-G: General Information 492 AB-E: Experiments at Cross Roads 496 AB-V: Viva 498

9. Hematology and Serology (HS) 499–540

HS-I: Introduction 501 HS-1: Bleeding Time 503 HS-2: Clotting Time 505 HS-3: Blood Group Testing 507 HS-4: Total Erythrocyte Count (TEC) 510 HS-5: Total Leukocyte Counts (TLC) 513 HS-6: Thin Blood Film 516 HS-7: Differential Leukocyte Count (DLC) 518 HS-8: Erythrocyte Sedimentation Rate (ESR) 522 HS-9: Hematocrit/PCV/EVF 524 HS-10: Hemoglobin Estimation 526 HS-11: Mean Erythrocyte Hemoglobin Concentration (MEHC) 530 HS-12: Mean Cell Hemoglobin Concentration (MCHC) 531 HS-13: Plasma Separation 532 HS-14: Serum Separation 534 HS-15: PBMCs Separation 536 HS-E: Experiments at Cross Roads 539 HS-V: Viva 540

10. Immunology (IM) 541–606 IM-I: Introduction 543 IM-1: Bleeding of Animals 545 IM-2: Histopathology of Spleen, Thymus,

Lymph-nodes and Bone-marrow 548 IM-3: Antigen Preparation with Complete

Adjuvant 550 IM-4: Antigen Preparation with Incomplete

Adjuvant 552 IM-5: Single Radial Immunodiffusion Test (SRID) Or Radial Immunodiffusion Test (RID)

554 IM-6: Ouchterlony’s Double Diffusion (ODD) 558 IM-7: Counter-Current Immunoelectrophoresis 562

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IM-8: Rocket Electrophoresis Test 565 IM-9: Immunoelectrophoresis 569 IM-10: IgG Digestion by Papain 572 IM-11: IgG Digestion by Pepsin 574 IM-12: IgG Purification 576 IM-13: Direct Hemagglutination 579 IM-14: Indirect Hemagglutination 581 IM-15: Indirect ELISA 584 IM-16: Sandwich ELISA 587 IM-17: DOT ELISA 590 IM-18: Lymphoproliferation Assay 593 IM-19: Immunohistochemistry 596 IM-20: Widal Test 599 IM-21: CH50 Assay 602 IM-E: Experiments at Cross Roads 605 IM-V: Viva 606

11. Bioprocess Engineering and Fermen-tation Technology (BP) 607–661

BP-I: Introduction 609 BP-1: Urease Production by Microorganisms 611 BP-2: Cellulase Production by Microorganisms 613 BP-3: Protease (Caseinase) Production by

Microorganisms 615 BP-4: Gelatinase Production by Microorganisms 617 BP-5: Catalase Production by Microorganisms 619 BP-6: Citrate Permease Production by

Microorganisms 621 BP-7: Effect of Dyes on Bacterial Growth 623 BP-8: Effect of pH on Bacterial Growth 625 BP-9: Effect of Salt Concentration on Bacterial Growth 627 BP-10: Ion-Exchange Chromatography 629 BP-11: Lyophilization/Freeze Drying 631 BP-12: Ultrafiltration 633 BP-13: Concentration of Protein Sample by

Sephadex 635 BP-14: Speed Vac 637 BP-15: Dialysis 639 BP-16: Desalting 642 BP-17: Bioreactor/Laboratory Fermentor 644 BP-18: Sterilization of Fermentor 646 BP-19: Alcohol Fermentation 647 BP-20: Sauerkraut Production 650 BP-21: Penicillin Production 652 BP-22: Loss of CO2 during Fermentation 654 BP-23: Thermal Death Point 656 BP-24: Thermal Death Time (TDT) 658

BP-E: Experiments at Cross Roads 660 BP-V: Viva 661

12. Plant Biotechnology (PB) 663–714 PB-I: Introduction 665 PB-1: Aseptic Conditions for Plant Tissue Culture 667 PB-2: MS Medium Preparation and Sterilization 669 PB-3: Micropropagation from Plant Leaf 672 PB-4: Organogenesis 674 PB-5: Callus Culture 676 PB-6: Growth Index 678 PB-7: Indirect Organogenesis 680 PB-8: Suspension Cell Culture 682 PB-9: Androgenesis 684 PB-10: Somatic Embryogenesis 686 PB-11: Isolation of Protoplast 689 PB-12: Plant Cell Viability 692 PB-13: Protoplast Fusion 694 PB-14: Germination of Orchid Seed 697 PB-15: Production of Virus Free Plants 701 PB-16: Agrobacterium Mediated

Transformation of Plants 703 PB-17: Cryopreservation of Plant Cells 706 PB-18: Thawing and Recovery of

Cryopreserved Plant Cells 708 PB-19: Artificial Seed Preparation 710 PB-E: Experiments at Cross Roads 713 PB-V: Viva 714

Appendix 715–735 Appendix–I: Scientific Notation: How to deal with them? 715 Appendix–II: Dilutions: How to Dilute? 717 Appendix–III: The Greek Alphabets 719 Appendix–IV: SI* Prefixes and their Etymologies 720 Appendix–V: Commonly Used Buffers 721 Appendix–VI: General Information 726 Appendix–VII: Common Reagents 730 Appendix–VIII: Graphs 731 Appendix–IX: List of Suppliers 734

Abbreviations 736–738

Reference Books 739–739

Index 740–743

Laboratory Manual For Biotechnology

Publisher : SChand Publications ISBN : 9789383746224Author : Ashish S. Verma,Surajit Das and AnchalSingh

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