Laboratory Protocols in Applied Life Sciences

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: BiotechnologyAnalytical biochemistryClinical biochemistryBiophysicsMolecular biologyGenetic engineeringBioprocess technologyIndustrial processesAnimalPlantMicrobial biologyComputational biologyBiosensors 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 ContentMicroscopy 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 I