SOP for Surface Water E.Coli
Contents
1. Fill bottle to the 100ml line indicated on first immersion pour off the excess and cap Do not under fill or over fill do not redunk If too much water is poured off redo sample with new 100 ml container 12 7 Do not touch bottle mouth or inside of cap Be careful not to contaminate the sample with surface film contact with human skin breathing in on the bottle or cap etc If stream is too shallow to immerse bottle fully collect as much as possible being very careful not to touch the bottom Note depth on field notes 12 8 Collect one duplicate sample every two weeks sampling frequency Sample sites chosen for duplicate sampling are selected at random among sites sampled When a duplicate sample is selected for the site repeat procedures as with normal stream samples The duplicate is the second sample when two samples are collected Duplicates document repeatability of individual sample collections and reproducibility of laboratory results 12 9 Samples are analyzed in the Karuk Tribe Microbiology Lab Keep samples cool while transporting Store at 4 C until analysis but do not freeze The maximum holding time is 6 hours 12 10 For each sample the location number bottle numbers used and time collected will be recorded in the field sample log 12 11 The samples will be kept in the possession of Karuk Tribe personnel who both collect and analyze the samples 13 Quality control 13 1 Accuracy Initial analyst demonstration of2. Drive Westbrook Maine 04092 USA Cuartl Tray and Defined Substrate Technology are trademarks or registered trademarks cf IDEXX Latoratars nc In the United States andor rar ww other countries US Patent Numbers 4 925 789 5 429 933 5 518 992 Other patents pending LF Ban SATO 12 bapnl sumed ug 2999 CR At guy WEY Sy Aq bag un pjo pp SOR pep ayu al SOIR OAC E Mib pobo eso yeuopen e sapa Apyu Cep GOW SLE rer one eer ene e SS Cer LWZ GSE Ir CvIZ org FWL Tel CGL oul OSS sei Ce rei emt ee cre ot Ce Ver ERE re eer CET Cer Lt Sa FG sp oe OSI CG Sat est Great reet Oort GIE eet FS G oz ee SOL OZ ONZ r Ue reg ee e OW Sei CL ra LOH et o ont Li reeL GOL Sei we En rt SUEZ rer FL ent SAE ee rei Ce TOL STALL on gg ee sei on V L oi Cen Lei oe ONZE Cu VELL got Cen cic F 7 ce oa Gell oes SE ra FE SS S CS S 7 ra D S S oa oD Soo Ce emm LAI LTAL GW Lee SESE LISE LOM Eet rn ri FEEL SEIL GST CL LA Ca Gu Se ren Com e ap Le gui THO zem ee Ce Ge G rt OSI ep re LICL on mei t OL mu ZH en oen BIOL Of oe oe rn om ZL Let Z Cell est Or ZAL GL FOS SL SL Lo eau JH FOL ap eso In ege os zee ore re zoe KG St ee Cl gei rei oe LL rei ZZ eu OML oer HOW Up Cru rw 296 ee c oe oe ee ve eng ow EA Ce Ce Lei ZEZE CL F2 eru Su Oe Cent So oun res ee re em re ose oe rie GeL LA rn Ei oO OZI Co PELL eau Gt Zu een ee ren rm cW ee ee oe ee e ee O19 rR Iu ei Lt OL FRO Ce Sau Gank CH ZOM ent 990 took oo E ge LC ro bee See Soe tr om om ole FS ec EH D
3. Tray 2000 100 trays containing 97 wells each part number WQT 2K IDEXX Laboratories Inc Westbrook ME 12 Sample collection preservation shipment and storage 12 1 Arrive at site and record site number date and time 12 2 Immerse the thermometer or YSI handheld in the water and leave immersed five minutes before reading temperature Avoid disturbing the bottom with the thermometer at the sample site 12 3 Label bottle with location geographic area name and stream or lake name date time water temperature and sampler s initials Label bottle before immersion using a black permanent marker or pre printed labels KARUK TRIBE Bacteria Lab State Certified Lab purchases only certified sterile 100 ml sealed containers from IDEXX 12 4 Use latex gloves when handling bottles during sampling Fingers contain contaminants such as nitrates Bug repellents or sunscreen are particularly troublesome as contaminants Once the gloves are on be careful not to touch your face the ground or anything but the bottles 12 5 The sample should be taken from flowing not stagnant water facing upstream positioned in the thalwag 12 6 Be sure to immerse the bottle completely 10 cm 4 inches deep with mouth of bottle pointing upstream so no water flows over your hand into the bottle Be sure the bottle does not get near the bottom of the stream where sediments can be disturbed Water samples should be collected 6 12 inches below the water surface
4. capability and for each new lot of Quanti Tray 2000 analyze the following Check each new lot of Colilert e Shine the ultraviolet lamp on the media snap packs If the lot is fluorescent it will be discarded e Dissolve one packet in 100 ml distilled water Do not incubate Check for fluorescents e Analyze sterile reagent water blank with each batch of samples to verify that there is a negative result from 24 28 hours e Gravimetrically check each new lot of sterile transparent non fluorescing 100 ml vessels to ensure the 100 mL fill line is accurately represented on the vessel a b c d e g h dD k m Quanti Cult Procedure Good laboratory practices will be used for this procedure Pre heat incubator to bring temperature up to 35 C Pre warm rehydration fluid vials to 35 37 C Use blue autoclavable foam vial holder to hold vials Discard blue cap from rehydration fluid vial Remove organism vial from pouch vial with colorless cap Transfer colorless cap onto pre warmed rehydration vial and discard vial containing the desiccant Place rehydration vials into the foam vial holder Invert and place in incubator for 10 minutes at 35 37 C Fill four sterile IDEXX 100 ml bottles with distilled water to fill line Label three bottles with each bacteria name and one bottle control Place in incubator until a temperature of 35 0 5 C is reached Remove vial from holder Hold vial upside down and
5. do this may result in the wells filling or sealing improperly 15 6 Record the sample s site code on the back of the well for identification purposes 15 7 Record the lot number of the reagents and the wells used on the bench sheet in the comments section 15 8 Incubate at 35 0 0 5 C for 24 hours 15 9 Count the number of small and large positive wells and refer to the MPN table to find the most probable number for Total coliform 15 10 E coli results are obtained by placing the wells under a black light and counting the number of fluorescent wells Refer to the MPN table to determine the E coli concentration 15 11 Report results on the bench sheet 15 12 The completed bench sheet should be reviewed by the analyst the laboratory director and the QA manager 16 Data acquisition calculations and reporting 16 1 For each sample analyzed including quality control samples record the number of small and large positive wells and the MPN in the appropriate places on the bench sheet see below Calculate precision for duplicate analyses using equation 1 16 2 Equation 1 Precision as RPD A B x 100 A B 2 Where A MPN from aliquot A and B MPN from aliquot B 17 Computer hardware and software Word This document and attached bench sheet are prepared using Microsoft Word The Word document file name for this SOP is Standard Operating Procedures for Surface Water_E coli doc 18 Method performance The Karuk T
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9. 4 6 8 D H s H z H o INY Od oansod SIAMA EUS SPA 81981 Nd 000Z Aea yuen XXI anwa 14 Karuk Tribe Water Resources Program Somes Bar CA Escherichia coli IDEXX System Analyst Project Date analyzed Incubator Data Start Day Time Data Reviewed By Start Temperature C End Day Time End Temperature C Sample Data Large Well Positive Count Small Well Most Probable Positive Number Count MPN 100ml Replicate Replicate Mean of A B Sample Date A B Identification Collected A B Comments See MPN tables 15 Karuk Tribe Water Resources Program Somes Bar CA Total Coliform IDEXX System Analyst Project Date analyzed Incubator Data Start Day Time Data Reviewed By Start Temperature C End Day Time End Temperature C Sample Data Large Well Positive Count Small Well Most Probable Positive Number Count MPN 100ml Replicate Replicate Mean of A B Sample Date A B Identification Collected A B Comments See MPN tables 16 Karuk Tribe Microbiology Laboratory Log for formally notifying client of a Total or E coli Positive Test Result Name of Phone Date of Date of T
10. Data assessment and acceptable criteria for quality control measures 19 1 The analyst should review all data for correctness e g use of MPN table 19 2 Precision values are calculated for pairs of duplicate analyses 19 3 Record the precision values as RPD on the bench sheet 19 4 The desired precision is 20 RPD 19 5 The desired detection limit is 1 MPN 100mL 19 6 The completed bench sheet is reviewed by the analyst s supervisor or the Karuk Tribe Lab Director 20 Corrective actions for out of control or unacceptable data 20 1 The results for precision and blank data are compared to the acceptable values for this analysis 20 and 1 MPN 100mL respectively 20 2 If a precision value exceeds 20 then the analyst should write in the comments section of the bench sheet These data are associated with an out of control duplicate analysis The UCL 20 Note UCL is the Upper Control Limit 1 e 20 20 3 If a blank value exceeds 1 MPN 100mL then the analyst should write in the comments section of the bench sheet These data are associated with a blank value that exceeds the detection limit of 1 MPN 100mL 20 4 The samples cannot be reanalyzed because the sample volume will be depleted after the initial analysis 20 5 If data are unacceptable for any reason the analyst should review their analytical technique prior to conducting this analysis again 21 Waste management The wastes generated in this method are not
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12. Standard Operating Procedure for Escherichia coli and Total Coliform using the IDEXX Quanti Tray 2000 System with Colilert Reagent Karuk Tribe Water Resources Department Prepared by Date Karuk Tribe Quality Assurance and Lab Director Reviewed by Date Environmental Laboratory Accreditation Program Table of Contents 1 Identification of the test method csssccccccssssssccssssscscssssssccscssssscssssssscssssssesD 2 Applicable matrix or matrices x J 3 Detection Limit 3 4 SCOPE of the test method 3 5 Summary of test method e ssesooesessocssossosssssoossosssessossosssessossoessessossocesessossoossesssse e 6DPeinitiooooonnnn nterfereneessssssssss eege 8Healih and safetftf f f f f 66 9 Personnel qualifications 4 1D Equipment and Tu UE 11 Reagents and Standards 4 12 Sample collection preservation shipment and torage 4 5 7 JREONETA REN OU ON 14 Calibration and stamdardization cccccccccscscssscscscscscscscscscscscscscscscseccccccscsescssees PSS PEOCOCULE fo ecocesesscasesscsscsdecsscnsscctededecdevoscsscececsccdteccedscsoecdsedtowsusctuvecdccstevcssctheusccssesees 1 16 Data acquisition calculations and reporting 8 17 Computer hardware and software O 18 Method performance 5 cicccecisiessicssscncesaicanssoonssocedeanestnesvenioseessonssehsschdadodssnssoevesseassnee 9 19 Data
13. assessment and acceptable criteria for quality control measure 9 20 Corrective actions for out of control or unacceptable data cccsssseseeee H 21 Waste managemMentt cccccccsccsccssscecscsecssssecscsseees 22 REGOLENCES vi cvcsccaceesicccccsscccccosdccscsecscachsssdscsccessccccessdccacsesdcesopnadscsccessccsessadcsacsecsccseeesdec SS te TEEN LO 24 Tables diagrams flowcharts and validation data sccsccccsssssssscscsecesreesee LO Appendit eerste Bench Sheet Roots b ugeiet desgeb ngh Eed Bench Sheet Total Coliforim cccccccccccscsssssssssssssssscscssscssssscscscssscssssscssscssssssssseees LO Log for formally notifying client of a Total or E coli positive Test Result 17 1 Identification of the test method Escherichia coli using the IDEXX Quanti Tray 2000 System with Colilert reagent Standard Methods 9223 B 2 Applicable matrix or matrices This method is suitable for use with surface water samples 3 Detection Limit The detection limit for this analysis is 1 Most Probable Number MPN per 100mL of sample 4 Scope of the test method This standard operating procedure describes the test method for the collection and analysis of water samples for the enumeration of Escherichia coli E coli and Total coliform bacteria 5 Summary of test method Surface water samples are collected in 120ml shrink banded sterile IDEXX bottles An undiluted water sample
14. cates precision associated with laboratory procedures but not with sample collection preservation or storage procedures 7 Interferences Water samples containing humic or other material may be colored If there is background color compare inoculated trays to a control tray containing only water SM 9223 A 8 Health and safety The analysis involves handling of freshwater samples that may contain live microorganisms and therefore pose some threat of infection Laboratory personnel who are routinely exposed to such water samples are encouraged to protect themselves from water borne illnesses by wearing clean disposable gloves and washing their hands frequently The Colilert reagent is not hazardous according to the manufacturer s material safety data sheet The manufacturer does recommend wearing gloves and safety glasses while using this reagent and washing hands after use 9 Personnel qualifications Laboratory and field personnel shall have a working knowledge of this analytical procedure and will have received training from a Karuk tribal employee knowledgeable of the proper sample analysis procedures 10 Equipment and supplies 10 1 Sterile shrink wrapped 100ml IDEXX bottles 10 2 Quanti Tray Sealer catalog number WQTS2X 115 IDEXX Laboratories Inc Westbrook ME 10 3 Incubator 11 Reagents and standards 11 1 Colilert reagent for 100 ml samples catalog number WP200 IDEXX Laboratories Inc Westbrook ME 11 2 Quanti
15. e OI OZ D OO 06 oe Di o9 os Dr DE D OF Le HU ki 2 z z Ki a A s Sk H KE ua a 6 8 D U s H z H o INY Od SAMSOd SAM EWS SPA 81921 NdIW 000Z Aea L pueno SO bell 13 bupuol saed apg arb ies CR o 7 ECO quy WARY Sy NM bus amer pjo ppw opt pen apu a van Pop CO E Arup app eao yeuapen e sapa meng Cep op SLE re ME eer GUS em SS Cer Let GSE LPZ EPZ org aen Tiel ei ou OS eet Ce ri g t ee cre OI Ce Irc car re owt CET Ce Lt Sex FL sept Got ORI CG Sat ent OSI Leet Oort GEI tet Fret GE OO ee Sel OWL o rm IE POR ee ei OWL SOY CL re LOS cet oe OSH Li re ere sei r Cat EPH ere rer FL SOZ SAE FI POEL ZL TOL SZAL OWL og ee eet LOM nt an Cen Lei oe ou ZAI ru gem Cen a F 7 F CC Cell ce cis EC S F ra 5 5 x x TL em LLI LTAL em Lee SEGL mt LO ei rt FLE FECL ee GSU CM LAL ESL OHI Se ren ZZ r ap e eu ZOHO om FECL Ce OLS Gei OSL ep ei e en seet Va Su Seu ZIM en Gen eum O86 oe oe roe e Zot Leet Z Ce est Spnpi ZIEL ot FOS SL SL VO eau H gou F20 gan CL See os zee ome vie zoe KG Sl ee CWI SWEL rei oe L L re ZZ VEN ou OSE CON pn EPO vw Z ee ce oe OMe ee ve eng om Ce Ze Lei Ze em ri eru eut oe Cen om oun r ee re ee re ose oe cle eu LM rn TUL oo Ol ZZL re gou Gt ZIU SSL eet ren FOL cW epp r oe F ze ee oe rR Iu end Lm eo rm Ce ooh OSH ECH 40H Loo O90 took 9 COL cee roo ro bee KA soe tr om em Fit FS ect El re a oso om eth Co 20 Ca em SOO e em ep IG ee L0 om es FOR ow ee eu H Je Li Je Le N9 ee tol os gel ET ow Tee ots cle T
16. gned to give quantitated bacterial counts of 100 ml samples using IDEXX Defined Substrate Technology reagent products Add the reagent sample mixture to a Quanti Tray seal it in a Quanti Tray Sealer and incubate per the reagent directions Then count the number of positive wells and use the MPN table attached to determine the Most Probable Number MPN Contents This package contains 100 sterile 51 well Quanti Trays User Instructions 1 Use one hand to hold a Quanti Tray 2 Squeeze the upper part of the 3 Open the Quanti Tray by pulling the upright with the well side facing the Quanti Tray so that the Quanti Tray foil tab away from the well side palm bends towards the palm Avoid touching the inside of the foil or tray 6 Seal according to Sealer instructions 7 Incubate according to reagent directions 8 Count positive wells and refer to the MPN table on the back of this instructions sheet to find the Most Probable Number MPN 9 Dispose of media in accordance 4 Pour the reagent sample mixture 5 Place the sample filled Quanti Tray directly into the Quanti Tray onto the rubber tray carrier of the avoiding contact with the foil tab Quanti Tray Sealer with the well Allow foam to settle side plastic of the Quanti Tray facing down to fit into the carrier For Technical Assistance visit www idexx com water or in the U S and Canada call 1 800 321 0207 or 207 856 0496 IDEXX Laboratories Inc One IDEXX
17. hazardous The water can be discarded in the laboratory sink Quanti Trays are hand delivered to Basic Lab in Redding CA were they are autoclaved and then discarded with the trash 22 References 22 1 IDEXX Laboratories Inc Westbrook ME 04092 Instruction manuals for use of Colilert Quanti Tray 2000 and Quanti Tray Sealer 22 2 Standard Methods for the Examination of Water and Wastewater Method 9223 B APHA 21st Edition 2005 23 Reporting 23 1 Procedure for notification of clients for drinking water positives It is required to formally notify water suppliers for Total or E coli positive test results as follows a Formally document notification see attachment data sheet b Notify an officially designated contact person with the water supplier c Arrange for re sampling within 24 hours Important the notification must be with a live voice The message should not be left on an answering machine when notifying the water supplier 23 2 A final report will be written by the Project Coordinator and QC Officer as well as sent to any funders local Boards EPA and other interested parties The final report will include the table and graphs that were developed for the web site and media and it will describe the program s goals methods quality control results data interpretation and recommendations Following notification of the Tribal Council the Karuk Water Resources Program would then inform the North Coast Regiona
18. ime of Date Staff Water Number Sample Notification notification scheduled Initials Supplier for Re Performing Sample Notification 17
19. l Water Quality Control Board staff and work cooperatively with that agency for abatement of problems 24 Tables diagrams flowcharts and validation data 24 1 See Appendix A for MPN tables and Quanti Tray 2000 instructions 24 2 See below for the bench sheet The analyst should make a copy of this form for each batch of samples analyzed 24 3 The following validation procedures will be established throughout the project Equipment will be calibrated at the start of the season and checked before each collection blind field replicates will be submitted to the laboratory which will also analyze lab duplicates blanks and Quanti Cult new lot checks chain of custody will be maintained field sheets and data entry will be checked by the QC Officer descriptive statistics and graphs will be produced 10 06 02060 09 Quanti Tray Insert amp MPN Table RICH IC HCH J a J al JS 1a Tam A 20820000 00 d En s 4 d WD d 1 t 3 UA be md Aen d ed n ka m wc J A JOE 3 A f Xh b Siks uan Wied 3333 SEIE aN ROO GC WK vocor WWW WW Quanti Tray Certificate of Sterility This certifies that the enclosed Quanti Trays have been sterilized with ethylene oxide For further information or documentation contact IDEXX Laboratories Inc IDEXX Laboratories One IDEXX Drive Westbrook Maine 04092 USA Phone 1 800 321 0207 Fax 207 856 0630 Wm mm g 11 Quanti Tray Introduction IDEXX Quanti Trays are desi
20. on the analyst should analyze a Field duplicates one field duplicate per every 10 samples or 10 randomly selected taken at the same time b Laboratory duplicates LD two replicates taken from the same collection bottle Analyze at least one LD for every 10 samples collected c Laboratory reagent blank LRB analyze one LRB per sample batch 13 3 Calculate Relative Percent Deviation RPD Section 16 2 14 Calibration and standardization There are no calibration or standardization procedures for this method 15 Procedure See Appendix A for the manufacturer s instructions 15 1 The 100ml duplicate water sample is shaken well just prior to preparation for analysis Samples over the 100 ml mark must not be poured to volume If there is at least 1 of headspace the sample may be shaken and excess volume taken out with a sterile pipet If there is insufficient headspace lt 1 for proper mixing do not pour off and discard a portion of the sample Rather pour the entire sample into a larger sterile container mix properly and proceed with the analysis 15 2 Open a Colilert ampule and pour contents into either the diluted sample or undiluted sample Repeat for the remaining sample 15 3 Mix thoroughly making sure the Colilert reagent is completely dissolved 15 4 Follow manufacturer s instructions for preparation of Quanti Tray 2000 and use of the Quanti Tray Sealer 15 5 Allow bubbles to settle or dissipate Failure to
21. ribe lab must successfully analyze at least one set of PT material once every 12 months for each method for which it is certified The only method that the Karuk Tribe lab is certified for is an enzyme substrate method using Colilert The test must contain 10 samples all shipped at the same time and must be lyophilized dehydrated or in aqueous state The test should contain total coliforms fecal coliforms E coli non coliforms and at least one blank An acceptable result is a correct analysis of 9 of the 10 samples with no false negative results The WS Water Supply Micro Standards and the WP Water Pollution Micro Standards from Wibby Environmental should both be used 18 1 Store PT standards in the refrigerator at 4 C Remember to record the temperature daily until samples are used 18 2 Follow the date of analysis requirements provided by Wibby Environmental 18 3 When ready to use place the samples in the incubator for 2 hours at 35 C 18 4 Homogenize the samples by shaking vigorously prior to beginning sample analysis 18 5 If using the Water Pollution standards be sure to complete sample dilutions before proceeding with analysis 18 6The Wibby Environmental WP Microstandard contains total Coliforms Fecal Coliforms and E coli within the EPA NELAC specified concentration range of 20 2400 CFU 100m1 18 7 Report results to Wibby Environmental within the specified dates 18 8 Include results in yearly laboratory report 19
22. tap cap gently to mix Remove cap and look at inside surface to ensure that no un dissolved black particles are present Inoculate an additional 10 minutes if present Add entire contents of each appropriate bacteria vial to pre warmed 100 ml labeled bottles Add Colilert reagent to sample bottles including control Place in incubator and follow incubation instructions outlined in section 7 Do not place in Quanti Trays See figure 5 1 for Quanti Cult datasheet Results The following results should be observed Organism Result Escherichia coli Yellow wells fluorescence Klebsiella pneumoniae Yellow wells no fluorescence Pseudomonas aeruginosa Clear wells no fluorescence Method Blank Clear wells no fluorescence Disposal i All materials must be autoclaved prior to disposal and workspaces thoroughly disinfected ii Dispose of media in accordance with Good Laboratory Practices Bibliography IDEXX Colisure Granulated Test Kit product instructions Number 06 03553 00 undated IDEXX Quanti Tray product instructions Number 06 02030 07 undated IDEXX Quanti Tray Sealer Model 2X User Manual Number 06 03128 02 undated EPA Region 9 Laboratory Standard Operating Procedure guidance Colilert Colilert 18 and Colisure Total Coliform and E coli Water Analysis Revised July 30 1998 Standard Method 9223 B Enzyme Substrate Test 20 ed rev 1998 13 2 Precisi
23. will be analyzed from the sample collected The Colilert reagent is added directly to the 100 ml undiluted sample Both are mixed thoroughly to dissolve the reagent The sample is transferred to Quanti Trays 2000 and sealed using the Quanti Tray sealer Samples are incubated at 35 0 0 5 C for 24 hours Results are reported as MPN 100mL 6 Definitions 6 1 Analytical batch The set of samples processed at the same time 6 2 Control cultures For each lot of medium check analytical procedures by testing with known positive and negative control cultures For example E coli is a positive control for this analysis and Staphylococcus aureus is a negative control 6 3 Field duplicate FD Two samples taken at the same time and place under identical circumstances and that are treated identically throughout field and laboratory procedures Analysis of field duplicates indicates the precision associated with sample collection preservation and storage as well as laboratory procedures 6 4 Laboratory reagent blank LRB An aliquot of sterilized water treated as a sample in all aspects except that it is not taken to the sampling site The purpose is to determine if the analytes or interferences are present in the laboratory environment the reagents or the apparatus 6 5 Laboratory duplicate LD Two aliquots of the same environmental sample treated identically throughout a laboratory analytical procedure Analysis of laboratory duplicates indi
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