A laboratory Text book of Biochemistry, Molecular Biology and Microbiology is intended to prepare the undergraduate, postgraduate and research students to perform basic experiments on various aspects of bioscience and biotechnology. Moreover, in the Semester system of teaching it is necessary to explore experiments which are not lengthy and easily completed within contact hours. Initially the book deals with dilutions, pH, buffers, units of measurements and calculations. This is followed by lab safety rules which is very important for any student working with chemicals for their and safety of others. This book emphasizes on principles, reagent preparations and procedures related to experiments, which will be handy for students from different scientific backgrounds. A number of methods are available in the literature for quantification of various molecules. This book does not present all the available methods but based on experience it contains commonly used methods, which students should know. The methods have been written in a manner for direct practical use in the laboratory.
This work has originated as a result of numerous requests from my students for eased out and explanatory methods pertaining to biochemistry, biotechnology, microbiology and others. The section on testing of adulterants is of much use for common mass because most of the food products we eat are adulterated. The approach is rather simple with the use of very easily available chemicals and the tests can be performed even in house. It is hoped that the reliable assays presented in this manual will help the students and research scholars to get to basics of experiments and various aspects associated with it.
Table of Contents
1. WATER, pH AND BUFFERS
1.1 PREPARATION OF ACETATE BUFFER
1.2 TESTING OF BUFFER CAPACITY
2. DILUTIONS, UNITS FOR MEASURING CONCENTRATION OF SOLUTIONS AND SPECTROPHOTOMETRIC CALCULATIONS
3. LAB SAFETY RULES
4. CARBOHYDRATES
4.2 DETERMINATION OF REDUCING SUGARS CONTENT (NELSON-SOMOGYI METHOD)
4.3 DETERMINATION OF SUGARS CONTENT (DNSA METHOD)
4.4 DETERMINATION OF TOTAL CARBOHYDRATES (ANTHRONE METHOD)
4.5 DETERMINATION OF TOTAL CARBOHYDRATES (PHENOL SULFURIC ACID METHOD)
4.6 DETERMINATION OF GLUCOSE CONTENT (GLUCOSE OXIDASE METHOD)
4.7 DETERMINATION OF STARCH CONTENT
4.8 DETERMINATION OF CELLULOSE CONTENT
4.9 DETERMINATION OF FRUCTOSE CONTENT
4.10 DETERMINATION OF INULIN CONTENT
5. LIPIDS
5.1 QUALITATIVE TESTS FOR LIPIDS
5.2 QUALITATIVE TESTS FOR CHOLESTEROL
5.3 DETERMINATION OF CHOLESTEROL CONTENT (ZAK'S METHOD)
5.4 DETERMINATION OF OIL/LIPID CONTENT IN OILSEEDS
5.5 DETERMINATION OF VOLATILE ACIDS CONTENT
5.6 DETERMINATION OF ACID VALUE AND % FREE FATTY ACIDS
5.7 DETERMINATION OF SAPONIFICATION NUMBER OF OIL/FAT
5.8 DETERMINATION OF IODINE NUMBER OF OIL/FAT
5.9 DETERMINATION OF PEROXIDE VALUE
5.10 SEPARATION OF PLANT PIGMENTS USING THIN LAYER CHROMATOGRAPHY
6. PROTEINS
6.1 QUALITATIVE TEST FOR AMINO ACIDS AND PROTEINS
6.2 PRECIPITATION OF PROTEINS (A GENERAL APPROACH)
6.3 DETERMINATION OF λMAX FOR A GIVEN PROTEIN SAMPLE
6.4 DETERMINATION OF PROTEIN CONTENT (LOWRY’S METHOD)
6.5 PRECIPITATION AND DETERMINATION OF PROTEIN CONTENT (TCA METHOD)
6.6 DETERMINATION OF PROTEIN CONTENT (BIURET METHOD)
6.7 DETERMINATION OF PROTEIN CONTENT (BRADFORD METHOD)
6.8 DETERMINATION OF PROLINE CONTENT IN PLANTS
6.9 ISOLATION AND PURIFICATION OF CASEIN FROM MILK
6.10 DETERMINATION OF TOTAL FREE AMINO ACIDS
6.11 DETERMINATION OF TRYPTOPHAN CONTENT
6.12 DETERMINATION OF METHIONINE CONTENT
6.13 DETERMINATION OF LYSINE CONTENT
6.14 SEPARATION OF AMINO ACIDS BY CIRCULAR PAPER CHROMATOGRAPHY
6.15 SEPARATION OF AMINO ACIDS USING PAPER CHROMATOGRAPHY
6.16 SDS-PAGE
6.17 PREPARATION OF STANDARD CURVE OF PARA NITRO PHENOL (PNP)
6.18 ENZYMATIC ASSAY OF ACID PHOSPHATASE
6.19 DETERMINATION OF THE TIME LINEARITY OF AN ENZYMATIC REACTION
6.20 DETERMINATION OF THE PROTEIN LINEARITY OF AN ENZYMATIC REACTION
6.21 TO DETERMINE THE PROPERTIES OF (KM AND VMAX VALUES) OF AN ENZYME
6.22 NITRATE REDUCTASE ASSAY
6.23 AMYLASE ASSAY
6.24 PHENYL AMMONIA LYASE ASSAY
7. NUCLEIC ACIDS
7.1 ISOLATION OF GENOMIC DNA
7.2 SOLATION OF PLASMID
7.3 ISOLATION OF PLANT GENOMIC DNA
7.4 DETERMINATION OF DNA CONTENT (DIPHENYL AMINE TEST)
7.5 DETERMINATION OF CONCENTRATION AND PURITY OF THE DNA SAMPLE
7.6 ELECTROPHORESIS
7.7 RESTRICTION DIGESTION OF DNA
7.8 SOUTHERN BLOTTING
7.9 ISOLATION OF TOTAL RNA FROM PLANT TISSUE
7.10 ISOLATION OF RNA FROM YEAST
7.11 DETERMINATION OF RNA CONTENT (ORCINOL REAGENT METHOD)
8. MICROBIOLOGY
8.1 PREPARATION OF MEDIA
8.2 PREPARATION OF SLANTS, STABS AND POURING ON PETRIPLATES
8.3 GRAM STAINING OF BACTERIA
8.4 STAINING OF BACTERIAL SPORES
8.5 STREAK PLATE TECHNIQUE
8.6 ASSESSMENT OF AIR MICROFLORA
8.7 ENUMERATION OF BACTERIA IN SOIL
8.8 PREPARATION OF Escherichia coli COMPETENT CELLS AND ITS TRANSFORMATION
8.9 STUDY OF DIFFERENT GROWTH PHASES OF BACTERIAL POPULATION AND PLOT A BACTERIAL GROWTH CURVE
8.10 ANTIMICROBIAL SUSCEPTIBILITY TESTING (DISC DIFFUSION METHOD)
8.11 MINIMUM INHIBITORY CONCENTRATION (BROTH DILUTION METHOD)
8.12 MINIMUM BACTERICIDAL CONCENTRATIONS (MBC)
8.13 BIOCONVERSION OF TANNIC ACID TO GALLIC ACID BY Aspergillus niger
8.14 COMPARISON BETWEEN AEROBIC AND ANAEROBIC PROCESS ON ETHANOL PRODUCTION
8.15 DETERMINATION OF BIOMASS AND PACKED CELL VOLUME
8.16 CONJUGATION: Hfr MAPPING TO DETERMINE THE GENETIC DISTANCE BETWEEN GENES IN E. coli
9. DETECTION OF COMMON FOOD ADULTERANTS
10. APPENDIX
10.1 MOLARITY OF CONCENTRATED ACIDS
10.2 SOME TROUBLES AND REMEDIES FOR SDS-PAGE ANALYSIS
10.3 COMPOSITION OF BUFFERS AND MEDIA
10.4 TYPICAL IODINE NUMBERS
10.5 SAPONIFICATION VALUE OF COMMON FATS OR OIL
Objectives and Topics
This laboratory textbook aims to provide undergraduate, postgraduate, and research students with clear, explanatory methods for performing basic experiments in biochemistry, molecular biology, and microbiology. The focus is on offering reliable, time-efficient assays that can be easily conducted within standard laboratory contact hours while emphasizing safety and practical scientific application.
- Fundamental laboratory techniques including solution preparation, pH management, and buffering.
- Methods for the quantitative and qualitative analysis of carbohydrates, lipids, and proteins.
- Molecular biology procedures such as DNA/RNA isolation, restriction digestion, and electrophoresis.
- Microbiological protocols for culture maintenance, bacterial staining, growth curve plotting, and antimicrobial susceptibility testing.
- Practical screening methods for common food adulterants.
Excerpt from the Book
1. WATER, pH AND BUFFERS
Water is the major component of living organisms. About 75-90% of a cell consists of water. The basis for much of these unusual properties is the presence of hydrogen bonds. The boiling point is 100°C, which is very high, compared to 161°C for methane having similar molecular weight as water. The density of water is highest at 4°C. This is unusual since most substances are denser in their solid state. Due to which the ice floats. Water has a high heat capacity, i.e. it will take a relatively large amount of energy to increase the temperature of water. As a result large bodies of water will experience far less severe temperature changes than the surrounding atmosphere. Water has high surface tension and considered as universal solvent.
pH
Because of the strong electronegativity of oxygen and H-bond formation the H-atom in water carries a partially positive charge. By chance the bonding pair in some water molecules may be shifted totally to the oxygen causing the ionzation of water, i.e. the formation of an OH and H+ ion. However, the likely hood for this to happen is very small, to be exact: 1 mol of H2O contains 10-7mol of OH- and 10-7mol of H+. Water is said to dissociate and the degree of dissociation is a constant. It can be expressed as the dissociation constant (Kdiss).
Summary of Chapters
1. WATER, pH AND BUFFERS: Discusses the fundamental properties of water and explains the theory behind pH and the preparation/testing of buffers.
2. DILUTIONS, UNITS FOR MEASURING CONCENTRATION OF SOLUTIONS AND SPECTROPHOTOMETRIC CALCULATIONS: Provides essential mathematical foundations for preparing experimental solutions and calculating concentrations.
3. LAB SAFETY RULES: Outlines critical safety protocols and hazard symbols for laboratory work.
4. CARBOHYDRATES: Details the chemical theory and laboratory methods for the identification and quantification of various sugar types and polysaccharides.
5. LIPIDS: Covers the classification and chemical analysis of lipids, including tests for cholesterol and the extraction of oil from oilseeds.
6. PROTEINS: Provides a comprehensive guide to protein tests, purification techniques (e.g., casein isolation), and enzyme assays.
7. NUCLEIC ACIDS: Explains the protocols for isolation of DNA and RNA and molecular biology techniques like electrophoresis and Southern blotting.
8. MICROBIOLOGY: Focuses on the preparation of growth media, staining, bacterial enumeration, and antimicrobial testing.
9. DETECTION OF COMMON FOOD ADULTERANTS: Offers practical, household-style tests to screen food items for common adulterants.
10. APPENDIX: Contains reference tables for acid molarity, troubleshooting for SDS-PAGE, and buffer compositions.
Keywords
Biochemistry, Molecular Biology, Microbiology, Laboratory Techniques, pH Buffers, Carbohydrates, Lipids, Proteins, Enzyme Assay, DNA Isolation, RNA Isolation, Bacterial Staining, Food Adulteration, SDS-PAGE, Spectrophotometry
Frequently Asked Questions
What is the primary purpose of this textbook?
The book is designed to equip students with the basic, practical skills required to conduct experiments in bioscience and biotechnology fields, focusing on methods that are efficient and suitable for classroom settings.
What core subjects are covered in this manual?
The manual covers a broad spectrum including biochemistry (carbohydrates, lipids, proteins), molecular biology (DNA/RNA extraction, electrophoresis), and microbiology (media, staining, antimicrobial testing).
What is the main objective of the experiments provided?
The primary goal is to teach students the fundamentals of experiment design, reagent preparation, and the reliable quantification of biological molecules.
Which scientific methods are primarily highlighted?
The book features spectrophotometric analysis, titration methods for acids/buffers, chromatographic techniques (TLC, paper chromatography), and enzymatic assays for kinetic properties like Km and Vmax.
What specific focus is placed on food safety?
The book includes a dedicated section on the detection of common food adulterants, providing simple, accessible tests that can be performed using basic lab equipment to screen food products.
What distinguishes this book from others in the field?
It emphasizes "eased out" and explanatory methods, making it highly suitable for students with different scientific backgrounds and for use within the constraints of semester-based teaching systems.
How does the book address DNA purification?
The book details the isolation of genomic DNA from bacterial cultures (E. coli) and plants, covering the crucial steps of cell lysis, protein removal, and nucleic acid precipitation.
How is protein concentration determined in this manual?
The manual presents several standardized methods for protein estimation, including the Lowry method, the Biuret method, and the Bradford assay, explaining the chemical principles behind each.
- Arbeit zitieren
- Sharad Vats (Autor:in), 2014, A laboratory Text book of Biochemistry, Molecular Biology and Microbiology, München, GRIN Verlag, https://www.grin.com/document/284414
