Though many practical books are available in the market but this Laboratory Manual of Microbiology, Biochemistry and Molecular Biology is an unique combination of protocols that covers maximum (about 80%) of the practicals of various Indian universities for UG and PG courses in Bioscience, Biotechnology, Microbiology, Biochemistry and Biochemical  Engineering.

While preparing this laboratory manual the efforts have been made into various aspects of laboratory practices for the beginners like; the do’s and don’ts of working in any laboratory, concepts and terminologies used, how to prepare the solutions/reagents. The protocols given here have been tested by authors during their long teaching experience. The book has been divided into four sections, the first one is Introduction which is subdivided into laboratory etiquette and safety, molecular, empirical and formula weight, planning a solution of a particular morality, accuracy and calibration, buffers etc. Second part is about Instruments: Principle and Precautions which elaborates various commonly used equipments needed to perform different experiments. The third part of the book is about Experiments which has all the traditional to latest experiments with principle, requirements, procedure, results and observation and precautions in three major areas of Bioscience and Biotechnology i.e. Microbiology, Biochemistry and Molecular Biology. At the end a rather comprehensive appendix is given as section four.

The microbiology section contains all the basic techniques used in laboratories and industries. It also consists of some advanced microbiological experiments related to industrial microbiology. The second section deals with the basic practicals associated with carbohydrates, lipids, amino acid analysis, chromatographic techniques. Besides, other advanced techniques such as isoelectric focusing etc have also been included. As molecular biology has become an integral part of almost all biological courses, the third section of this book dedicated to all the techniques related to and used in molecular biology. Some experiments related to mitochondrial and chloroplast DNA isolation, recombinant DNA transformation and selection, blotting and hybridization techniques which are generally not found in most practical books, have also been included here. All the three sections of the book i.e. microbiology, biochemistry and molecular biology have been written by authors who have long experience and are well versed with conducting practicals related to their expertise. 

I. Introduction

i. Laboratory Etiquette and Safety
ii. Good Laboratory Practices
iii. Maintenance of Distillation Apparatus
iv. Washing of Glassware
v. Dry Sterilization
vi. Handling of Pipette
vii. Molecular, Empirical and Formula Weight
viii. Planning a Solution of a Particular Molarity
ix. Making Solutions from Hydrated Compounds
x. Measurement of Liquids with Normality / Molarity
xi. Numbers found on Chemical Bottles
xii. Accuracy and Calibration
xiii. Buffers

II. Instruments: Principle and Precautions
i. Weighing Balance
ii. Spectrophotometer
iii. Laminar Flow
iv. Autoclave
v. Centrifuge
vi. pH meter
vii. Incubator
viii. Hot air oven
ix. Compound microscope 
III. EXPERIMENTS

PART 1 MICROBIOLOGY
1.1 Micrometry and Measurement of Microorganisms
1.1.1. Calibration of microscope by using ocular and stage micrometer
1.1.2. Measurement of size of microorganism / spore
1.2 Preparation of Culture Media
1.2.1. Liquid culture medium (broth)
1.2.2. Solid medium
1.2.3. Agar plates and agar slants
1.2.4. Selective and differential media
1.2.4.1. Eosin Methylene Blue (EMB) Method
1.2.4.2. Mannitol Salt Agar
1.2.4.3. Pikovskaya Agar
1.3 Aseptic Methods
1.3.1. Physical methods of sterilization
1.3.2. Chemical methods of sterilization
1.3.3. Gaseous sterilization
1.4 Cultivation Techniques
1.4.1. Streak plate method
1.4.2. Pour plate technique
1.4.3. Spread plate technique
1.4.4. Serial dilution technique
1.5 Preservation and Maintenance of Cultures
1.5.1. Maintenance by sub-culturing
1.5.2. To prepare agar slants with mineral oil
1.5.3. Storage in soil
1.5.4. To store the cultures in liquid nitrogen
1.5.5. To store the cultures at -70°C
1.5.6. To preserve the cultures by lyophilization (Freeze drying)
1.6 Staining Techniques
1.6.1. Preparation of bacterial smear and fixation
1.6.2. Simple staining of bacteria
1.6.3. Negative staining of bacteria
1.6.4. Gram’s staining of bacteria
1.6.5. Acid Fast staining
1.6.6. Endospore staining
1.6.6.1. By malachite green
1.6.6.2. Dorner’s method
1.6.7. Capsule staining to detect the capsule or slime layer in bacteria
1.6.8. Flagella staining
1.6.8.1. Lee’s method
1.6.8.2. Leifson’s method
1.6.9. To test viability of bacteria by staining.
1.6.10. Staining of fungi
1.6.11. Staining of Arbuscular Mycorrhiza (AM)
1.7 Isolation and Eneumeration of Microbes
1.7.1. Isolation and enumeration of fungi from soil
1.7.1.1. Isolation of fungi by dilution plate method
1.7.1.2. Isolation of fungi by Warcup method
1.7.2. Isolation of seed microflora
1.7.2.1. Agar plate method
1.7.2.2. Blotter method
1.7.3. Isolation of aeromycoflora
1.7.4. Isolation of vesicular arbuscular mycorrhazia (VAM) spores from the soil
1.7.4.1. Wet- Sieving method
1.7.4.2. Floatation method
1.7.5. Isolation and enumeration of microorganisms from rhizosphere
1.7.6. Isolation of microorganisms from rhizoplane
1.7.7. Isolation of protozoa from soil
1.7.8. Isolation of cyanobacteria
1.7.9. Isolation of aquatic fungi by baiting method
1.7.10. Isolation of yeast
1.7.11. Isolation of rhizobia from the root nodules
1.7.12. Isolation and cultivation of anaerobic bacteria
1.7.12.1. Candle Jar method
1.7.12.2. Gaspak Anaerobic Jar method
1.7.13. Analysis and enumeration of bacteria in milk
1.7.14. Presumptive test for coliforms to check the quality of milk
1.7.15. Microbiological examination of cheese
1.7.16. Microbiological examination of butter
1.8 Counting of Cells/ Spores
1.8.1. Measurement of cells/ spores by counting chamber
1.8.2. Counting of cells by serial dilution technique
1.8.3. Counting of bacterial population spectrophotometrically
1.9 Microbial Growth
1.9.1. Determination of bacterial growth by spectrophotometric method
1.9.2. Measurement of fungal growth by colony diameter
1.9.3. Measurement of fungal growth by dry weight of mycelium
1.9.4. Estimation of biomass
1.9.4.1. Dry cell weight estimation
1.9.4.2. Packed cell volume determination
1.9.5. Effect of temperature on growth
1.9.6. Effect of pH on growth
1.9.7. Determination of antibiotic sensitivity by disc method
1.10 Fermentation Technology
1.10.1. Estimation of lactic acid
1.10.2. Estimation of citric acid production by Aspergillus niger
1.10.3. Bioconversion of tannic acid to gallic acid
1.10.4. Determination of alcohol production in the fermented broth
1.10.5. Alpha amylase biosynthesis and measurement of its activity
1.11 Mushroom Cultivation
1.11.1. Production of spawn for white button mushroom (Agaricus bisporus)
1.11.2. Cultivation of white button mushroom

PART 2 BIOCHEMISTRY
2.1. Carbohydrates
2.1.1. Qualitative test for Carbohydrates
2.1.1.1. Molisch’s Test
2.1.1.2. Iodine Test
2.1.1.3. Barfoed’s Test
2.1.1.4. Seliwanoff’s Test
2.1.1.5. Fehling’s Test
2.1.1.6. Benedict’s Test
2.1.1.7. Bial’s Test
2.1.1.8. Osazone Test
2.1.1.9. Test for non reducing sugars
2.1.2. Quantitative test for carbohydrates
2.1.2.1. Determination of total carbohydrate by Anthrone reagent
2.1.2.2. Determination of reducing sugar by Nelson-Somogyi method
2.1.2.3. Determination of cellulose
2.2. Amino Acids and Proteins
2.2.1. Color reaction of proteins and amino acids
2.2.2. Titration curve of amino acids
2.2.3. Protein assay methods
2.2.3.1. The l max for proteins and amino acids
2.2.3.2. Determination of molar absorbance coefficient (ε) of L-Tyrosine
2.2.3.3. Lowry’s method of protein assay
2.2.3.4. Bradford protein assay
2.2.3.5. Bicinchoninic acid protein assay
2.2.3.6. Biuret assay
2.2.4. Isolation of casein from milk
2.2.5. Ammonium sulphate fractionation of proteins
2.3. Enzymes
2.3.1. Extraction and purification of enzymes: General strategies
2.3.2. Expression of enzyme activity
2.3.2.1. To study the effect of pH and temperature on activity of acid phosphatase

2.3.2.1.1. To determine the activity of acid phosphatase
2.3.2.1.2. To study the effect of pH on activity of acid phosphatase
2.3.2.1.3. To study the effect of temperature on activity of acid phosphatase
2.3.2.2. Purification of acid phosphatase from raw wheat germ
2.3.2.3. To determine the Km and Vmax value of acid phosphatase
2.3.3. Extraction and assay of enzymes
2.3.3.1. Malate dehydrogenase
2.3.3.2. Peroxidase
2.3.3.3. Polyphenol oxidase
2.3.3.4. Salivary Amylase
2.3.3.5. Papain
2.3.4. Immobilization of an enzyme
2.4. Lipids
2.4.1. Qualitative test for lipids
2.4.1.1. Solubility test for lipids
2.4.1.2. Grease spot test
2.4.1.3. Acrolein test for glycerol
2.4.1.4. Liebermann-Burchard test for cholesterol
2.4.2. Determination of free fatty acids
2.4.3. Determination of Iodine number
2.4.4 Determination of Saponification value
2.4.5 Estimation of serum cholesterol (total)
2.5. Nucleic acids
2.5.1. Determination of l max of nucleic acids
2.5.2. Determination of DNA by Diphenylamine method
2.5.3. Determination of RNA by Orcinol method
2.6. Cell organelles
2.6.1. Isolation of mitochondria
2.6.1.1. Isolation of mitochondria from plant leaves
2.6.1.2. Isolation mitochondria from rat liver
2.6.2. Isolation of chloroplast from plant leaves
2.7. Plant Pigments and Phenolics
2.7.1. Estimation of Chlorophyll
2.7.2. Estimation of carotene
2.7.3. Estimation of lycopene
2.7.4. Estimation of Total phenol by Folin-Ciocalteau method
2.8. Vitamins
2.8.1. Estimation of Thiamine
2.8.2. Estimation of Ascorbic acid in lemon juice
2.8.3. Estimation of Riboflavin
2.8.4. Estimation of Vitamin A
2.9. Biochemical Separation Techniques
2.9.1. Paper chromatography
2.9.2. Thin layer chromatography
2.9.3. Gel-filtration chromatography
2.9.4. Polyacrylamide sodium dodesyl sulphate gel electrophoresis (SDS-PAGE)
2.9.5. Silver staining
2.9.6. Isoelectrofocusing.

PART 3 MOLECULAR BIOLOGY
3.1. Isolation of Nucleic acids
3.1.1. Isolation of DNA
3.1.1.1. Isolation of E. coli plasmid DNA
3.1.1.2. Isolation of E. coli genomic DNA
3.1.1.3. Isolation of phage DNA
3.1.1.4. Isolation of plant DNA (CTAB method)
3.1.1.5. Isolation of mammalian DNA
3.1.1.5.1. Isolation of DNA from mammalian tissue
3.1.1.5.2. Isolation of DNA from blood cells
3.1.1.6. Isolation of chloroplast DNA
3.1.1.7. Isolation of mitochondrial DNA
3.1.1.8. Agarose gel electrophoresis
3.1.2. Isolation of RNA
3.1.2.1. Isolation of total RNA
3.1.2.2. Isolation of messenger RNA
3.1.2.3. Agarose gel electrophoresis of RNA
3.1.2.4. Quantification of DNA/RNA
3.1.2.5. Determination of DNA quality
3.2. Denaturation and Renaturation of DNA
3.2.1. Determination of melting temperature (Tm value) of DNA
3.2.2. Determination of GC/AT content of DNA
3.3. Restriction Digestion
3.3.1. Restriction digestion of DNA.
3.3.2. Agarose gel electrophoresis of digested DNA samples.
3.4. Polymerase chain Reaction (PCR)
3.4.1. DNA amplification by PCR.
3.4.2. RAPD analysis of Plant DNA.
3.4.3. PCR amplification of 16s ribosomal RNA.
3.5. Blotting Techniques and Hybridization
3.5.1. Southern blotting and hybridization
3.5.2. Northern blotting and hybridization
3.5.3 Western blotting and hybridization
3.6. Ligation and Transformation of DNA
3.6.1. Ligation
3.6.2. Preparation of competent E. coli cells
3.6.3. Transformation of E. coli cells
3.6.4. Transformation of plant cells using Agrobacterium tumifaciens
3.6.5. Isolation of plant protoplasts.
3.7. Selection of Recombinant Cells
3.7.1. Selection by antibiotic resistance
3.7.2. Lac Z’ gene selection
3.7.3. Gus assay
REFERENCES

APPENDIX
Appendix IVA: Microbiology
Appendix IV B: Biochemistry
Appendix IV C: Molecular Biology