Laboratory Protocols in Applied Life Sciences.pdf
Analytical Instrumentation Microscopy Introduction Light microscopy Atomic force microscopy (AFM) Bright-field microscopy Dark-ground microscopy Phase contrast microscopy Confocal scanning laser microscopy (CSLM) Differential interference microscopy (DIC) Polarisation microscopy Fluorescence microscopy Experiments on microscopy Electron microscopy Sample Preparation for Electron Microscopy The Crewe Microscope The backseatter Scanning Microscope Scanning Electron Microscopy of Cyanobacteria Transmission electron microscopy (TEM) of phages (Bacteriophage and Cyanophage)Suggested Readings Important Links Microtome Introduction Traditional Histology Sectioning Ultramicrotome The Cryostat Incubation Electron Microscopy Technique Botanical microtomy Processing of tissueSuggested Readings Important links 3 pH and Buffers Introduction Acids and bases Hydrogen ion concentration and pH Measurement of pH Titration curves 6 Buffer solutions What is the pH of a mixture of 5 ml of 0.1 mol/litre sodium acetate and 4 ml of 0.1 mol/litre acetic acid? How the pH changed on adding 1 ml of 0.1 mol / litre HCl in the above mixture? Buffers used in biochemical experiments pH and life The care and use of the pH meter Titration curves The 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 The determination of 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 sampleSuggested Readings Important LinksAppendix: Indicators of hydrogen ion concentration Spectrophotometer Introduction Colorimeter Spectrophotometry Infrared Spectroscopy Fluorescence spectroscopy The sensitivity of fluorescence assays Fluorescence quenching Measurement of alpha-naphthyl phosphatase activity Suggested Reading Important Links Centrifugation Introduction Centrifuges Ultracentrifugation Suspending medium Rotors The fractionation of rat liver The 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 The determination of lysosomal integrity The effect of detergents on the stability of the lysosomal membrane Density gradient centrifugation The fractionation of pig brain by density gradient centrifugation The 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 Reading Important Links Radiation biology Introduction Radioisotopes Sample preparation Autoradiography Distribution of 14C02 in Different Plant Parts Photosynthetic reduction of 14CO2 to primary metabolic product Incorporation of labelled acetate into leucoplastic fatty acids of developing Brassica seeds Determination of 2-carboxyarahinitol-l-P in plant leaves by isotope dilution techniqueSuggested Readings Important Links Separation Techniques 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 Separate and Identify the Amino Acids in a Given Mixture by Two Dimensional Paper Chromatography Thin Layer Chromatography Thin Layer Gel Chromatography Separation and Identification of Sugars by Adsorption TLC Identify 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 The separation of amino acids by ion exchange chromatography The separation of proteins from human serum by ion exchange chromatography Gel Filtration/ Size Exclusion Column Chromatography The separation of blue dextran and cobalt chloride on Sephadex G25 Desalting of Protein Sample by Gel Filtration Determination Molecular Weight of a given Protein Gel Filtration Concentration of Dilute Protein Solutions using Sephadex G25 Affinity chromatography Hydrophobic interaction chromatography (HIC) or affinity elution Purification by affinity chromatography The preparation of an affinity column Preparation of rat brain acetylcholinesterase Acetylcholinesterase assay Purification of acetylcholinesterase by affinity chromatography Suggested Readings Important Links Electrophoresis Principle Types of Electrophoresis Separation of amino acids by paper electrophoresis Cellulose acetate strip electrophoresis Gel electrophoresis Polyacrylamide gel electrophoresis Polyacrylamidedisc gel electrophoresis SDS-gel electrophoresis Slab Gel electrophoresis Native Disc-Gel Electrophoresis of Proteins SDS-Polyaerylurnide Slab Gel Electrophoresis of Proteins under reducing Conditions Slab gel Gradient electrophoresis of protein and multiple molecular forms of Protein (Western) Blotting Isoelctric 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 ElectrophoresisSuggested Readings Important Links Cell Biology Histochemical Techniques Introduction Protein Acidic compounds Carbohydrates - Polysaccharides Lipids Minor compounds Autoradiography Enzyme Localization in Embedded MaterialSuggested 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 sqush techniques from onion root tips Perform carmine or orcein staining techniques Perform 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 ? radiation on chromosomes To prepare slides showing diplotene stages in spermatocytes of desert locusr (Schistocerca gregaria) or in oocytes of the newt (Triturus viridescens) showing lampbrush chromosomes Introduction Materials Method Procedure 1 (For Triturus -newt- or any other Amphibian) Procedure 2 (For Schistocerca gregaria) Study karyotypes of Allium cepa and Vicia faba To demonstrate reciprocal translocation in pollen mother cells of Rhoeo discolor 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 Test for nutritional requirement Suggested media for Nurospora, Sordaria and Aspergillus experiments Study Of heterokaryosis in Neurospora from the nutritionally deficient mutants 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 Study of Blood Groups Autoradiography Photomicrography Suggested Readings Important links Biochemistry Biomolecules Introduction Carbohydrates Amino acids and proteins Lipids Nucleic acidsSuggestedReading Important Links Enzymology Introduction Enzymes as catalysts Catalysis Classification Measurement of enzyme activity Time course studies of the reaction catalysed by alkaline phophatase (EC 184.108.40.206 ) The effect of enzyme concentration on the rate of enzyme catalyzed reaction Determination of temperature optima for alkaline phosphatase Determination ofeffect 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, 220.127.116.11) Effect of variation of serum alkaline phosphatase activity with enzymes concentration The effect of substrate concentration and inhibitors on ox heart lactate dehydrogenase The effect of temperature on the activity of alpha-amylase The determination of the molecular weight and purity of chymotrypsin from the enzyme Yeast isocitrate dehydrogenase: an allosteric enzymes Purification and characterization of enzymes The isolation of muramidase (mucopeptide N-acetylmuramyl hydrolase, 3.21.17) from egg white Separation of the isoenzymes of lactate dehydrogenase by electrophoresis on polyacrylamide-gel Enzyme Engineering Artificial enzymesSuggested Readings Important Links Membranes Introduction Composition of membranes Membrane Transport The effect of lipid composition on the permeability of a lipid monolayer The effect of detergents and other membrane-active reagents on the erythrocyte membrane The permeability of model membrane (liposome) The effect of cholesterol on the anion permeability of a phospholipid membrane Effect of insulin on the transport into isolated fat cell The transport of amino acids across the small intestine The absorption of xylose from the gut in manSuggested readings Important Links Photosynthesis and Respiration Introduction Measurement Photosynthesis The isolation of chloroplasts from spinach leaves Determination ofchlorophyll concentration in the chloroplast suspension The evolution of oxygen by isolated chloroplast using Hill oxidants Methyl viologen as a terminal electron acceptor The spectrophotometric assay of the Hill reaction and the estimation of chlorophyll The respiration of mitochondria and oxidative phosphorylation The effect of inhibitors on the respiratory chain energy state o...
This manual includes details of methodologies used in diverse disciplines of life sciences including biotechnology, pharmaceutical sciences, medical sciences, and related fields. It provides the comprehensive information required to build a firm foundation in the life sciences. Due emphasis is given to techniques encountered both in practical classes but also in high throughput techniques involved in modern eras of commercialization and product inventories. Well illustrated to explain the theories behind each exercise, this book covers both basic techniques and also new, advanced techniques that continue to evolve and change.