Lab Exercises:

Results:#8 Quantification lab#9 Aerobic/ Anaerobic#12 UV radiation lab#22 Normal Skin BiotaNew Labs:#14 Antibiotics#15 Disinfectants

Viable cell counts: cells capable of multiplyingCan use selective, differential media for particular speciesPlate counts: single cell gives rise to colonyPlate out dilution series: 30300 colonies ideal4.8. Methods to Detect and Measure Microbial GrowthCopyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.Adding 1 ml of culture to 9 ml of diluent results in a 1:10 dilution.Original bacterialcultureto 9 ml diluent1:10,000 dilutionto 9 ml diluent1:1,000 dilutionto 9 ml diluent1:10 dilutionto 9 ml diluent1:100 dilution5cells/ml50,000cells/ml5,000cells/ml500cells/ml50cells/mlToo many cellsproduce toomany coloniesto count.Too many cellsproduce toomany coloniesto count.Too many cellsproduce toomany coloniesto count.Between 30300cells produces acountable plate.Does not produceenough coloniesfor a valid count.1 ml1 ml1 ml1 ml1 ml

Plate counts determine colony-forming units (CFUs)4.8. Methods to Detect and Measure Microbial GrowthCulture, dilutedas needed0.11.0 ml0.10.2 mlSpread cells onto surfaceof pre-poured solid agar.Pour-plate methodSpread-plate methodIncubateBacterial coloniesappear only on surface.Melted cooled agarIncubateSome colonies appear onsurface; many are below surface.Add melted cooled agarand swirl gently to mix.Solid agarCopyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Dilutions: Determining number of organism in original culture# colonies x (1/volume plated) x (1/dilution)Want 30-300 colonies (countable range)Helpful things to remember:We plated 1 mL of each dilutionDilutions ranged from A(10-4-10-7) or B (10-6-10-9)1 over a negative exponent = a positive exponentExample: 256 colonies, 1 mL plated, at 10-7256 x (1/1) x (1/10-7)=256 x 1x 107=256 x 107= 2.56x109Remember to move the decimal for proper scientific notation (then add that number to the exponent)

Boil nutrient agar to drive off O2; cool to just above solidifying temperature; innoculate; gently swirlGrowth demonstrates organisms O2 requirements Oxygen Requirements

Radiation: two typesUltraviolet irradiation forms thymine dimersCovalent bonds between adjacent thyminesCannot fit into double helix; distorts moleculeReplication and transcription stall at distortionCell will die if damage not repairedMutations result from cells SOS repair mechanismX rays cause single- anddouble-strand breaks in DNADouble-strand breaksoften produce lethaldeletionsX rays can alter nucleobases

8.3. Induced MutationsCopyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.Sugar-phosphatebackboneUltravioletlightThymineThymineThymine dimerCovalentbonds

StaphylocciS. aureus- pathogen, many virulence factorsS. epidermidis- commensal, not typically pathogenicDifferentiating Staphylococci species:Mannitol Salt Agar (MSA) platesS. aureus can ferment mannitolResults in yellow color changeS. epidermidis can NOT ferment mannitolResults in no color change (agar remains pink/red)Coagulase testS. aureus coagulates rabbit plasmaS. epidermidis can NOT coagulate rabbit plasma

Potency of Germicidal Chemical FormulationsSterilants destroy all microorganisms Heat-sensitive critical instrumentsHigh-level disinfectants destroy viruses, vegetative cellsDo not reliably kill endosporesSemi-critical instrumentsIntermediate-level disinfectants destroy vegetative bacteria, mycobacteria, fungi, and most virusesDisinfect non-critical instrumentsLow-level disinfectants destroy fungi, vegetative bacteria except mycobacteria, and enveloped virusesDo not kill endospores, naked virusesDisinfect furniture, floors, walls5.5. Using Chemicals to Destroy Microorganisms and Viruses

Selecting the Appropriate Germicidal ChemicalToxicity: benefits must be weighed against risk of useActivity in presence of organic materialMany germicides inactivatedCompatibility with material being treatedLiquids cannot be used on electrical equipmentResidues: can be toxic or corrosiveCost and availabilityStorage and stabilityConcentrated stock decreases storage spaceEnvironmental riskAgent may need to be neutralized before disposal5.5. Using Chemicals to Destroy Microorganisms and Viruses

Classes of Germicidal Chemicals

20.3. Mechanisms of Action of Antibacterial DrugsAntibacterial drugs target specific bacterial processes and structuresCell wall synthesisProtein synthesisNucleic acid synthesisMetabolic pathwaysCell membranes ABNucleic acid synthesisFluoroquinolonesRifamycinsCell wall(peptidoglycan)synthesis-lactam drugsVancomycinBacitracinCell membraneintegrityPolymyxin BDaptomycinMetabolic pathways(folate biosynthesis)SulfonamidesTrimethoprimProtein synthesisAminoglycosidesTetracyclinesMacrolidesChloramphenicolLincosamidesOxazolidinonesStreptograminsCopyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

20.4. Determining Susceptibility of Bacterial StrainConventional Disc Diffusion MethodKirby-Bauer disc diffusion test routinely used to determine susceptibility of bacterial strain to drugsStandard concentration of strain uniformly spread on agar plate; discs containing different drugs placed on surfaceDrugs diffuse outward, establish gradientResulting zone of inhibition compared with specially prepared charts to determinewhether strain is susceptible, intermediate, or resistantDrug characteristics mustbe taken into account (e.g., molecular weight, stability,amount)

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