Challenges of the Unseen World

Solving real-world health challenges in a learning environment You are at an exciting gateway into the world of microorganisms. With nothing more than basic lab equipment such as microscopes, Petri dishes, media, and a handful of reagents, you will learn to isolate, grow, and identify bacteria that live all around us. This is no ordinary microbiology laboratory course; not only will you learn how to streak plates, use a microscope, perform a Gram stain, and prepare serial dilutions and spread plates—fundamental skills found in every microbiologist's toolkit—you will solve a series of public health–related challenges that many professional microbiologists encounter in their work. By the end of this course, you will: Determine the origin of a nosocomial infection. Using foundational and molecular methods, you will determine whether the infections occurring in hospitalized patients are the result of contaminated medical items. Select the antibiotic to treat a patient with Crohn's disease. You will find minimum inhibitory concentrations of various antibiotics for a Pseudomonas strain associated with Crohn's disease. Pinpoint the source of lettuce contaminated with E. coli. Using molecular tools you will investigate a common food safety challenge, antibiotic-resistant E. coli and the potential for spread of this resistance in the environment. Find the farm releasing pathogens into a stream used for drinking water. Using bacteriophage load in water samples, you will locate the source of fecal contamination in the water supply of a village in an underdeveloped country. Evaluate the potential of bacteria to cause a urinary tract infection. You will test for biofilms, quorum sensing behavior, and chemotaxis and assess which disinfectants would be most effective for sanitizing contaminated surfaces. Microbiology educators and researchers Richard Meyer and Stacie Brown have created this hands-on, engaging introduction to the essential laboratory skills in the microbial sciences that is sure to change the way you view the world around you.

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