Laboratory Methods in Microbiology and Molecular Biology

Dr. Salwan is currently an Assistant Professor (Microbiology) at the College of Horticulture and Forestry (Dr. YS Parmar University of Horticulture and Forestry), Neri, Hamirpur, Himachal Pradesh, India. Dr. Salwan’s research interests and contributions are on the diversity of psychrotrophic bacteria from the Western Himalayas and their utilization for industrial applications. She has also worked on the exploration of extremophiles for industrially relevant enzymes and plant beneficial microbes for agricultural benefits. She has published two books and numerous research papers in several international journals. Dr. Salwan serves as an Academic Editor for PLOS ONE and is also a recognized reviewer for several journals including MDPI Genes, MDPI Diversity, MDPI Foods, BMC Microbiology, Journal of Plant Growth Regulation, and Microbial Ecology. Dr. Vivek Sharma is currently an Assistant Professor at the University Centre for Research and Development at Chandigarh University, Mohali (PB). He has more than 12 years of research experience exploring molecular attributes of Trichoderma. His research also involves examining the molecular aspects of microbes beneficial to plants such as Streptomyces, and Bacillus. He has published several research papers in international journals, serves as an Academic Editor for PLOS ONE, the review editor for Frontiers in Bioengineering and Biotechnology, an Associate Editor of Chemical and Biological Technologies in Agriculture, and is a member of the editorial board of Current Proteomics. He is also a recognized reviewer for journals such as the Journal of Advanced Research, Applied Microbiology and Biotechnology, Environmental Research, the Journal of Proteomics, BMC Genomics, BMC Plant Biology, AMB Express, Molecular Biotechnology, MDPI Pathogens, Folia Microbiology, Physiological and Molecular Plant Pathology and Archives of Microbiology.

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