Laboratory Protocols in Applied Life Sciences By Prakash Singh Bisen

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Laboratory Protocols in Applied Life Sciences

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Features

  • Offers a complete solution of protocols for graduate students and practitioners in life science technology/general and applied biology
  • Provides comprehensive coverage and relevance as a unique and valuable guide
  • Includes details of methodologies used in different disciplines e.g. animal science, plant science, biotechnology, biochemistry, general biology, life sciences and pharmaceutical sciences
  • Presents a firm foundation on which to build further studies in different disciplines of life sciences
  • Contains illustrations

Summary

 

As applied life science progresses, becoming fully integrated into the biological, chemical, and engineering sciences, there is a growing need for expanding life sciences research techniques. Anticipating the demands of various life science disciplines, Laboratory Protocols in Applied Life Sciences explores this development.

This book covers a wide spectrum of areas in the interdisciplinary fields of life sciences, pharmacy, medical and paramedical sciences, and biotechnology. It examines the principles, concepts, and every aspect of applicable techniques in these areas. Covering elementary concepts to advanced research techniques, the text analyzes data through experimentation and explains the theory behind each exercise. It presents each experiment with an introduction to the topic, concise objectives, and a list of necessary materials and reagents, and introduces step-by-step, readily feasible laboratory protocols.

Focusing on the chemical characteristics of enzymes, metabolic processes, product and raw materials, and on the basic mechanisms and analytical techniques involved in life science technological transformations, this text provides information on the biological characteristics of living cells of different origin and the development of new life forms by genetic engineering techniques. It also examines product development using biological systems, including pharmaceutical, food, and beverage industries.

Laboratory Protocols in Applied Life Sciences presents a nonmathematical account of the underlying principles of a variety of experimental techniques in disciplines, including:

  • Biotechnology
  • Analytical biochemistry
  • Clinical biochemistry
  • Biophysics
  • Molecular biology
  • Genetic engineering
  • Bioprocess technology
  • Industrial processes
  • Animal
  • Plant
  • Microbial biology
  • Computational biology
  • Biosensors

Each chapter is self-contained and written in a style that helps students progress from basic to advanced techniques, and eventually design and execute their own experiments in a given field of biology.


 

Table of Contents

Microscopy

Introduction

Light Microscopy

Atomic Force Microscopy

Bright Field Microscopy

Dark Ground Microscopy

Phase Contrast Microscopy

Confocal Scanning Laser Microscopy

Differential Interference Contrast Microscopy

Polarization Microscopy

Fluorescence Microscopy

Experiments on Microscopy

Electron Microscopy

Sample Preparation for Electron Microscopy

Dark Field Electron Microscopy

Crewe Microscope

Backscatter Scanning Microscope

Experiment on Scanning Electron Microscopy of Cyanobacteria

Transmission Electron Microscopy of Phages (Bacteriophage and Cyanophage)

Suggested Readings

Important Links

Microtome

Introduction

Sectioning

Traditional Histology

Ultramicrotome

Cryostat

Incubation

Electron Microscopy Technique

Botanical Microtomy

Processing of Tissue

Suggested Readings

Important Links

pH and Buffers

Introduction

Acids and Bases

Hydrogen Ion Concentration and pH

Measurement of pH

Titration Curves

Buffer Solutions

What Is the pH of a Mixture of 5 mL of 0.1 mol/L Sodium Acetate and 4 mL of 0.1 mol/L Acetic Acid?

How the pH Changed on Adding 1 mL of 0.1 mol/L HCl in the Previous Mixture?

Buffers Used in Biochemical Experiments

pH and Life

Care and Use of the pH Meter

Titration Curves

Determination of pH Using Indicators

Titration of a Mixture of a Strong and a Weak Acid

Titration of a Strong Acid with a Strong Base

Titration of a Weak Acid with a Strong Base

Determination of pKa

pKa Values of a Dicarboxylic Acid

Acetate Buffers

Titration Curves of Amino Acids

Determination of pH of the Given Water/Soil Sample

Determination of pH of Fruit Juice

Measurement of pH of Soil Sample

Appendices

Suggested Readings

Important Links

Spectrophotometer

Introduction

Beer–Lambert Law

Limitations of the Beer–Lambert Law

Measurement of Extinction

Colorimeter

Spectrophotometry

Infrared Spectroscopy

Fluorescence Spectroscopy

Sensitivity of Fluorescence Assays

Fluorescence Quenching

Measurement of α-Naphthyl Phosphatase Activity

Suggested Readings

Important Links

Centrifugation

Introduction

Centrifuges

Ultracentrifugation

Suspending Medium

Rotors

Fractionation of Rat Liver

Estimation of DNA, RNA, and Protein in the Isolated Cell Fractions

Enzyme Distribution in the Cell

Glucose-6-Phosphatase

Glutamate Dehydrogenase

Acid Phosphatase

Mitochondrial Swelling

Determination of Lysosomal Integrity

Effect of Detergents on the Stability of the Lysosomal Membrane

Density Gradient Centrifugation

Fractionation of Pig Brain by Density Gradient Centrifugation

Distribution of Enzymes in the Fractions Obtained from Pig Brain

Isolation of Chloroplasts

Isolation of Chloroplast DNA

Isolation of Mitochondria and Assay of a Marker Enzyme

Isolation of Genomic DNA

Isolation of Chloroplasts from Spinach Leaves by Differential Centrifugation

Isolation of Plasmid DNA by Centrifugation

Suggested Readings

Important Links

Radiation Biology

Introduction

Radioisotopes

Sample Preparation

Autoradiography

Distribution of 14CO2 in Different Plant Parts

Photosynthetic Reduction of 14CO2 to Primary Metabolic Product

Incorporation of Labeled Acetate into Leucoplastic Fatty Acids of Developing Brassica Seeds

Suggested Readings

Important Links

Chromatography

Chromatography

Paper Chromatography

Ascending Chromatography

Descending Chromatography

Separation and Identification of Amino Acids by Descending Paper Chromatography

Separation and Identification of Amino Acids by Ascending Paper Chromatography

Separation and Identification of Amino Acids in a Given Mixture by 2 D Paper Chromatography

Thin Layer Chromatography

Thin-Layer Gel Chromatography

Separation and Identification of Sugars by Adsorption Thin Layer Chromatography

Identification of Lipids in a Given Sample by Thin Layer Chromatography

Identification of Sugars in Fruit Juices Using Thin Layer Chromatography

Column Chromatography

Gradient Preparation

Adsorption Chromatography

Separation of Pigments from Leaves or Flowers by Adsorption Column Chromatography

Ion-Exchange Chromatography

Separation of Amino Acids by Ion-Exchange Chromatography

Separation of Proteins from Human Serum by Ion-Exchange Chromatography

Gel Filtration/Size-Exclusion Column Chromatography

Separation of Blue Dextran and Cobalt Chloride on Sephadex G-25

Desalting of Protein Sample by Gel Filtration

Determination of Molecular Weight of a Given Protein by Gel Filtration

Concentration of Dilute Protein Solutions Using Sephadex G-25

Affinity Chromatography

Hydrophobic Interaction Chromatography or Affinity Elution

Purification by Affinity Chromatography

Preparation of an Affinity Column

Preparation of Rat Brain Acetylcholinesterase

Acetylcholinesterase Assay

Purification of Acetylcholinesterase by Affinity Chromatography

MALDI-TOF

Nuclear Magnetic Resonance Spectroscopy

Maintenance of Enzyme Activity

Addition of Chaotropes

Role of Polyols/Hydrophilic Polymers in Stabilizing Structures

Stabilization by Side-Chain Modification

Criteria of Purity

Biuret

Folin (Lowry’s Method)

Bradford

Suggested Readings

Important Links

Separation Technology

Electrophoresis

Principle

Types of Electrophoresis

Separation of Amino Acids by Paper Electrophoresis

Cellulose Acetate Strip Electrophoresis

Gel Electrophoresis

Polyacrylamide Gel Electrophoresis

Polyacrylamide Disc Gel Electrophoresis

SDS Gel Electrophoresis

Slab Gel Electrophoresis

Native Disc Gel Electrophoresis of Proteins

SDS-Polyacrylamide Slab Gel Electrophoresis of Proteins Under Reducing Conditions (SDS-PAGE)

Slab Gel Gradient Electrophoresis of Protein and Multiple Molecular Forms of Acetylcholinesterase

Protein (Western) Blotting

Isoelectric Focusing

Agarose Gel Electrophoresis

Determination of Molecular Weight of Plasmid DNA by Agarose Gel Electrophoresis

Electrophoretic Evaluation of Quality of High Molecular Weight DNA Isolated from Plant Material

Large-Scale Preparative Electrophoresis

Suggested Readings

Important Links

Histochemical Techniques

Introduction

Protein

Acidic Compounds

Carbohydrates–Polysaccharides

Lipids

Minor Compounds

Autoradiography

Enzyme Localization in Embedded Material

Suggested Readings

Important Links

Cytogenetics

Introduction

Study Different Stages in Root Tip Cells of Plant

Preparation of Root Tips for Mitotic Studies

Preparation and Study of Slides for Mitosis Using Acetocarmine Squash Techniques from Onion Root Tips

Perform Carmine or Orcein Staining Techniques

Feulgen Staining Techniques

Preparation of Onion Flower Buds for Meiosis Studies

Fixation

Mounting and Making Temporary Slides Permanent

Study the Effect of Colchicines or Acenaphthene or Chloral Hydrate on Chromosomes of Allium cepa

Estimate the Chiasma Frequency at Diplotene–Diakinesis

Study the Effect of γ Radiation on Chromosomes

To Prepare Slides Showing Diplotene Stages in Spermatocytes of Desert Locust (Schistocerca gregaria) or in Oocytes of the Newt (Triturus viridescens) Showing Lampbrush Chromosomes

Study Karyotypes of Allium cepa and Vicia faba

Micrometry

To Demonstrate Reciprocal Translocation in Pollen Mother Cells of Rhoeo discolor (Tradescantia)

To Study the Spiral Nature of Chromosome Using Any Plant Material

To Show the Different Banding Patterns in Eukaryotic Chromosomes

Fungal Cytogenetics

Harvesting of Neurospora

To Study the Ordered (Tetrad) and Random Ascospore Analysis in Neurospora sp

To Demonstrate the Tetrad and Random Analysis

Tetrad Analysis

Pigmentation and Nutritional Requirement Analysis

Random Spore Analysis

Test for Nutritional Requirement

Suggested Media for Neurospora, Sordaria, and Aspergillus Experiments

Study of Heterokaryosis in Neurospora from the Nutritionally Deficient Mutants

General Information and Methods for Drosophila Experiments

Study of Salivary Gland Chromosomes of Drosophila melanogaster for Nature of Polyteny and Regions of Chromosome Puffs

Human Genetics

Human Sex Chromosomes and Barr Bodies

Autoradiography

Photomicrography

Suggested Readings

Important Links

Biomolecules

Introduction

Carbohydrates

Amino Acids and Proteins

Lipids

Nucleic Acids

Suggested Readings

Important Links

Enzymology

Introduction

Enzymes as Catalysts

Classification

Measurement of Enzyme Activity

Time Course Studies of the Reaction Catalyzed by Alkaline Phosphatase (EC 3.1.3.1)

Effect of Enzyme Concentration on the Rate of Enzyme-Catalyzed Reaction

Determination of Temperature Optima for Alkaline Phosphatase

Determination of Effect of pH on the Activity of Alkaline Phosphatase

Effect of Substrate Concentration on Activity of Alkaline Phosphate and Determination of the Km and Vmax of the Reaction

Progress Curve of Serum Alkaline Phosphatase (Orthophosphoric Acid Monoester Phosphohydrolase, EC 3.1.3.1)

Effect of Variation of Serum Alkaline Phosphatase Activity with Enzyme Concentration

Effect of Substrate Concentration and Inhibitors on Oxheart Lactate Dehydrogenase

Effect of Temperature on the Activity of α-Amylase

Determination of the Molecular Weight and Purity of Chymotrypsin from the Enzyme Kinetics

Yeast Isocitrate Dehydrogenase: Allosteric Enzyme

Purification and Characterization of Enzymes

Isolation of Muramidase (Mucopeptide N-Acetylmuramyl Hydrolase, ) from Egg White

Separation of the Isoenzymes of Lactate Dehydrogenase by Electrophoresis on Polyacrylamide Gel

Enzyme Engineering

Artificial Enzymes

Suggested Readings

Important Links

Membranes

Introduction

Composition of Membranes

Membrane Transport

Effect of Lipid Composition on the Permeability of a Lipid Monolayer

Effect of Detergents and Other Membrane-Active Reagents on the Erythrocyte Membrane

Permeability of Model Membrane (Liposome)

Effect of Cholesterol on the Anion Permeability of a Phospholipid Membrane

Effect of Insulin on the Transport into Isolated Fat Cell

Transport of Amino Acids across the Small Intestine

Absorption of Xylose from the Gut in Man

Suggested Readings

Important Links

Photosynthesis and Respiration

Introduction

Measurement

Photosynthesis

Isolation of Chloroplasts from Spinach Leaves

Determination of Chlorophyll Concentration in the Chloroplast Suspension

Evolution of Oxygen by Isolated Chloroplast Using Hill Oxidants

Methyl Viologen as a Terminal Electron Acceptor

Spectrophotometric Assay of the Hill Reaction and the Estimation of Chlorophyll

Respiration of Mitochondria and Oxidative Phosphorylation

Effect of Inhibitors on the Respiratory Chain

Compounds That Affect the High-Energy State of Mitochondria

Suggested Reading

Important Links

Microbiology

Introduction

Good Microbiological Practice

Culture Media

Types of Media

Preparation of Culture Media

Maintenance and Preservation of Cultures

Preparation of Basic Liquid Media (Broth) for the Cultivation of Bacteria

Solid Media for Growth of Microorganisms

Isolation and Maintenance of Organisms

Isolation of Organisms by Pour Plate Method and Serial Dilation Method

Spread Plate Technique

Isolation of Vibrio parahaemolyticus from Seafood Sample

Isolation of Lactic Acid Bacteria from Milk and Buttermilk Sample

Isolation of Staphylococcus aureus from Food Sample

Sauerkraut Fermentation

Isolation of Microorganisms from Air

Phyllosphere/Phylloplane Microbiology

Measurement of Microbial Growth

Methods to Measure Bacterial Growth

Growth Curve Measurement of Bacterial Population by Serial Dilution Method

Serial Dilutions

Measurement of Fungal Growth by Colony Diameter Method

Biomass Measurement of Fungal Growth (Dry Weight Method)

Effect of Incubation Temperature on Growth

Effect of pH on Microbial Growth

Microscopic Examination of Bacteria

Microscopic Examination of Pathogens by One Tube One Plate Method

Smear Preparation

Staining

Biochemical Tests

Microbial Genetics

Bacterial Recombination

Suggested Readings

Important Links

Soil Biology

Introduction

Estimation of Humus in Soil/Manure

Determination of Microbial Carbon Biomass

Estimation of Algal (Photosynthetic) Biomass in Soil Cores

Soil Decomposition and Microbial Community Structure (Winogradsky Technique)

Soil Algae Enumeration

Phosphate Solubilizing Bacteria

Isolation of Sulfur Oxidizing Bacteria from Soil

Demonstrate the Process of Ammonification in Soil

Demonstrate the Process of Nitrification in Soil

MPN Method for Nitrosomonas and Nitrobacter

Isolation of Azospirillum sp from Soil and Plant Root

Enumeration of Azospirillum by MPN Method

Demonstration of Microbial Denitrification in Soil

Isolation of Plant Growth Promoting Rhizobacteria

Isolation of Photosynthetic Bacteria

Cyanobacteria

Nitrogen Fixation

Mycorrhizal Symbiotic Association

Actinomycetes

Aquatic Molds

Fungi

Rhizosphere

Microbial Production of Citric Acid by Aspergillus niger

Isolation of Industrially Important Antibiotic Producing Microorganisms

Assay of Antibiotics and Demonstration of Antibiotic Resistance

Characterize and Identify Aflatoxin by Thin-Layer Chromatography

Protozoa

Observation of Microscopic Invertebrates from Pond Water

Observation of Algae from Pond Water

Microscopic Examination of Soil Microbes

Bacteria Frequently Isolated from Soil

Fungi Frequently Isolated from Soil

Appendices

Suggested Reading

Important Links

Virology

Introduction

Isolation of Bacteriophage from Sewage

Preparation of High Titer of Bacteriophage of Isolated Bacterial Virus

One-Step Growth Curve of Coliphage and Determining the Number of Phage Particles Released by the Lysis of the Single Infected Bacterial Cell (Burst Size)

Demonstration of Lysogeny and Isolation of Rhizobium Phage

Isolation of Cyanophage LPP from Pond Water

Demonstration for the Presence of Actinophages in Soil

Isolation of Phage from Flies

Virus Culture in Embryonating Chicken Eggs

Extraction and Isolation of Tobacco Mosaic Virus

Cultivation of Viruses in Tissue Culture

Viral Hemagglutination Test for the Presence of Antigens

Suggested Readings

Immunochemical Methods

Introduction

Immunity

Ags

Antigenic Determinant or Epitope

Haptens

Specificity

T-Cell Dependent (TD) and T-Cell Independent (TI) Ags

Immunoglobulins

IG Determinants

Ig Classes

Monoclonal Antibodies

Polyclonal Antibodies

Ag–Ab Reaction

Determination of Ag–Ab Reactions

Collection of Blood Sample

Experiment: To Determine the ABO Blood Group by Using Slide Agglutination Test

Experiment: Determination of RH Factor in Human Being

Experiment: Demonstration of Agglutination Reaction of Unknown Bacterial Culture by Slide Agglutination Technique

Experiment: Estimate the Total Leukocyte Count by Hemocytometer

Experiment: Determine the Differential Count of WBCs (Leucocyte) by Staining Method

Experiment: Determine the Total Erythrocyte Count by Hemocytometer

Experiment: Lymphoid Cells Identification in Blood Smears

Experiment: Spleen Cell Preparation from Mouse

Experiment: Identification of Lymphocyte Populations

Experiment: Isolation of Monocytes by Adherence

Experiment: Isolation of Neutrophils

Experiment: Isolation of Human Basophils

Experiment: Isolation of Tissue Mast Cells

Experiment: Measurement of Percent Β-Glucuronidase Secretion

Experiment: Generation of Polyclonal Antibodies in Rabbits

Experiment: Gel Diffusion/Ouchterlony Immunodiffusion

Experiment: Demonstration of Radial Immunodiffusion

Experiment: Counter Current Immunoelectrophoresis

Experiment: Serum Electrophoresis

Experiment: Demonstration of Immunoelectrophoresis

Experiment: Determination of Albumin by Laurell Rocket Immunoelectrophoresis

Experiment: Purification of Igs BY ION Exchange Chromatography

Experiment: The Detection and Assay of a Myeloma Protein

Experiment: ELISA

Experiment: Pregnancy Testing by Using the Immunological Methods

Experiment: HIV DOT Test of AIDS Patients

Experiment: Demonstration of Immunofluorescence Assay

Experiment: Identification of Bacteria by Using Fluorescent Ab Technique (FAT)

Experiment: Immunofluorescence Labeling of Cultured Cells

Experiment: Isolation of IgG from Serum

Experiment: Purification of IgM

Suggested Reading

Important Links

Genetic Engineering

Introduction

Tools of Genetic Engineering

DNA Sequencing

Isolation of Genomic DNA from E coli

Rapid Method for Isolating Plasmid DNA from Yeast

Isolation of Plasmid DNA from Yeast (Protocol 1)

Isolation of Yeast Plasmid DNA (Protocol 2)

Isolation of Drosophila Genomic DNA

Determination of λmax Value of the Isolated DNA Sample

Determination of the Purity of the Isolated DNA Sample

Determine the Quantity of DNA in the Sample

To Perform Agarose Gel Electrophoresis for the Given DNA Sample

Isolation of Plasmid DNA from Given Bacterial Culture

Electrophoresis of DNA: Linear, Circular, and Super Coiled

To Perform Restriction Digestion of the Given DNA Sample

To Demonstrate Ligation

Elution of DNA from Agarose Gel

Purification of Phage λ DNA

To Perform Restriction Digestion of Plasmid DNA with Different Restriction Endonucleases and to Determine the Position of Restriction Site Using Restriction Mapping

To Perform Transformation

Directional Cloning Into Plasmid Vectors

To Assay Reporter Chloramphenicol Acetyltransferase Gene

To Assay β-Gal in Extracts of Mammalian Cells

Determination of Nucleotide Sequence of DNA by Dideoxy Chain Termination Method

Amplification of 16S rDNA Genes by the Polymerase Chain

PCR from Fungal Spores/AMF (Arbuscular Mycorrhizal Fungi)

To Demonstrate the Nested PCR Reaction

Suggested Readings

Important Links

Molecular Biology

Introduction

Isolation of Nucleic Acids from Biological Samples

Demonstration of Southern Blotting

Demonstration of Experiment on RFLP Analysis

Restriction Fragment Length Polymorphism

Isolation of RNA from Biological Sample

Extraction of Polysomal RNA of Nuclear Sequences

Separation of RNA Species by Ion-Exchange Column Chromatography on Methylated Albumin Kiesselguher Columns

Northern Hybridization of RNA Fractionated by Agarose Formaldehyde Gel Electrophoresis

Preparation of Radiolabeled Probe by Random Primer Method

Detection of Specific Fragment of Plant DNA from Restriction Digest by Hybridization with a Labeled Probe

In Vitro Transcription

In Vitro Translation of RNA

Reticulocytes and Their Use in the Study of Protein Synthesis

Protein Synthesis in Intact Rabbit Reticulocytes

Metabolic Labeling of Proteins and Immunoprecipitation

Induction of β-Galactosidase in Strains of E coli

Effect of Different Inducers on the Induction of β-Galactosidase

Effect of Protein Synthesis Inhibitors on the Induction of β-Galactosidase

Turnover of β-Galactosidase

Suggested Readings

Important Links

Biosensors

Introduction

Biosensor

Classification of Biosensors

Characteristics of Biosensors

Components of Biosensors

Biochip

Immunobiosensor

Aptamers

SPR Technology

Working Principle of Biosensors

Immunosensors

Calorimetric Biosensors

Potentiometric Biosensors

Amperometric Biosensors

Optical Biosensors

Piezoelectric Biosensors

Second-Generation Biosensors

Third-Generation Biosensors

Biosensor for Healthcare

Conclusion and Future Challenges

Suggested Readings

Important Links

Enzyme Immobilization

Introduction

Selection of Carrier and Support before Immobilization

Carriers for Enzyme Immobilization

Limitations and Advantages of Enzyme Immobilization

Methods of Immobilizations

Cross-Linking

Entrapment Method

Whole-Cell Immobilization

Industrial Application of Enzymes

Experiment Cell Immobilization by Using Alginate

Experiment Immobilization of Enzyme Horseradish

Peroxidase (Donor: H2O2Oxidoreductase; EC 1.11.1.7) by

Aminoantipyrine–Phenol Assay

Suggested Readings

Plant Tissue Culture

Introduction

Facilities

Glassware Preparation

Sterilization

Wet Heat Sterilization

Dry Sterilization

Filter Sterilization

Media Preparation

Preparation of Stock Solution

Protocol

Sterilization of Media

Aseptic Manipulation

Sterile Transfer Facilities

Temperature

Light

Aeration

Temperature

Plant Cell Culture System

Explants

Calluses

Cell Suspension

Protoplast

Surface Sterilization

Common Culture Media

Sterilization of Plant Materials

Introduction

Materials

Protocol

Observation and Result

Preparation of Tissue Culture Media

Organ Culture

Callus Propagation

Single Cell Culture

Preparation and Fusion of Protoplasts

Protocols on Protoplast Fusion

Fusion Products: Hybrids and Cybrids

Methods of Somatic Hybridization

Protoplast Fusion (Somatic Cell Hybridization)

Electrofusion Isolation of Oat and Corn Protoplast

Culture of Protoplasts

Other Protocols

Somatic Embryogenesis

Production of Haploid Plants

Embryo Encapsulation: Production of Artificial Seeds

Cytological Examination of Regenerated Plants

Demonstration of Root Hair Culture of Different Explants Source of Cereal Using Agrobacterium rhizogenes

Experiment Demonstration of Root Hair Culture of Different Explants Using Arbuscular Mycorrhizal Fungi

Useful Secondary-Metabolite Production from Plant Tissue Culture

Genetic Transformation of Plants through Agrobacterium tumefaciens as Cloning Vector

Assay of β-Glucuronidase in Transformed Plant Tissue

Random Amplified Polymorphic DNA Analysis

Efficiency of Rhizobia for Nodulation in Leguminous Crops

Seed Inoculation with Rhizobia

Suggested Readings

Important Links

Animal Cell Science and Technology

Introduction

Chronology of Cell Culture Development

Cell Culture Laboratory

Culture Medium

Cell Subculturing

Cell Quantification

Seeding Cells onto Culture Plates

Maintenance of Cells in Culture

Growth Kinetics of Animal Cells in Culture

Cryopreservation of Cells

Resuscitation of Frozen Cells

Determination of Cell Viability

Safety Considerations in Cell Culture

Aseptic Techniques and Good Cell Culture Practice

Preparation of Tissue Culture Medium

Preparation of Single Cell Suspension of Spleen and Thymus

Cell Counting

Cell Viability Assay

Estimation of Viability by Dye Uptake

Macrophage Monolayer from PEE and Measurement of Phagocytic Activity

Trypsinization of Monolayer and Subculturing

Cryopreservation

Thawing

Role of Serum in Cell Culture

Metaphase Chromosome Preparation from Cultured Cells

Isolation of DNA and Demonstration of Apoptosis of DNA Laddering

Demonstration of Apoptosis of DNA Laddering

MTT Assay for Cell Viability and Growth

Cell Fusion by PEG

Suggested Readings

Important Links

Bioprocess Engineering

Introduction

Isolation of Industrially Important Microorganisms

Determination of Thermal Death Point and Thermal Death Time of Microorganisms for Design of a Sterilizer

Determination of Ethanol Production Using Different Substrates

Sugar Fermentation by Yeast (S. cerevisiae) for Production of Ethanol and Cell Biomass Using Laboratory Fermenter of 2 L capacityMeasurement of Yeast Biomass Using Hemocytometer Technique

Study Fermentation Process for Lactic Acid Production by Streptococcus thermophilus and Cell Biomass

Microbial Production of Citric Acid Using Aspergillus niger

Microbial Production of Antibiotics (Penicillin)

Production of Vinegar (Acetic Acid)

Demonstration of Wine Production Using Grape Wine Juice

Production and Manufacture of Beer

Production and Estimation of Alkaline Protease

Mushroom Production Technology

Identification of Mushrooms by Spore Print Method

Isolation and Maintenance of Mushroom Culture

Production of Spawn for White Button Mushroom (A. brunnescens syn A.bisporus)

Cultivation of White Button Mushroom (A. brunnescens = A. bisporus)

Cultivation of G. lucidum (Ling Zhi)

Cultivation of L. edodes

Cultivation of Paddy Straw Mushroom (V. volvacea)

Cultivation Method of Oyster Mushroom

Environmental Biology

Wastewater Treatment Process

Measurement of pH

Measurement of Alkalinity of Water

Measurement of DO of Water

Measurement of Chloride in Water

Measurement of Nitrate in Water

Measurement of Nitrite in Water

Measurement of Ammonia in Water

Measurement of Total Phosphorus in Water

Measurement of Inorganic Phosphate in Water

Measurement of Sulfate in Water

Measurement of Sulfide in Water

Measurement of Calcium and Magnesium in Water

Detection of Coliforms for the Determination of the Purity of Potable Water

Determination of Total Dissolved Solids of Water

Determination of DO Concentration of Water by Winkler’s Iodometric Method

Determination of Biochemical Oxygen Demand of Sewage Water

Determination Chemical Oxygen Demand Sewage Sample

Determination of the Efficiency of Removal of Air Pollutant, Using Fibrous Air Filter

Isolation of Xenobiotic-Degrading Bacteria

Degradation of Aromatic Hydrocarbons by Bacteria

Survey of Degradative Plasmids in Microbes Growing in Polluted Environment

Effect of SO2 on Crop Plants

Estimation of Heavy Metals in Water/Soil by Atomic Absorption Spectrophotometry

Preliminary Digestion for Metals

Estimation of Metals

Flame AAS

Measurement of Aluminum in Soil/Water Sample

Measurement of Arsenic in Soil/Water Sample

Measurement of Cadmium in Soil/Water Sample

Measurement of Mercury in Soil/Water Sample

Study on Biogenic Methane Production in Different Habitats (Biogas)

Suggested Readings

Important Links

Bioinformatics

Introduction

Development

Nucleotide Sequence Databases

Protein Sequence Databases

Biomolecular Structure Databases

Pair-Wise Sequence Alignment

Detection of CpG Island for Nucleotide Sequence

Detection of Gene Structure of DNA Sequence

Translation of Nucleotide Sequence to Protein Sequence

Reports of Various Chemical and Physical Properties of Protein Sequence

Database Search "Blast"

Phylogenetic Analysis

Molecular Pathways Database

Proteomics

Metabolomics

Genomics

Bioinformatics Approaches for DNA-Based Signatures of Species/Populations/Breeds/Races/Variety/Strains

DNA-Based Signature of Plant Variety (Example, Basmati Rice)

DNA-Based Bar-Coded Signature of Fishes

Bioinformatics Tool and Protocol for SNP/STR Signatures

FastPCR

Suggested Readings

Important Links

Appendices

Glossary

Index

 

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