Challenges of the Unseen World: A Laboratory Course in Microbiology

ISBN-13: 9781555819927 / Angielski / Miękka / 2018 / 203 str.

Richard J. Meyer; Stacie A. Brown

Spis treści

PrefaceAbout the AuthorsIntroductionThe scientific methodExperimental designBig dataDocumentationSafetyStudent Laboratory Safety ContractAppendixChallenge One: Identifying the bacteria causing infections in hospital patientsLab OneBackgroundDiversity and pure culturesBright field and phase contrast microscopyLearning outcomesObjectivesPart 1: Isolate bacteria from a mixed culture: Procedure: Streaking for isolated coloniesPart 2: Examine bacterial cells under the microscopeProcedure: Making a wet mountProcedure: Using the microscopePreparation for next labQuestionsLab TwoBackgroundColony morphology and optimum temperature for growthCell shape and bacterial sporesThe cell envelopeLearning outcomesPart 1: Describe the colony morphology of the unknownPart 2: Describe the characteristics of an individual cell viewed under themicroscopePart 3: Determine the optimum temperature for growthPart 4: Determine if the unidentified microorganism is Gram-positive or Gram-negative.Procedure: Doing a Gram stainPreparation for next labQuestionsLab ThreeBackgroundModes of energy generation in bacteriaLearning outcomesPart 1: Can the unidentified microorganism grow in the presence of bile salts and ferment lactose?Procedure: Streaking cells on MacConkey-lactose platesPart 2: Can the unidentified microorganism ferment glucose?Procedure: Glucose fermentation testPart 3: Does the unidentified microorganism use cytochrome C duringrespiration (Gram-negative bacteria)?Procedure: Oxidase testPart 4: Does the microorganism make catalase (Gram-positive bacteria)?Procedure: Catalase testPart 5: Is the microorganism motile?Procedure: Soft agar motility assayQuestionsSolving Challenge OnePreparing for Challenge TwoQuestionsBibliographyChallenge Two: Confirming the identification of a microorganism by sequencing the 16S rRNA geneQuestions before you begin the challengeLab One: BackgroundClassification of bacteria and 16S rRNA genePolymerase chain reaction (PCR)Lab One: Learning outcomesLab One: ObjectivePart 1: Obtain enough DNA for sequencing: amplify the 16S rRNA gene by PCRProcedure:Diluting from stock solutions:Lab One: QuestionsLab Two: BackgroundAgarose gel electrophoresisDideoxy DNA sequencingLab Two: Learning outcomesLab Two: ObjectivesPart 1: Visualize the PCR product by agarose gel electrophoresisProcedure:Making an agarose gel and carrying out gel electrophoresisPart 2: Submit sample for DNA sequencingLab Two: QuestionsSolving Challenge Two: BackgroundSolving Challenge Two: Learning outcomesSolving Challenge Two: ObjectiveIdentifying the unknown microorganism from the 16S rRNA gene sequenceProcedure: Preparing the sequence for analysisProcedure: Doing a BLAST searchQuestionsBibliographyChallenge Three: Choosing an antibiotic to alleviate the symptoms of Crohn's diseaseQuestions before you begin the challengeLab One: BackgroundExponential growthThe bacterial growth curvePure cultures in liquid medium and the real world of bacteriaLab One: Learning outcomesLab One: ObjectivesPart 1: Construct a growth curve and calculate the generation timeProcedure: Recording the optical density of a growing culturePart 2: Determine viable cell counts during exponential growthProcedure: Serial dilution of samplesProcedure: Spreading cells on agar mediumLab One: QuestionsLab Two: BackgroundAssaying for antibiotic sensitivityLab Two: Learning outcomesLab Two: ObjectiveDetermine the MICs of different antibiotics for the Pseudomonas isolate.Procedure: Setting up a MIC dilution assayLab Two: QuestionsSolving Challenge ThreeBibliographyChallenge Four: Tracking down the source of an E. coli strain causing a local outbreak of diseaseQuestions before you begin the challengeLab One: BackgroundGenomic diversity and horizontal gene transferThe shifting genome of many bacteriaConjugation and other mechanisms of horizontal gene transferLab One: Learning outcomesLab One: ObjectivesPart 1: Determine if chloramphenicol resistance can be transferred byconjugationProcedure: Doing a conjugation experiment on TSA mediumPart 2: Determine if the donor strain for conjugation contains a plasmidProcedure: Rapid isolation of plasmid DNALab One: QuestionsLab Two: BackgroundStrain typingLab Two: Learning outcomesLab Two: ObjectivesPart 1: Determine if the plasmid DNAs from the lettuce isolate and the pathogenic strain are relatedProcedure: Doing a restriction digestPart 2: Determine if the donor strain for conjugation contains a plasmidProcedure: Rapid isolation of plasmid DNAProcedure: Agarose gel electrophoresis of the DNA fragmentsSolving Challenge FourQuestionsBibliographyChallenge Five: Using bacteriophage to identify the farm releasing pathogenic bacteria intoa village streamQuestions before you begin the challengeLab: BackgroundHistory and properties of bacteriophageTesting water purityLab: Learning outcomesLab: ObjectiveDetermine the load of bacteriophage at each collection siteProcedure: Filter the water samples to remove all the bacteriaProcedure: Titer the phages in the sterile filtratesSolving Challenge FiveLab: QuestionsBibliographyChallenge Six: Evaluating the pathogenic potential of bacteria causing urinary infectionsQuestions before you begin the challengeLab One: BackgroundQuorum sensingBiofilmsLab One: ObjectivesPart 1: Determine if the hospital isolates form biofilmsProcedure: Staining biofilms with crystal violetPart 2: Quantitatively analyze biofilm formationProcedure: Quantifying the amount of biofilm by spectrophotometryPart 3: Determine whether the hospital strains produce quorum sensingcompoundsProcedure: Using a reporter strain to detect quorum sensingLab One: QuestionsLab Two: BackgroundSwimmingLab Two: ObjectivesPart 1: Complete the analysis of quorum sensingPart 2: Assay the hospital strains for chemotaxis to different compoundsProcedure: Testing for chemotaxis with the "plug-in-soft agar" testPart 3: Determine the effectiveness of chemical cleanersProcedure: Testing for chemical effectiveness with the Kirby-Bauer disk diffusion assayQuestionsSolving Challenge SixBibliography

O autorze

Richard J. Meyer, Ph.D., is a professor in the Department of Molecular Biosciences at the University of Texas at Austin. He joined the Department of Microbiology in the University of Texas at Austin in 1978, and has been at that institution ever since. From the beginning of his career, Meyer has been interested in the hands-on aspect of teaching biology to undergraduates. He developed the introductory microbiology laboratory course currently used at the University of Texas at Austin. It was the pedagogical approaches that are used in this course that inspired him to develop the manual you hold in your hands. Over more than forty years, Meyer's research was on the molecular mechanisms of replication and conjugative transfer of broad host-range plasmids.Stacie A. Brown, Ph.D., is director of first year biology laboratories and a member of the biology department at Southwestern University. Prior to her current position, she taught microbiology courses for biology majors and pre-nursing students while also overseeing the microbiology labs, at Texas State University. For several years, she also taught microbiology labs and courses at the University of Texas at Austin. Her experience teaching microbiology labs to thousands of undergraduates ensures that the challenge-based microbiology labs in this manual will work in any introductory laboratory course in undergraduate microbiology.