Challenges of the Unseen World. A Laboratory Course in Microbiology. Edition No. 1. ASM Books

Table of Contents

Preface

About the Authors

Introduction

The scientific method

Experimental design

Big data

Documentation

Safety

Student Laboratory Safety Contract

Appendix

Challenge One: Identifying the bacteria causing infections in hospital patients

Lab One

Background

Diversity and pure cultures

Bright field and phase contrast microscopy

Learning outcomes

Objectives

Part 1: Isolate bacteria from a mixed culture: Procedure: Streaking for isolated colonies

Part 2: Examine bacterial cells under the microscope

Procedure: Making a wet mount

Procedure: Using the microscope

Preparation for next lab

Questions

Lab Two

Background

Colony morphology and optimum temperature for growth

Cell shape and bacterial spores

The cell envelope

Learning outcomes

Part 1: Describe the colony morphology of the unknown

Part 2: Describe the characteristics of an individual cell viewed under themicroscope

Part 3: Determine the optimum temperature for growth

Part 4: Determine if the unidentified microorganism is Gram-positive or Gram-negative.

Procedure: Doing a Gram stain

Preparation for next lab

Questions

Lab Three

Background

Modes of energy generation in bacteria

Learning outcomes

Part 1: Can the unidentified microorganism grow in the presence of bile salts and ferment lactose?

Procedure: Streaking cells on MacConkey-lactose plates

Part 2: Can the unidentified microorganism ferment glucose?

Procedure: Glucose fermentation test

Part 3: Does the unidentified microorganism use cytochrome C duringrespiration (Gram-negative bacteria)?

Procedure: Oxidase test

Part 4: Does the microorganism make catalase (Gram-positive bacteria)?

Procedure: Catalase test

Part 5: Is the microorganism motile?

Procedure: Soft agar motility assay

Questions

Solving Challenge One

Preparing for Challenge Two

Questions

Bibliography

Challenge Two: Confirming the identification of a microorganism by sequencing the 16S rRNA gene

Questions before you begin the challenge

Lab One: Background

Classification of bacteria and 16S rRNA gene

Polymerase chain reaction (PCR)

Lab One: Learning outcomes

Lab One: Objective

Part 1: Obtain enough DNA for sequencing: amplify the 16S rRNA gene by PCR

Procedure:Diluting from stock solutions:

Lab One: Questions

Lab Two: Background

Agarose gel electrophoresis

Dideoxy DNA sequencing

Lab Two: Learning outcomes

Lab Two: Objectives

Part 1: Visualize the PCR product by agarose gel electrophoresis

Procedure:Making an agarose gel and carrying out gel electrophoresis

Part 2: Submit sample for DNA sequencing

Lab Two: Questions

Solving Challenge Two: Background

Solving Challenge Two: Learning outcomes

Solving Challenge Two: Objective

Identifying the unknown microorganism from the 16S rRNA gene sequence

Procedure: Preparing the sequence for analysis

Procedure: Doing a BLAST search

Questions

Bibliography

Challenge Three: Choosing an antibiotic to alleviate the symptoms of Crohn’s disease

Questions before you begin the challenge

Lab One: Background

Exponential growth

The bacterial growth curve

Pure cultures in liquid medium and the real world of bacteria

Lab One: Learning outcomes

Lab One: Objectives

Part 1: Construct a growth curve and calculate the generation time

Procedure: Recording the optical density of a growing culture

Part 2: Determine viable cell counts during exponential growth

Procedure: Serial dilution of samples

Procedure: Spreading cells on agar medium

Lab One: Questions

Lab Two: Background

Assaying for antibiotic sensitivity

Lab Two: Learning outcomes

Lab Two: Objective

Determine the MICs of different antibiotics for the Pseudomonas isolate.

Procedure: Setting up a MIC dilution assay

Lab Two: Questions

Solving Challenge Three

Bibliography

Challenge Four: Tracking down the source of an E. coli strain causing a local outbreak of disease

Questions before you begin the challenge

Lab One: Background

Genomic diversity and horizontal gene transfer

The shifting genome of many bacteria

Conjugation and other mechanisms of horizontal gene transfer

Lab One: Learning outcomes

Lab One: Objectives

Part 1: Determine if chloramphenicol resistance can be transferred byconjugation

Procedure: Doing a conjugation experiment on TSA medium

Part 2: Determine if the donor strain for conjugation contains a plasmid

Procedure: Rapid isolation of plasmid DNA

Lab One: Questions

Lab Two: Background

Strain typing

Lab Two: Learning outcomes

Lab Two: Objectives

Part 1: Determine if the plasmid DNAs from the lettuce isolate and the pathogenic strain are related

Procedure: Doing a restriction digest

Part 2: Determine if the donor strain for conjugation contains a plasmid

Procedure: Rapid isolation of plasmid DNA

Procedure: Agarose gel electrophoresis of the DNA fragments

Solving Challenge Four

Questions

Bibliography

Challenge Five: Using bacteriophage to identify the farm releasing pathogenic bacteria intoa village stream

Questions before you begin the challenge

Lab: Background

History and properties of bacteriophage

Testing water purity

Lab: Learning outcomes

Lab: Objective

Determine the load of bacteriophage at each collection site

Procedure: Filter the water samples to remove all the bacteria

Procedure: Titer the phages in the sterile filtrates

Solving Challenge Five

Lab: Questions

Bibliography

Challenge Six: Evaluating the pathogenic potential of bacteria causing urinary infections

Questions before you begin the challenge

Lab One: Background

Quorum sensing

Biofilms

Lab One: Objectives

Part 1: Determine if the hospital isolates form biofilms

Procedure: Staining biofilms with crystal violet

Part 2: Quantitatively analyze biofilm formation

Procedure: Quantifying the amount of biofilm by spectrophotometry

Part 3: Determine whether the hospital strains produce quorum sensingcompounds

Procedure: Using a reporter strain to detect quorum sensing

Lab One: Questions

Lab Two: Background

Swimming

Lab Two: Objectives

Part 1: Complete the analysis of quorum sensing

Part 2: Assay the hospital strains for chemotaxis to different compounds

Procedure: Testing for chemotaxis with the “plug-in-soft agar” test

Part 3: Determine the effectiveness of chemical cleaners

Procedure: Testing for chemical effectiveness with the Kirby-Bauer disk diffusion assay

Questions

Solving Challenge Six

Bibliography

Authors

Richard J. Meyer University of Texas at Austin, TX. Stacie A. Brown Southwestern University, Georgetown, TX.