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??