Laboratory Methods in Microbiology and Molecular Biology

Contents
List of contributors
Preface
Acknowledgements
Introduction

1. General guidelines, expectations, and responsibilities for inculcating good lab practices
Richa Salwan and Vivek Sharma

1. Distributing/sharing lab responsibilities

2. Awareing students/researchers for research ethics/Authorship claims

3. Celebrations

4. Lab meetings

5. Reproducibility and data management

6. Scientific collection/work life balance

7. Handling reagents/Solutions and visiting fields

8. Laboratory safety

9. Personal safety

10. Preparing standard solutions

2. Preparations of laboratory solutions
Richa Salwan, Aditi Rana and Vivek Sharma

1. Guidelines for preparing reagents, buffers, etc

2. General considerations for working with RNA

3. General terms and description for the preparation of reagents

4. Selection and preparation of buffer solution
References
Further reading

3. Basic concepts of microbiology
Richa Salwan, Aditi Rana and Vivek Sharma

1. Aim: preparation of culture media for the routine cultivation of bacteria

2. Aim: general techniques for the isolation and cultivation of bacteria

3. Aim: to preserve bacterial and fungal cultures by using different methods
Further reading

4. Microscopy and imaging analysis of microorganisms
Richa Salwan, Aditi Rana and Vivek Sharma

1. Aim: to prepare wet mount slide of the given sample

2. Aim: to observe motility of bacteria using hanging drop experiment

3. Aim: preparation of bacterial smear

4. Aim: to prepare a slide for simple staining

5. Aim: to perform negative staining of bacteria

6. Aim: to prepare a slide for Gram’s staining

7. Aim: capsule staining by anthony staining method

8. Aim: to perform capsule staining of the bacterial culture

9. Aim: to stain flagella of a bacterial culture

10. Aim: to perform spore staining of the given culture

11. Aim: perform spore staining using Dorner’s method

12. Aim: to stain the nuclear material and study the detailed morphology of the nucleus

13. Aim: to perform lactophenol cotton blue staining of the give fungal culture

14. Aim: to perform staining of a given actinomycetes culture

15. Aim: to perform acid-fast staining of a given culture
Further reading

5. Characterization of microbes using molecular methods
Richa Salwan, Aditi Rana and Vivek Sharma

1. Morphological characterization using Biolog

2. Microbial identification based on FAME

3. Electron microscopy

4. Molecular characterization of bacteria
Further reading

6. Soil microbiology
Vivek Sharma and Richa Salwan

1. Aim: Isolation of Rhizobium from the given sample

2. Aim: To isolate Actinomycetes from the given soil sample

3. Aim: Isolation of Azotobacter from soil

4. Aim: To estimate soil microbial biomass by Chloroform fumigation extraction method

5. Aim: To estimate the quantity of various enzymes in soil samples

6. Aim: To study the effect of pH on the growth of microbes

7. Aim: To study the effect of temperature on growth of microbes

8. Aim: To study the effect of salt concentration on growth of microbes

9. Aim: To perform dual culture assay or direct confrontation assay
References
Further reading

7. Basic experiments in food microbiology
Richa Salwan, Aditi Rana and Vivek Sharma

1. Aim: Evaluation of milk quality using methylene blue reduction test (MBRT) assay

2. Aim: Isolation of lactic acid bacteria

3. Aim: To prepare yogurt under laboratory conditions

4. Aim: Preparation of bread from wheat flour

5. Aim: To prepare alcohol from grape juice under laboratory conditions

6. Aim: To prepare sauerkraut from cabbage

7. Aim: To prepare cheese under laboratory conditions
Further reading

8. Basic experiments in microbial biochemistry
Richa Salwan, Aditi Rana and Vivek Sharma

1. Aim: Qualitative screening of bacteria for amylase production

2. Aim: Qualitative screening of bacteria for lipase production

3. Aim: Qualitative screening of bacteria for protease production

4. Aim: Qualitative screening of bacteria for indole production

5. Aim: To perform methyl red (MR) test for detection of coliforms

6. Aim: To perform Voges-Proskauer test

7. Aim: Assessment of bacteria for utilization of sodium citrate as carbon source

8. Aim: To perform catalase test

9. Aim: Qualitative assessment of oxidase production

10. Aim: Assessment of carbohydrate fermentation

11. Aim: To perform oxidation-fermentation test

12. Aim: To perform nitrate reduction test

13. Aim: Qualitative assessment of urea hydrolysis

14. Aim: To perform triple sugar iron agar (TSIA) test
Further reading

9. Analytical techniques in microbial genomics
Vivek Sharma and Richa Salwan

1. Isolation of nucleic acid (DNA and RNA) from microorganisms

2. Analysis of nucleic acid (DNA and RNA) from microorganisms
References

10. Analytical techniques in proteomics
Richa Salwan and Vivek Sharma

1. Basics of protein electrophoresis

2. SDS-PAGE

3. Consumables

4. Equipments

5. SDS-PAGE staining using Coomassie R250 or G250

6. Silver staining

7. Chemicals

8. Equipments and per-requisite

9. Method

10. Fluorescent silver staining

11. Chemicals

12. Equipments and per-requisite

13. Method
Further reading

11. Analytical techniques in proteomics
Richa Salwan and Vivek Sharma

1. Aim: Isolation and characterization of extracellular protein

2. Protein dialysis and ultrafiltration

3. Protein fractionation and desalting using gel filtration chromatography

4. Use of bioinformatics in planning a protein purification
Further reading

12. Expression of recombinant proteins in prokaryotic host
Richa Salwan and Vivek Sharma

1. Aim: Cloning and transformation of gene of interest in expression vector

2. Background of pET vector system and expression host

3. Equipment and reagents for cloning and expression of genes

4. Protocol

5. Optimization of insert: vector molar ratios
Further reading

13. Sero-diagnostics in microbiology
Richa Salwan and Vivek Sharma

1. Enzyme-linked immunosorbent assay (ELISA) and variants of ELISA

2. Western blotting

3. Immunoprecipitation/agglutination assay using blood sample

4. To perform ouchterlony double diffusion or passive double immunodiffusion

5. To perform radial immunodiffusion assay
Further reading

14. Basic tools for next-generation DNA sequencing of bacterial genome
Richa Salwan and Vivek Sharma

1. Background

2. Protocol

3. Reagents for illumina

4. Equipments

5. Protocol

6. Bioinformatic analyses
Further reading
Index