Microbiology Midterm Notes Lab 1-6 PDF

Lab 1 Notes Types of microscope Lightmicroscope DarkFeild microscope phasecontrastmicroscope Florescent microscope Electron microscope Refinish.it ijm a lensto distinguishsmallobjectsthatareclosedtogether is determined by wavelength shorterwavelength1400soonanomet...

Lab 1 Notes Types of microscope Lightmicroscope DarkFeild microscope phasecontrastmicroscope Florescent microscope Electron microscope Refinish.it ijm a lensto distinguishsmallobjectsthatareclosedtogether is determined by wavelength shorterwavelength1400soonanometer greaterresolution Working Distance distancebetween thefrontsurfaceoflensandthesurfaceof coverglass objectivetenseand workingdistance inversley proportional Parfocal microscope remains in focus whenobjects are changed Iris Diaphram control theamount of lightpassingthrough an object Condenser focusesmaximum of object lightupon Magnification scanning 4 lowpower 10x HighPower40 oilImmersion100x Bacterial morphology morphology sizeshapeandarrangement i cocci spherical 2 Bacilli cylindrical 3 Spiral corkshrew Why is oil Immersionnecessary allows a consistentopticaldensityof lightflow morelightfromthe object willreachtheviewerseye helpslight not bend refractdueto air resistance What is Limit ofResolution refers tothesmallestobjectthatcan be distinguished clearly How can youincreasetheresolution on microscope qualityof thelensdeterminesthemaximum resolution can achieve optimalresultsthrough properlenscleaningdiaphramandcondenser adjustmen Lab 2 Notes In general at whichpositionshouldyoukeepyoumicroscopesubstage condenserlens Thesubstagecondenserlens willneedto beadjusted to itsappropriatesettingforeachlens Types of staining i simplestaining forvisualization of morphologicalshapeand arrangement 2 Differentialstaining a Identification 82 isFasining spore staining b visualization ofstructure capsulestaining Smear a thin uniform film of bacterialgrowthon a glassslidein order to proceedwith furtherstainingfor microscopicexamination Fixationprocesswhichinternalandexternal structures arepreservedandin a fixedposition theorganism is killedandfirmlyattached to microscopeslide 1 HeatFixation anemia aim sin sit.in io iariiisiive ng staining 1 make asmear 2 airdrysmear 3Heat fix thesmear 4 Take basicdyeandcover the smear for 60sec safrain crystalvioletmethylneblue 5 Rinsewith water 6 Blotdryandobserveundermicroscope ax 10x40 1007 Basic Positive Dyes methlyne blue basic fuchin crystalviolet safranin malachite gree Negative Acid Dyes rosebengal nigrosini eosin acid fuchin Lab 3 Notes Gram staining invented by Christian Gram in the 1800s The dye retention is based on the bacterialcellwall density thickness porosity Grampositive more thickcellwall Gramnegative thinnercellwalls Gram Stain Procedure 1 Prepare a smear a putsome of the materialontodrop of water on a microscope slide thenspread thedrop b allow to air then heat fix gently 2 Apply the Primary off a flood the smear with crystalviolet 3 Apply themordant fixingagent a flood the smear withIodine solution 4 Rinse off mordant 5 Decolorize Alcohol a Drip 95 Alcohol across the slideabout is sec b effluet should appear clear 6 Rise off Alcohol declorizer 7 counterstain a Flood the slide with safrainfor 60sec 8 Rinse and Dry Gram positive should be crystalviolet Gramnegativeshouldbesafrain Why use Iodine when crystalviolet mixes with iodine theycreate a new substancecalle crystal violet iodine complexThis new substancecannot dissolve inwaterandmakes bigger particles than original ones thesebigparticles helpthestainstayon and not washaway What happens during Alcohol washing when alcohol remove water frompeptidoglycan layer it dehydratesshrinks The dehydration makes the layer tighter since the crystal idonine mix is big it can't easily pass through the shrunkenlayer sothe dyegets stuck inside thickcellwall of GramPositive What happens to Gram Negative CellDuring Decolorizing when alcohol is used on Gram negative they breakdownthe outerlayer of the cell this shows the driedup peptidoglycan layer underneathThecrystalviolet iodine mix can escape throughbig holes in thelayer so the color getswashed awayleavingthe cell clear andwithout any stain Gram variable somebacteria yeild a Gram variable pattern a mix of pink and purple caused by 1 breakage of cell wall 2 decrease in petidoglycan 3 of the culture age Acid Fast staining used for mycobacterium high lipid content in cell wall responsible for staining characteristic Lab 4 and 5 se support the growthreproduction of microorganism the media can be liquid or solid important for the study of microorganism AsepticTechnique thesteriletechniqueis away ofworkingthatreducesthechancesofcontaminationthisisimportantsincebacter ismicroscopic thecontamination is notvisibletonakedeye 1 Have a clear workspace 2 wipedown lab areawith 70 ethanol have  I al pl.fi me h b a 1 I mach e m p a to kill harmful bacteria 4 Usethe Bursenburner whentransferringbacteria 5 Donot touchthemouth of atesttube 6 Do notcough sneeze near labbench 7 make sure petridish is closed 8 Donotnotplaceglasspipetteonthebench 9 Opensterilecottonswabpackages fromstickend sterilefreefrommicroorganism sterilization thecompletedestruction removal ofmicroorganism sterillant an agent that kills all types of microorganism Commensals non pathogenicmicroorganism that are livingand reproducing as human or animalparasites 2 Principalsterilization method 1 Physical dry heat or saturated steam steamsterilization alsoknown as autoclaving depends on theuse of steamabove100C temperatures rangingfrom 121134C at is sopsi 2 chemical ethyleneoxidegas or chemicalliquids iseffective all types microorganism against of thepurpose ofthegas is to kill livingcellsbyattatchingcertainchemical groupscalled alkylgroups oralkylation to the geneticmaterial DNA in the cellsThis damagesgeneticmaterial which canstopcellsfrom functioning or reproducingleading to death Synthetic or Defined Media Complex Media yeast extract Isolation of Pure culture Pure culture population of singlecellarisingfroma singlecell Techniquesused to isolatepureculture spreadplatestreakplate pourplate Spread Plate andStreak Plate involvesspreading a mixtureof cells on an agarsurface so thatindividualcellsare well separated fromeachother Eachcell can reproduce to form aseparatecolonylavisiblegrowthorclusterofmicroorganism Pour Plate sample is diluted to reduce the microbialpopulation sufficiently to obtainseparatecolonies Colony Morphology and Growth individualspecies form characteristic colonies i  Lab 6 Identification of Bacteria Selectivemedia Differential media Physiology study of lifeprocess or study of thingsthatlivingorganismdoinorder to stay alive why is thestudy ofphysiology important we must know some biochemicalpropertyof bacteria Selective media selectiveculturemedium allowscertaintype of organismtogrowandinhibit thegrowth of otherorganism addingantibioticsheavy metals ordyesto the medium whichwill kill orblock thegrowthofthe unwantedorganism allowingthe Enrichmentmediagrowthofoneorganism is fa ii nni nfme w.it growthofotherorganism DifferentialMediaallowsbacteria to bedistinguishedbetweenoneanother makesbacterialookdifferent worksbestwithcloselyrelatedorganismthedifferentialagentiswhatcausesbacteriatolookdifferent exampleBlood Agar nonhemolytic us hemolytic In Lab 6 themedia we used is 1 SA sugaragarplate 2 TSI triplesugarironslant 3 EMB eosinmethyleneblueagarplate 4 MSA mannitol saltagarplate Organism Used SA Bacillusand Ecoli TSI EcoliBacillus PseudomonasfluorescensPt Proteusvulgaris EMBEcoliBacillus PseudomonasfluorescensPt Proteusvulgaris pv MSA staphylococcus and Ecoli mannitolsugaragarplate isbothselectiveanddifferentialgrowthmedium It isusedtodifferentiatepathogenic staphylococcusspeciesfrom nonpathogenicmembers of the genus selectshighsalt bacteria as well if the organismdoesnot usemannitolthemedium willremain red nochange if the organismdoes ferment usemannitol it willcreate a metabolic byproduct whichareacidic themedium willturnyellow  Iatrogenic EosinMethyleneBlue Agar selects for Gramnegativeorganism thereforeGramPositive doesn'tgrow Differentiatesbetweenlactosefermenting vs nonlactosefermenting Ecoli willdevelop a metalicgreensneen Startch Agar used togrowbacteria bacteriain the Bacillusgenus releasespecialenzymethat breaksdownlargemolecules likestarchOneof theseenzymescalled exoamylase breaksdownstrach to smallerpeices medium containsstarchand a nutrientpeptonethathelpsmanyorganismsgrow But not all bacteria can breakdown the starch after usurs of incubation addGramsIodinestain TripleSugar Iron Agar TSI purpose 1 Differentiatebacteriabasedontheirabilitytofermentlactose glucoseand orsucroseAndtoreducesulfurtohydrogensulfide 2 usedprimarily todistinguishthemorphologicallysimilarbacteria of Enterobacteriaceaewhichallfermentglucoseto anacidendproduct 3 Thismedia isimportanttodifferentiateorganismcloselyrelatedto E coli andthosein salmonelashigellagroup Principle of TSI contains peptone glucosesucroselactoseandthiosulfate phenolredis thepHindicator yellow pHlessthan6.8 red pHabove6.8 prepared on agarslant deepbutttoprovide anaerobicgrowth inoculated by a stab in the butt the streak on theslant Community Acquired Diseases i Klebsiellapneumoniae respitoryinfection by EcoliandProteus Enterobacteriaceae this group of organism in set infused primaryinfections of thehumangastrointestinal tract Coliform bacteria that can breakdownlactoseand arerodshapedlike theyare Gramnegative usually live in theintestinesofhumans andanimals Also live in enviorments like so examples E coli Klebsiella Enterobacter citrobacter SerratiaandEdwardsiella