Laboratory Methods in Microbiology and Molecular Biology

<p>Contents<br>List of contributors<br>Preface<br>Acknowledgements<br>Introduction</p> <p>1. General guidelines, expectations, and responsibilities for inculcating good lab practices<br>Richa Salwan and Vivek Sharma</p> <p>1. Distributing/sharing lab responsibilities<br>2. Awareing students/researchers for research ethics/Authorship claims<br>3. Celebrations<br>4. Lab meetings<br>5. Reproducibility and data management<br>6. Scientific collection/work life balance<br>7. Handling reagents/Solutions and visiting fields<br>8. Laboratory safety<br>9. Personal safety<br>10. Preparing standard solutions</p> <p>2. Preparations of laboratory solutions<br>Richa Salwan, Aditi Rana and Vivek Sharma</p> <p>1. Guidelines for preparing reagents, buffers, etc <br>2. General considerations for working with RNA <br>3. General terms and description for the preparation of reagents <br>4. Selection and preparation of buffer solution <br>References<br>Further reading</p> <p>3. Basic concepts of microbiology<br>Richa Salwan, Aditi Rana and Vivek Sharma</p> <p>1. Aim: preparation of culture media for the routine cultivation of bacteria <br>2. Aim: general techniques for the isolation and cultivation of bacteria <br>3. Aim: to preserve bacterial and fungal cultures by using different methods <br>Further reading </p> <p>4. Microscopy and imaging analysis of microorganisms <br>Richa Salwan, Aditi Rana and Vivek Sharma</p> <p>1. Aim: to prepare wet mount slide of the given sample<br>2. Aim: to observe motility of bacteria using hanging drop experiment<br>3. Aim: preparation of bacterial smear<br>4. Aim: to prepare a slide for simple staining<br>5. Aim: to perform negative staining of bacteria<br>6. Aim: to prepare a slide for Gram’s staining<br>7. Aim: capsule staining by anthony staining method <br>8. Aim: to perform capsule staining of the bacterial culture<br>9. Aim: to stain flagella of a bacterial culture<br>10. Aim: to perform spore staining of the given culture<br>11. Aim: perform spore staining using Dorner’s method<br>12. Aim: to stain the nuclear material and study the detailed morphology of the nucleus<br>13. Aim: to perform lactophenol cotton blue staining of the give fungal culture <br>14. Aim: to perform staining of a given actinomycetes culture <br>15. Aim: to perform acid-fast staining of a given culture <br>Further reading </p> <p>5. Characterization of microbes using molecular methods <br>Richa Salwan, Aditi Rana and Vivek Sharma</p> <p>1. Morphological characterization using Biolog <br>2. Microbial identification based on FAME <br>3. Electron microscopy <br>4. Molecular characterization of bacteria <br>Further reading </p> <p>6. Soil microbiology <br>Vivek Sharma and Richa Salwan</p> <p>1. Aim: Isolation of Rhizobium from the given sample <br>2. Aim: To isolate Actinomycetes from the given soil sample <br>3. Aim: Isolation of Azotobacter from soil <br>4. Aim: To estimate soil microbial biomass by Chloroform fumigation extraction method <br>5. Aim: To estimate the quantity of various enzymes in soil samples <br>6. Aim: To study the effect of pH on the growth of microbes <br>7. Aim: To study the effect of temperature on growth of microbes <br>8. Aim: To study the effect of salt concentration on growth of microbes <br>9. Aim: To perform dual culture assay or direct confrontation assay <br>References <br>Further reading </p> <p>7. Basic experiments in food microbiology<br>Richa Salwan, Aditi Rana and Vivek Sharma</p> <p>1. Aim: Evaluation of milk quality using methylene blue reduction test (MBRT) assay <br>2. Aim: Isolation of lactic acid bacteria <br>3. Aim: To prepare yogurt under laboratory conditions <br>4. Aim: Preparation of bread from wheat flour <br>5. Aim: To prepare alcohol from grape juice under laboratory conditions <br>6. Aim: To prepare sauerkraut from cabbage <br>7. Aim: To prepare cheese under laboratory conditions <br>Further reading </p> <p>8. Basic experiments in microbial biochemistry <br>Richa Salwan, Aditi Rana and Vivek Sharma</p> <p>1. Aim: Qualitative screening of bacteria for amylase production <br>2. Aim: Qualitative screening of bacteria for lipase production <br>3. Aim: Qualitative screening of bacteria for protease production <br>4. Aim: Qualitative screening of bacteria for indole production <br>5. Aim: To perform methyl red (MR) test for detection of coliforms <br>6. Aim: To perform Voges-Proskauer test <br>7. Aim: Assessment of bacteria for utilization of sodium citrate as carbon source <br>8. Aim: To perform catalase test <br>9. Aim: Qualitative assessment of oxidase production <br>10. Aim: Assessment of carbohydrate fermentation <br>11. Aim: To perform oxidation-fermentation test <br>12. Aim: To perform nitrate reduction test <br>13. Aim: Qualitative assessment of urea hydrolysis <br>14. Aim: To perform triple sugar iron agar (TSIA) test <br>Further reading </p> <p>9. Analytical techniques in microbial genomics <br>Vivek Sharma and Richa Salwan</p> <p>1. Isolation of nucleic acid (DNA and RNA) from microorganisms <br>2. Analysis of nucleic acid (DNA and RNA) from microorganisms <br>References </p> <p>10. Analytical techniques in proteomics<br>Richa Salwan and Vivek Sharma</p> <p>1. Basics of protein electrophoresis<br>2. SDS-PAGE <br>3. Consumables <br>4. Equipments<br>5. SDS-PAGE staining using Coomassie R250 or G250<br>6. Silver staining<br>7. Chemicals<br>8. Equipments and per-requisite<br>9. Method <br>10. Fluorescent silver staining<br>11. Chemicals<br>12. Equipments and per-requisite<br>13. Method<br>Further reading</p> <p>11. Analytical techniques in proteomics<br>Richa Salwan and Vivek Sharma</p> <p>1. Aim: Isolation and characterization of extracellular protein <br>2. Protein dialysis and ultrafiltration <br>3. Protein fractionation and desalting using gel filtration chromatography <br>4. Use of bioinformatics in planning a protein purification <br>Further reading</p> <p>12. Expression of recombinant proteins in prokaryotic host<br>Richa Salwan and Vivek Sharma</p> <p>1. Aim: Cloning and transformation of gene of interest in expression vector<br>2. Background of pET vector system and expression host<br>3. Equipment and reagents for cloning and expression of genes<br>4. Protocol<br>5. Optimization of insert: vector molar ratios<br>Further reading</p> <p>13. Sero-diagnostics in microbiology<br>Richa Salwan and Vivek Sharma</p> <p>1. Enzyme-linked immunosorbent assay (ELISA) and variants of ELISA<br>2. Western blotting<br>3. Immunoprecipitation/agglutination assay using blood sample<br>4. To perform ouchterlony double diffusion or passive double immunodiffusion<br>5. To perform radial immunodiffusion assay<br>Further reading</p> <p>14. Basic tools for next-generation DNA sequencing of bacterial genome<br>Richa Salwan and Vivek Sharma</p> <p>1. Background <br>2. Protocol <br>3. Reagents for illumina <br>4. Equipments <br>5. Protocol <br>6. Bioinformatic analyses <br>Further reading <br>Index </p>