MICROBIOLOGY
Laboratory Exercises Third Edition
Keddis & Rauschenbach 2020
Photo Credits (in order of contribution): Diane Davis, Ines Rauschenbach & Ramaydalis Keddis
Acknowledgements: Many thanks to those in the Department of Biochemistry and Microbiology, Rutgers University, who have through the years inspired our enthusiasm for the science and teaching of microbiology, with special thanks to Diane Davis, Douglas Eveleigh and Max Häggblom.
Safety: The experiments included in this manual have been deemed safe by the authors when all necessary safety precautions are met. The authors recommend maintaining biosafety level 2 in the laboratory setting and using risk level 1 organisms for all exercises.
License: This work is licensed under a Creative Commons Attribution- NonCommercial-NoDerivatives 4.0 International License
Microbiology Laboratory Exercises Third Edition 2020
Ramaydalis Keddis, Ph.D. Ines Rauschenbach, Ph.D.
Department of Biochemistry and Microbiology Rutgers, The State University of New Jersey
CONTENTS PAGE
Introduction Schedule ii Best Laboratory Practices Iii Working in a Microbiology Laboratory iv
Exercises Preparation of a Culture Medium 1 Culturing and Handling Microorganisms 3 Isolation of a Pure Culture 5 Counting Bacterial Populations 8 Controlling Microorganisms 10 Disinfectants 10 Antimicrobial Agents: Susceptibility Testing 12 Hand Washing 14 The Lethal Effects of Ultraviolet Light 15 Selection of Fungi from Air 17 Microscopy 21 Morphology and Staining of Bacteria 26 Microbial Metabolism 30 Enzyme Assay 32 Metabolic Capabilities 34 Microbial Genetics 44 Transformation of Escherichia coli 44 Isolation of Plasmid DNA 47 Restriction Enzyme Digestion of Plasmid DNA and Gel Electrophoresis 49
Appendices A – List of Organisms Used Throughout the Semester 53 B – Vocabulary 54 C – Media 56 D – Intro to Micropipettes 59 E – Microscopy Pocket Guide 63 F – References and Supplemental Materials 65
Index 66
i
LABORATORY SCHEDULE
WEEK TOPIC
1 Biosafety; Best Lab Practices; LabArchives Notebook Preparation of a culture medium Culturing and handling microorganisms
2 Follow Up: Culturing and handling microorganisms Isolation of a Pure Culture Counting bacterial populations: Plate counts
3 Follow Up: Isolation of a Pure Culture Follow Up: Counting Bacterial Populations Control Of Microorganisms – Experimental Planning
4 Microscopy – Introduction Control Of Microorganisms – Lab Work
5 Microscopy – Phase Contrast (Yeast and Bacteria) Control of Microorganisms (Poster Work)
6 Microscopy – Phase Contrast (Unknowns) Control of Microorganisms (Poster Work) Selection of Fungi or Bacteria
7 Follow Up: Selection of Fungi or Bacteria Microbial Staining Control of Microorganisms (Poster Work)
8 Microbial Metabolism: Enzymes, Metabolic pathways Control of Microorganisms (Finish Poster)
9 Follow Up: Microbial Metabolism Poster Presentations
10 Transformation Plasmid Isolation Poster Presentations
11 Follow Up: Transformation Microbial Genetics: Electrophoretic analysis of restriction digest products (Plasmid Isolation)
12 Lab Practical
ii
Best Laboratory Practices The organisms used in General Microbiology are classified as Risk Level 1 organisms by the ABSA (American Biological Safety Association) as they are not known to cause disease in healthy humans. You will learn the techniques of safe microbiology using risk level 1 organisms; however, we will maintain bio safety 2 standards when working with all organisms. The following list of best practices is printed here to remind you of the safety discussion presented during the first laboratory meeting.