Post on 16-Apr-2022
Bacteria101‐ Scope
• BacteriaBackground• Sourcesoffecalbacteria
• IndicatorOrganisms• Pathogens• Healthrisks• Standardsforbacteria
WhyMonitorBacteria?
• Nearly40%ofassessed(700,000miles)riversandstreamsimpairedbyoneormoreuses1
• Bacteria/Pathogensidentifiedasamajorcauseofwater‐qualityimpairment– 93,431riverandstreammiles– ~3millionlakeacres(23%ofassessed)– 3,245squaremilesofestuarinewaters(15%ofassessed)
• Ecologicalandhealthimplications
12000 National Water Quality Inventory, U.S. Environmental Protection Agency, August 2002
Bacteria
• Unicellular organisms (procaryotes)• Microscopic• Can live in all types of environments• Many bacteria beneficial• Some bacteria of concern
Focus today:• E. coli and enterococci bacteria
and their role as an indicator of the presence of pathogenic organisms
Coliforms- gram negative, non-spore forming rod
shaped bacteria which ferment lactose
FecalColiformBacteria• Bacteria from feces of warm-blooded
animals• Most are nonpathogenic• Present in higher number than pathogens
EnterococciBacteria• Bacteria from feces of warm-blooded animals• Separate bacterial group from coliforms• Formerly classified as a fecal streptococcus• EPA concluded were better than coliforms as indicators of
pathogens causing gastrointestinal illness to swimmers in marine waters, useful in freshwater too.
Enterococci- gram positive, sphere shaped lactic acid
bacterium
Sourcesoffecalbacteria
• HumanSources– anytimefecalmatterreacheswatertherewillbebacteria– Wastewatertreatment– inadequateorleakysepticsystemsordischargefrommunicipalsystems
– Swimming“accidents”,diapers– Boatdumping,waterrecreation
MoreBacteriaSources• Animalsources
– Livestock– instreams,manureappliedtofields,manurepitsorlagoons
– Pets– Wildlife– geese,ducks,deer,etc.
Indicatorbacteriasurvivalinenvironment
• Sunlight(UVradiation)*– Turbidity
• Temperature• Insediment**• Inalgalmats
Waterbodyconditionsthatenhancesurvival– lowlightpenetration,highturbidity,lowsalinity,presenceofelevatednutrientsandorganicmatter
*Davies-Colley et al. 1994 Appl Environ Microbiol. 60 (6):2049-2058**Jamieson et al. 2005 J. Environ. Qual. 34:581-589
Persistenceintheenvironment(AcademyCreek–Brunswick,GA)
Moistsediment 3,160
Condition Enterococci(MPN)– FecalStrepBacteria
Dried2days andrewet 16,98024hafterrewet 23,440
Colony‐formingunitsg‐1 ofdriedsediment
Dried30days andrewet 51024hafterrewet 16,980
Dried60days andrewet 1,20024hafterrewet 28,840
*ProvidedbyPeterHartel
Bacterialevelsarealsoaffectedby:
• Sourceandamountofloading
• Rainfallandrunoff*
*Hill et al. 2006. Int. J. Environ. Res. Public Health 3(1), 114-117
Discharge and Fecal Coliform Bacteria Bloody Run Creek, Clayton Co., IA
Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec
Discharge (cubic feet per second)
10
100
1000
Fecal Coliform Bacteria (CFU/100 ml)
1
10
100
1000
10000
100000
1000000
1993
Discharge
Fecal Coliform Bacteria
Bacteria levels can be related to flow: More runoff = Higher bacteria counts
E.Coli&enterococciareusedasindicatorsbecausethey:
• Indicaterecentfecalcontamination
• Suggestthepresenceofpathogens
• Areeasytocollectandanalyze
• Arerelativelysafetohandleandgenerallyharmless
Pathogens• Pathogensarediseasecausingmicroorganismswhichcauseavarietyofillnesses;theycanbe– Virus– Bacteria– Fungus– Parasites
• Symptomsofwaterbornepathogenillnesssometimesconfusedwithotherdiseases
From USEPA 2001, Protocols for Developing Pathogen TMDLs. EPA 841-R-00-002.
SomeBacterialPathogens
Why not just sample for pathogens?
• Therearemanydifferentpathogens
• Fewlaboratorieshavethecapacity
• Costs• Time• Watervolume• Mosttestsidentifyonlyonepathogen
• Pathogenicorganisms‐ difficulttoisolateandidentify
From Bitton, 2006From: Marylynn V. Yates, Department of Environmental Sciences, University of California, Riverside
MinimalInfectiveDosesinSomeOrganisms
E.coliBodycontactstandard• Indicatorofpotentialhealthrisksfrombodycontact(swimming)
• Variesbystate– checkYOURstate’sstandards
• EPAsinglesamplestandardis235cfuper100mlforswimmingbeachadvisories
• GeometricMeanshallnotexceed126E.coli /100ml
EnterococciBodycontactstandard
• GeometricMeanshallnotexceed35cfu /100mlformarinebeachadvisories,33cfu/100mlforfreshwaterbeachadvisory
• Variesbystate– checkYOURstate’sstandards• EPAsinglesamplestandardis104cfu /100mlformarinebeachadvisories,62cfu/100mlforfreshwaterbeachadvisory
GeometricMeanMethodrecommendedbyEPA.Basedon5samplescollectedovera30‐dayperiod.Minimizesinfluenceofaone‐timehighresult.
Example:SunshineLakewithbacteriareadingsof5,10,120,20,2700
Averagewouldbe
GM 502700201201055
5715
270020120105
2
CurrentMonitoringApproachLeadstoErrors
Courtesy Richard Whitman - USGS
Equipment• Fieldlistprovided(p.11manual)• Supplierslist(provided)
SiteSelection• Choosesitesbasedonprojectgoals
• Consider– Tributariesorotherinflowstothestream
– Landusesalongsidethestream
• Alsoconsider:– Accessrights
• Knowtribalandstatelaws
– Budget– Numberofvolunteers– Safety
SiteSelectionResources• EPA’sVolunteerStreamMonitoringMethodsManualhttp://www.epa.gov/volunteer/stream/stream.pdf
• WashingtonDept.ofEcology’s,“ACitizen’sGuidetoUnderstandingandMonitoringStreamsandLakes”http://www.ecy.wa.gov/Programs/wq/plants/management/joysmanual/selectingstreamlocations.html
• Citizen’sGuidetoBacteriaMonitorign inVermontWatershttp://www.vtwaterquality.org/lakes/docs/lp_citbactmonguide.pdf
FrequencyofMonitoring• Choosefrequencybasedonprojectgoals– Checkyourlocalrecommendations– Monthlyforscreening– Fivetimeswithin30daystodeterminegeometricmean
• Beconsistentintimeofday• Haveregularintervalsbetween
samplingdates
QualityAssurance(p.14)• Maintainqualityinpracticesandprocedures– Defininganddocumentingmethodologies
– Trainingvolunteers– Keepingaccuraterecords– Checkingexpirationdates
QualityControl(p.15)
• Ensuresamplesarecollectedconsistentlyandaccurately– Nocontaminationofsample– Fieldblanks– DIH2Ointosterilebottlesat10%sites
– Fieldreplicates– 2nd samplecollectedat5‐10%ofsites– Labreplicates– Plate2ormoresamplesfrom1bottle– Controlplates– PlateDIH2Otoensurenolabcontamination
– Splitsamples– e.g.,splittingasampleandsendingto2labs– Regularinspectionofequipment
Fieldsamplingandpreparingthetest
plates
FieldSampling– Beforeyougo
• TakePetrifilmoutoffridgetowarmtoroomtemperature
• Filltraysinincubatorwithdistilledwater
• Turnonincubatorto35oC
• Collectsupplies,includingice(preferred)oranicepack
FieldSamplingattheSite
• Hangthermometerforairtemp• Completetopsectionofdatasheet• Takewatertemp,recordondatasheet
• Taketransparencytubereading(optional)
• Putongloves• Labelsamplebottles
CollectingaSample1. Wading:Trytodisturbaslittlebottomsedimentaspossible.Inany
case,becarefulnottocollectwaterthathassedimentfrombottomdisturbance.Wadetothedepthwheremostuserstypicallyswim.Boatordock:Carefullyreachoverthesideandcollectthewatersampleawayfromthesideoftheboatordockandatthedepthwheremostusersswim.
2. Removethecapfromasterilecollectionbottlewithouttouchingtheinsideofthecaportheinsideofthebottle.
3. Gripthebottleatthebaseandplungeitinadownwardmotionintothewatertoadepthof12to18inches.
4. Usingaforwardsweepingmotion(sowaterisnotwashedoverthehandintothebottle),invertthebottleandbringittothesurface.
5. Emptyitslightlytoleaveapproximatelyoneinchofairatthetop.6. Re‐capthecontainer,thenlabelandstoreitoniceatatemperature
between39° and45° F.Itisbettertouseweticeratherthancoldpacks.7. Transportthebottletothelaboratoryassoonaspossibleafter
sampling.
AccuracyandvariabilityResultsmaynotbeaccuratewhen:
• Samplesaren’tkeptcold
• Samplesdon’treachlabwithin24hours
• Samplesaren’tfromasinglesplit
• Workspaceorequipmentisn’tsterile
• Bottles,lids,pipettesarecontaminated
TimeforSomeHands‐OnWork
3MTM PetrifilmTM
Preparingtoplatesamples• Disinfectworkspaceandcollectsupplies
• Checkincubatortemp,adjustto35oC
• MakesurePetrifilmplatesareatroomtemperature
Preparation continued• Label the back of Petrifilm using a
permanent marker– Label should include:
• Site name• Date of collection• Replicate number
• Note the incubation temperature and time that samples were placed in incubator
- --
1. Pipette water sample (1 ml only)
2. Lift film and dispense sample onto pink
3. Carefully roll down film
4. Gently use spreader, if needed
5. Let sit 1 minute
6. Put in incubator right side up (can be stacked)
Plating3MTM PetrifilmTM
IDEXX
• Colilert(Coliform/E.coli24hrs)
• Colilert‐18(Coliform/E.coli18hrs)
• Enterolert(Enterococciin24hrs)
PreparingIDEXXsamples• Poursampleintomixingcontainer(addsterilewatertobringvolumeupto100mlifsampledilutionisnecessary)
• Pourmedia(Colilert,Colilert‐18orEnterolert)inthemixingcontainer
• Shakewell,allowfoamtosettle
LabeltheQuanti‐TraywithsampleIDandvolumeinfo
FillingtheQuanti‐Tray1. Use1handtoholda
sterileQuanti‐Trayuprightwiththewellsidefacingthepalm
2. Squeezetheupperpartofthetray,carefullypulltabsothetrayopens(Donottouchtheinside!!)
3. Pourthereagent/samplemixtureintothetray
4. Tapthetraytohelpremovebubbles– allowanyfoamtosettle
Detailed instructions available at: http://www.waterboards.ca.gov/water_issues/programs/swamp/docs/cwt/guidance/3410.pdf
SealingtheQuanti‐Tray1. SettheQuanti‐Trayinthe
properrubberinsert.2. PlacetheQuanti‐
Tray/Insertontotheinputshelfandfeeditintothesealer.
3. RemoveQuanti‐Tray/Insert.Iftheywerefedcrookedly,stopandreverse,andreinsert.
4. Incubatetrays– Colilert :35°Cfor24hrs– Colilert‐18:35°Cfor18hrs– Enterolert:41°Cfor24hrs
The Quanti-Tray Sealer
Reading&InterpretingPetrifilmPlates
• Countcoloniesafter24hoursincubation
• CountbluecoloniesWITHgasbubbles(theseareE.coli)
• Don’tcountbluecolonieswithoutgasbubbles
• Don’tcountpinkorwhitecolonieswithorwithoutgasbubbles
Is this an E. coli colony?
YES
Is this an E. coli colony?
NO, unless there’s a gas bubble with it –may need to hold up to light!
3M PetrifilmTM
Sample Plates & Graphic
#2a
#2c
#2b
#2d
PreparingforReadingIDEXX
• ResultsNotebook/datasheets• UVLamp• Anti‐UVgogglesorcabinet• Quanti‐trayMPNtables
ReadingIDEXX• Counttheyellowcellsthatarepositive,markingthecellwitha“Sharpie”.
• Usingthecabinetorwearinganti‐UVglasses/goggles,usea6‐watt365nmUVlightwithin5inchesofthesampleinadarkenvironmentandcountthepositive(fluorescing)cells.
• RecordresultsandcomparetotheMPNtable
Disposingofplates• Addateaspoonofbleachtoaziplock bag• PutPetrifilmplatesintheziplock bag• Ziptightlyandthrowintrash• Dumpremainingwatersampleandthrowsamplebottleinrubbish
IDEXXSampleDisposal• SincetheQuanti‐Traysarenowfilledwithbacteria,aftertheyhavebeenread,theyshouldbetreatedashazardouswaste.
• UsedQuanti‐Trayscanbesterilizedbyputtingtheminanautoclaveafterwhichtheycanbetreatedasnormalwaste.
• Seeyourstateagencyforspecificregulations.
Calculateaverageoftriplicates
Petrifilm:• Simpleaverage
mlsUsed
Coloniescounted
Calculatedcfu/100mls
1 6 600
1 7 700
1 4 400
Average = (600+700+400) / 3= 566.66 cfu/100 ml
IDEXXDatasheets
Bacterial Sample Log & Worksheet: December CollectionMedia Batch
# __________
IDEXX Multiple Well Method Expiration __________
Time in Time Out MQ Batch __________
Sample/Setup Setup Dilution Incubator incubator Count # pos. lg # pos. sm Table
Monitoring Location Date tech (mls) temp. start temp. end tech wells wells ValueSR – Watch Hill Harbor
10SR – Watch Hill
10SR – Misquam DEM Surf
10SR – Fenway Beach
10SR – Deep Hole
10SR – The K’s
10SR – Conant Ave.
10SR – Scarborough South
10SR – Scarb DEM Surfing
10SR – Monahan’s
10SR – Narr Pier Steps
10SR – First Beach
10SR – Second Beach
10SR – Third Beach
10E.feacalis Blank
Shouldincludespacefor:• SampleVolume• Incubationtempsandtime
• Reagentinfo• Positivecells• MPNTablevalues
Questions??