general microbiology miramar college biology...

GeneralMicrobiologyMiramarCollege

Biology205

LabManualSpring2012

PreparedbyMicrobiologyfacultyandstafffromtheBiologyDepartmentofMiramarCollegeforourstudents

Microbiology Laboratory Manual Spring 2012– Miramar College Biology Department

Acknowledgements:ThismanualhasbeenacooperativeeffortwiththeMicrobiologyFacultyandStaff.ContributingfacultymembersareDr.LauraMurphy,CristaWagner,andDr.DanTrubovitz.LaboratorysupervisorandVuongNguyenandourskilledMicrobiologyinstructionallabtechnicianEmiliaManalastashavebeeninvaluableaswell.AlexisBarker,TommyThamesandDavidR.Caprettedrewtheartworklinedrawings.


TableofContents

LabExercise TopicorNumber PageSpeciesList Microbesusedincourse 4Introduction 1 6LabMaterialCheckoff Materialneededforlab 10TheMicroscope 2 11MediaandAsepticTechnique 3 16MicrobialUbiquity 4 22PureCultureTechniques 5 28TheSmearandSimpleStaining 6 33TheGramStain 7 37DifferentialStains–SporeStain 8a 41DifferentialStains–Acid‐FastStain 8b 44TheNegativeandCapsuleStains 9 47MicrobialMotility 10 51GrowthCurveandSerialDilutions 11 55EffectsofOxygenonGrowth 12 65EffectsofpHonGrowth 13 70EffectsofTemperatureonGrowth 14 73LethaleffectsofUVlight 15 76LethaleffectsofTemperature 16 79AntisepticsandDisinfectants 17 82EvaluationofAlcoholAntisepsis 18 85AntibioticsEvaluations 19 88Transformation 20 92DNAFingerprintingPCRofAlu 21a 99DNAFingerprintingRestrictionDigest 21b 109Diversity–Bacteriophages 22 116Diversity–EukaryoticMicrobes 23 120Morphological&CulturalCharacteristics 24a 124DescriptiveCharts/MinorUnknownProject 130‐131PhysiologicalTests–Carbohydrates 24b 132PhysiologicalTestes–Proteins&Lipids 24c 137PhysiologicalTests–Multi‐testmedia 24d 143PhysiologicalTests–summary MinorUnknowns 147ColorPlatesandphotographs 150–156Staphylococcusspeciesandunknowns 25 157Streptococcusspeciesandunknowns 26 162EntericsandtheEntericunknowns 27 171MajorUnknownproject 28 182ELISA 29 187WaterQualityAnalysis 30 191 DentalmicrobiologytheSnyderTest 31 197Foodmicrobiology–the5secondRule 32a 200DiseaseTransmission–Epidemic 33 202


SpeciesListofMicrobesusedourMicrobiologylaboratories

# ORGANISMNAME GRAMRXN

1 Alcaligenesfaecalis Negative

2 Bacilluscereus Positive

3 Bacillusmegaterium Positive

4 Bacillussphaericus Positive

5 Bacillusstearothermophilus Positive

6 Bacillussubtilis Positive

7 Bacillusthuringiensis Positive

8 Branhamella(Moraxella)catarrhalis Negative

9 Chromobacteriumviolaceum Negative

10 Citrobacterdiversus Negative

11 Citrobacterfreundii Negative

12 Clostridiumsporogenes Positive

13 Clostridiumtetani Positive

14 Corynebacteriumdiphtheriae Positive

15 Corynebacteriumpseudodiphtheriae Positive

16 Corynebacteriumxerosis Positive

17 Edwardsiellatarda Negative

18 Enterobacteraerogenes Negative

19 Enterobactercloacae Negative

20 Escherichiacoli Negative

21 Flavobacteriumcapsulatum Negative

22 Kurthiazopfii Positive

23 Klebsiellapneumoniae Negative

24 Lactobacillusacidophilus Positive

25 Lactobacilluscasei Positive

26 Lactococcus(Streptococcus)lactis Positive

27 Micrococcusluteus Positive

28 Micrococcusroseus Positive

29 Moraxellacatarrhalis Negative

30 Morganellamorgani Negative

31 Mycobacteriumphlei Positive

32 Mycobacteriumsmegmatis Positive

33 Neisseriaflava Negative

34 Neisseriasicca Negative

35 Proteusmirabilis Negative

36 Proteusvulgaris(hauseri) Negative

37 Pseudomonasaeruginosa Negative

38 Pseudomonasfluoresens Negative

39 Rhodococcusrhodochrous Positive


# ORGANISMNAME GRAMRXN

40 Rhodospirillumrubrum Negative

41 Salmonellacholeraesuis Negative

42 Salmonellaenteritidis Negative

43 Salmonellagallinarum Negative

44 Salmonellatyphimurium Negative

45 Serratiamarcescens Negative

46 Shigellaflexneri Negative

47 Shigellasonnei Negative

48 Spirillumserpens Negative

49 Sporosarcinaureae Positive

50 Staphylococcusaureus Positive

51 Staphylococcusepidermidis Positive

52 Staphylococcussaprophyticus Positive

53 Streptococcusagalactiae Positive

54 Streptococcus.(Enterococcus)faecalis Positive

55 Streptococcusmutans Positive

56 Streptococcuspyogenes Positive

57 Streptococcussalivarius Positive

58 Streptomycesgriseus Positive

59 Vibrioanguillarum Negative FUNGIANDYEASTS MORPHOLOGY

60 Aspergillusniger Fungus

61 Mucorhiemalis Fungus

62 Neurosporacrassa Fungus

63 Penicillumnotatum Fungus

64 Rhisopusstolonifer Fungus

65 Rhodotorularubra Yeast

66 Saccharomycescerevisiae Yeast


LabExercise1:IntroductiontotheMicrobiologyLab

WelcometoMicrobiologyatMiramarCollege!Welookforwardtoarewardingandenlighteningexperiencewithyouall.Becauseourlabsandprotocolsinvolvenotonlylivingorganisms,butalsosomethatarepotentialpathogens,youmustadheretostrictsafetyguidelinesandrules,inordertoensuresafetyforyourself,yourcolleagues,andyourfamilyandfriendsathome.Thesafetyrulesandguidelinesaresetforthbelow:

1. Labcoatsareencouragedbutnotrequiredandmustbekeptinpersonalboxes.2. Nofoodordrinksareallowedinthelab.Itisnotsafetohavefoodinthelab.Ifyouwanttoeatordrink,

gooutsideofthelab.3. Closed‐toedshoesarerequiredforprotectionfromspillsandaccidents.4. Longhairmustbetiedbackduringlab.5. Washhandsbeforeyoubeginworkandbeforeyouleave.Inaddition,itisagoodideatowearglovesfor

mostlabexercises.6. Washdownlabbenchwithdisinfectantbeforeandafterwork.7. Noextraneousbooks,folders,purses,orbookbagsonlabbenchtop.8. Reportallspillsandaccidents.Bepreparedtoidentifythemicrobeastheinstructorrequests.(See

HazardousWasteProtocols)9. Noculturesinboxesatanytime,andnoremovalofanyculture,plate,ortesttubefromthelab.10. Testtubeswillbetransportedinatesttuberackatalltimes.

Theseruleswillensureagoodworkingenvironmentforeveryone.Additionalprotocolsandequipmentlistsareoutlinedbelow.

HAZARDOUSWASTEDISPOSALAllmaterialsthathavecomeincontactwithamicrobialorganismareconsideredhazardouswasteandmustbetreatedbythefollowingguidelines.Hazardouswastedisposal(TheMorgue)‐Testtubes:Alwaysplacetesttubesintheappropriatetesttuberackinthemorgue.Thereare2typesoftubes

used(13mmand16mm)andseparatesizeracksareprovidedforeach.Completelyremoveallmedialabelsbeforeplacingintherack.

‐Glass:GlassitemssuchasslidesandcoverslipsneedtobeplacedintheSharpscontainerinthemorgue.‐Additionalsharps:Othersharpobjects,suchaspipettesandpipettetips,toothpicks,andcottonswabs,need

tobediscardedinthewhitetub.‐Non‐sharpsmaterial:Allothercontaminatedmaterial,includingplates,gloves,andpapertowelsarediscarded

inthelarge,plasticBio‐Baginthemorgue.‐Stains:AllstainsmustbecollectedfordisposalandNOTpoureddownthesink.Stainingtrayscanbeemptied

intothelargewhitecarboylabeled“StainsCollection”inthemorgue.Traysshouldthenberinsedliberallywithwaterbeforebeingstored.

‐Non‐hazardouswaste:Materialthathasnotcomeintocontactwithamicrobialorganismcanbedisposedofintheregulartrashorbrokenglassreceptacle.


SpillsIfyouspillanylivingorganismonyourselforanyothersurface,youmusttreatitasabiohazard: ‐Informinstructororlabtechnicianofspill ‐Saturatesurfacewithdisinfectant ‐Letsitfor5minutes ‐WipeupanddisposeoftowelsinBio‐Baginthemorgue

‐Thoroughlywashhandsand/oraffectedareawithlargequantityofwarmwaterandsoapCHEMICALSTAINS/REAGENTSChemicalstainsandreagentsarekeptinacommonareaandcanberefilledandusedbystudentsonanongoingbasis.Stainkits‐Whenstainkitsarerunninglow,pleasebecourteoustootherstudentsandtakeafewsecondstorefillany

bottlesinthestainingkitsthatmayneedit.‐Whenrefillingstainingkits,besuretousethesinksandnotthecounters.Afterrefilling,pleasewashanyspilled

stainsdownthesinkwithplentyofrunningwater.‐Ifrefillbottlesareempty,pleaseaskyourinstructororlabstaffsowecanfillthemorgetnewbottles.Chemicalreagents‐Inthereagentscabinet,eachrowandindividualcanisterislabeledbutbesuretodoublecheckthatyouare

takingthecorrectreagent.‐Whenyou’refinishedwitheachreagent,pleasepromptlyreturnittothecorrectcabinetandrow.Ifoneis

empty,giveittotheinstructororlabstaffsowecanreplenishthestock.COMMONAREASPleasefollowthefollowingguidelineswithrespecttocommonareas.Instructor’sBench‐Keepallyourlabmaterials(tubes,plates,loops,needles,etc.)offfrontbenchatalltimes.‐Ifyouhavequestionsregardingyourlabwork,asktheinstructororlabstafftocometoyourseattohelpyou.BackBench‐Necessarymedia,reagents,andotherlabequipmentwillbeplacehereat thebeginning ofeachlab.This

areaneedstobekeptcleanandclearsothateveryonecangetthesuppliestheyneed.‐Takeyoursuppliesbacktoyourseatandcontinueyourworkthere.Donotdoanyinoculationsorotherlab

proceduresonthebackbenchunlessotherwiseinstructed.IncubatorRoom‐Incubators:Eachsectionwillhaveadesignated25°Cand37°Cincubator.‐Incubatorracks:Ineachsection’sincubator,thereisadesignatedrackforeachlabtable.Besurethatyouare

placingyourtestsintothecorrectrackinsidethecorrectincubator. Alwaysdouble‐checkthatyouareusingthecorrectincubator.(labsectionandtemperature)‐Culturemediaremoval:Whenyouaredoneincubatingyourtests,promptlymovethemintothe4°Cfor

storageorproperlydiscardofthem.Donotletoldmediapileup!‐NEVERmoveanotherstudent’smediaorchangetheincubatortemperature.


CULTUREIDENTIFICATIONYoumustproperlylabeleachandeverycultureyouworkwithinthelab.Testtubes:Usingprovidedpaperlabel,writeyourname,date,andlab#/organismandsecuretothetesttubeusingarubberband.DONOTwriteontesttubesorglasswarewithmarkersorpens.Petriplates:Usingamarker,labeltheouteredgeofthebottomsideofthePetriplatewithyourname,date,andlab#/organism.EQUIPMENTANDTOOLSOFTHEMICROBIOLOGYLAB:Youwillbeusingvariousequipmentandtoolsinthelab.Youneedtoknowwhattheyarecalledandtoproperlyusethem. Tools:

‐Microscopes–compoundandstereolightscopesareusedinourlabs‐Stainsandstainingpans–usedtomakespecimenseasiertosee‐Bunsenburner–usedtoheattransfertoolstosterilize

Transfertools‐inoculatingloop:usedtotransferbacteriafromonesourceto

another‐inoculationneedleusedtotransferbacteriafromasolidmediato

anothermediaMediaforms:- broths–liquidgrowthmedia- agar–usedtosolidifybrothmedia- slants–solidmediacooledinatubeataslant- agarplate–solidmediapouredintoaplate- Deep–solidmediainatube(notslanted)- Pour–largertubewithagarmediaforpouringintoaPetriplate


Incubators:usedtokeeptemperatureconstantforbacterialgrowthRefrigerators:usedtostorebacteriaforextendedtimeAutoclave:usedtokillbacterianolongerneededMICROSCOPEPROTOCOLSTheABCs(andDEFGs…)ofmicroscopecare.Everydaywhenfinishedwiththemicroscopeyoumust:A. MovemicroscopestagetolowestpositionB. Move4xobjectiveintoplaceC. Wipeimmersionoilofflens/scopewithlenspaperonlyD. TurnofflightE. TurnlightintensitydialallthewaydownF. WrapcordaroundbaseG. Returntocorrectlocation

IMPORTANT:Pleasereportanyproblems.Youareresponsibleforcheckingthestatusofthemicroscopeatthebeginningoflabeachday.Asyouwillbesharingmicroscopes,thisisVERYimportant.Ifyounoticeaproblem,informyourinstructorimmediatelyandtheappropriatelyguiltystudentwilllosepoints.

COMMUNITYANDPERSONALSUPPLYLISTS

Belowisalistofitemsyouwillfindinyourshared,communitybox,alistofitemsyouneedtokeepinyourownpersonalbox,andamicroscopestatuslist.Beginningnextlabsession,youmustgetthesecheck‐offsheetsapprovedbyaninstructorortechbeforeleavingclass.Propercompletionmightjustearnyousomeextracreditpoints!Pleasenote:Youaresharingthecommunitybox,soDONOTputitemsfromthecommunityboxanywhereexceptinthecommunitybox.Youarealsosharingmicroscopes,soyoumustproperlystoreandcleanthemicroscopeattheendofeachclassperiod.Pointswillbedeductedifitemsarelostormissingandifthemicroscopeisnotproperlystoredandclean.


NAME__________________________________LabNumber______________________ COMMUNITYBOXandSTAININGKITSLabDay1234 Item ________________ Steamingbeaker________________ Tuberack________________ Striker________________ Stainingpin________________ Bunsenburner________________ Gashose________________ Washbottle________________ Immersionoil________________ Gramstainkit________________ Acid‐fastkit________________ StainingtrayApproval________________

PERSONALBOXLabDay1234 Item ________________ Inoculatingloop________________ Inoculatingneedle________________ Lenspaper________________ Bibulouspaper________________ Slides________________ MarkerApproval________________

MICROSCOPELabDay1234 Status ________________ Lightoff________________ Lightpowerturneddown________________ Cordwrapped________________ Stageinlowestposition________________ 4xobjectiveinplace________________ Noslidesonstage________________ NooilonlensesApproval________________


LabExercise2:TheMicroscopeOBJECTIVESOnceyouhavecompletedthisexercise,youshouldbeabletodothefollowing:

1. Knowthepartsofthecompoundscopeandtheirfunctions.2. Usethescopetofocusonstainedorganisms.3. Useastagemicrometertomeasurethefieldofview(FOV)andestimatesizeoforganisms.4. Properlycleanandstorethescope.

PartsoftheCompoundMicroscopeStageThisisaplatformonwhichthemicroscopeslidewillrestandiscenteredoverthelightsource.ThemechanicalportionofthestagecanbeadjustedeitherverticallyorhorizontallytocenterthespecimenoverthelightsourceLightsource.Alightmicroscopeusesvisiblelighttoviewthespecimen.Theintensityoflightprovidediscontrolledbybothanon/offswitchandalightintensitydial.Formostexperimentsinthislaboratory,wewillhavetheintensitydialsettomaximum.Usuallythehigherthemagnification,themorelightisneeded.CondenserThecondenseriscomposedof2setsoflensesfounddirectlybelowthestage,whichcollectandconcentratethelightupwardintothelenssystems.Thecondenseralsohasanirisdiaphragm,whichcanbeusedtoregulatetheamountoflightenteringthelenssystem.Formostexperimentsinthislaboratory,thecondenserwillbesetjustaquarterturnshortofit’smaximumheight(closesttothestage).Usethediaphragmtoregulatetheamountoflightpassingthroughtheslide.Asaruleofthumb,asthemagnificationincreases,thediaphragmmustbeopenedtoallowmorelightintoviewthesample.ObjectiveLensesThisisthefirstsetoflensesthatwillmagnifythespecimenforviewing.Thesearefoundonthenosepiecethatrotates,whichallowsyoutochangethemagnificationpowerbasedtheobjectiveinplace.Forthislaboratory,wewilluseobjectivelenses,whichmagnify4x,10x,40x,or100x.Ourmicroscopesarealsowhatarecalledparfocal,whichmeansthatwhenonelensisinfocus,theothershavethesamefocallengthandcanberotatedintopositionwithoutmakinganymajoradjustmentstoachievefocus(usuallyahalf‐turnofthefinefocusknobwilldo).BodyTubeThebodytubeattachestothearmofthemicroscopeandhousestheobjectivelensesandtheocularoreyepiecelenses(seebelow).Thebodytubemayberaisedorloweredbyturningthecourse‐adjustmentandfine‐adjustmentknobsthatarelocatedbelowandtotheleftofthestage.EyepiecesorOcularLensesTheeyepieces,orocularlenses,arewhatareusedtoviewthesample,aswellasprovide10xmagnification.


MicroscopyPrinciplesTotalmagnificationThisisachievedbymultiplyingtheocularlenspowerbytheobjectivelenspower.Thus,thetotalmagnification=10x(4or10or40or100).ResolvingPowerWithallmicroscopes,thereisalimittotheextentofmagnificationthatcanbeachieved.Thisisdependantonwhatiscalledtheresolvingpowerofthelens.Resolvingpowerreferstotheabilityofalenstoshowtwocloselyspacedobjectsasdiscreteandseparate.Whenmagnificationincreasesbeyondthispoint,theimagewilllookblurry.Resolvingpowerisdependantonthewavelengthoflightused,aswellasthenumericalapertureofeachlens.Smallerwavelengthsoflightcontainmoreenergyandprovidebetterresolvingpower.Ourlightmicroscopesachieveamaximumresolvingpower(RP)ofapproximately0.2µmusingvisiblelightandamaximumobjectivelenspower.

UseandCareofMicroscopeBelowisabasicguidelineofstepstobeginusingyourmicroscope:

1. Pluginmicroscope,takingcaretoinsurethecordisfirmlyattachedtothebackofthescope.2. Turnonthelightsourcepowerswitch.SLOWLYincreasethelightintensitydialuntilitisatitsmaximum

position.3. UsingLENSPAPERONLY,gentlycleantheobjectivelenses.Ifthereisanyresidualoilremainingfroma

previousstudent,pleaseinforminstructor.4. Placemicroscopeslidewithspecimenonstage.Centertheslideoverthelightsourceusingthestage

adjustmentknobs.5. Rotatenosepiecesothe4xor10xobjectivelensisinplace.Raisestagetothetopposition.6. Whilelookingthroughtheocularlenses,slowlylowerthestageusingthecourseadjustmentknobuntil

thespecimencomesintofocus.Itisoftenhelpfultoveryslightlymovethestagebackandforthduringthisprocess.Thiswillhelpyoureyetracktothespecimenontheside.

7. Usefinefocusknobtobringthespecimenintosharpfocus.8. Becausethemicroscopesareparfocal,youcannowmovethenextobjective(i.e.10xor40x)into

position.Useahalf‐turnofthefinefocusknobonlytobringthespecimenintosharpfocus.Besuretoadjusttheiris‐diaphragmtothepropersettingfortheobjectivelensyouareusing.

9. Onceyourspecimenisinfocusat40x,youarereadytoobservethesampleundertheoilimmersion100xlens.

10. Rotatethenosepiecesothattheslideisbetweenthe40xand100xlenses.Addonedropofimmersionoildirectlytotheslide.

11. SLOWLYmovethe100xlensintoplace.The100xlensshouldbefullyimmersedintheoil.(NOTE:Onceyouhaveaddedoiltotheslide,youcannolongeruseyour40xobjective.Ifyoucannotfindyourspecimen,youmustrotateto10xonlyandnotallowthe40xtopassbytheslideagain,oritwillbecontaminatedwithoil.)

12. UsingFINEFOCUSONLY,bringtheobjectintosharpfocus.13.FormoreinformationfromNikononthisscope,it’scomponentsandfunctionsgoto:

http://faculty.sdmiramar.edu/dtrubovitz/microscope/


LabExercisesSuppliesIndividualSupplies GrouporTableSuppliesMicroscope AssortedeukaryoticspecimensLenspaperandcleaner Stagemicrometer

Protocol1.Placetheobjectivemicrometeronthestageandcenterinthelightpath.2.Focusonthemicrometerusingthe10xobjective.3.DeterminetheFieldofView(FOV)forthe10xobjective

‐Thesmallestlinesonthestagemicrometerare10µmapart‐Determinehowmanylineswouldfitacrossthefieldofview(lightcircle)at10x

4.YoumayusethemicrometertomeasuretheFOVfortheotherobjectivesaswell.‐AlternativelyyoucanextrapolatefromtheFOVfor10x,sincethechangeinmagnificationisinverselyproportionaltothechangeinFOV. ‐Example:Ifthemagnificationincreasesby10,theFOVdecreasesby10.5.Removethemicrometerandstorecarefullyintheobjectivemicrometerbox.6.Placepreparedslidesonthestage,focus,anduseyourknowledgeofFOVtoestimatethesizeofthemicrobes.7.Brieflysketchmicrobesandrecordanyobservations.

Whendonewithyourmicroscopemakesureitiscleanandputawayproperly!

DATAANDOBSERVATIONS1.RecordFOVmeasurementsforeachobjectivelens:

OcularLens TotalMagnification FOV 4x 40x ____________mmor____________µm 10x 100x ____________mmor____________µm 40x 400x ____________mmor____________µm 100x 1000x ____________mmor____________µm


2.Sketchpreparedeukaryoticmicrobesandestimatesize.

Organismname Estimatedsizeinµm DrawingName________________

Size_________________

Totalmagnification______

Name________________

Size_________________


Name________________

Size_________________


DISCUSSION1.Describethepositionofyourhandswhencarryingthemicroscopetoandfromyourlaboratorybench.


2.Forwhatpurposewouldyouadjusteachofthefollowingmicroscopecomponentsduringamicroscopeexercise? a.Irisdiaphragm b.Coarse‐adjustmentknob c.Fine‐adjustmentknob d.Mechanicalstagecontrol3.Whyisitadvisabletostartfirstwiththelowpowerlenswhenviewingaslide?4.Whyisitnecessarytouseoilinconjunctionwiththe100xlensbutnottheotherobjectives?5.HowdolightandFOVchangewhenyouincreasemagnification?6.WhatistherelationshipbetweenFOVandchangesintotalmagnification?7.Whatcharacteristicsarevisibleineukaryoticcells,butnotinprokaryoticcells?8.Howdoesthesizeprokaryoticcellscomparewitheukaryoticcells?Giveexamples.

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