Sampling Quality Assurance Plan for Henry County, Georgia Atlanta Regional Commission Henry County, GA Submitted by: Henry County Stormwater Management Department 347 Phillips Dr. McDonough, GA 30253 August 2011
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TABLE OF CONTENTS INTRODUCTION & STUDY OBJECTIVES 1.1 Project Organizational Structure. 1 1.2 Background Information 1 1.3 Project Objective 2 SAMPLING PLAN 2.1 Study Area... 2 2.2 Sampling Parameters and Schedule. 3 2.3 Personnel and Resources... 3 QUALITY ASSURANCE PLAN 3.1 Regulatory Compliance... 3 3.2 Field Quality Assurance.. 3 3.2 a. Sample Collection Technique (by Parameter) and Sample Representativeness... 4 3.2 b. Sample Containers, Preservatives, Refrigeration, and Holding Time Limitations 5 3.2 c. Field Instrument Calibration, Quality Assurance Measures.. 5 3.2 d. Sampling Personnel Training. 5 3.3 Laboratory Analyst Certification/Laboratory Accreditation. 5 3.3 a. Certified Laboratory Analyst(s). 5 3.3 b. Accredited Laboratories. 5 3.3 c. Laboratory Quality Assurance/Quality Control... 5 REFERENCES 7 APPENDIX A 2010 List of Non-Supporting Waters in Henry County, GA... 11 Water Quality Control Laboratory Fecal Coliform SOP 13 Laboratory Analyst Accreditation Certificate.. 19 APPENDIX B Map of Henry County SQAP Monitoring Locations. 23 TABLES Table 1: Contact Information. 1 Table 2: Henry County, Georgia Sampling Locations. 3 Table 3: Sample Container, Preservation Method, and Holding Time.. 6 Table 4: Laboratory Analytical Methods.. 6
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PART ONE: INTRODUCTION & STUDY OBJECTIVES The Atlanta Regional Commission (ARC), in cooperation with Henry County, Georgia (the County) has developed this Sampling Quality Assurance Plan (SQAP). The plan was developed in accordance with the Georgia Environmental Protection Division s (Georgia EPD) Guidance on Submitting Water Quality Data for Use by the Georgia Environmental Protection Division in 305(b)/303(d) Listing Assessments (October 2002) as well as Guidelines for SQAP Monitoring (revised November 2008). 1.1 Project Organizational Structure ARC will develop the SQAP document in coordination with the County s Stormwater Management Department. The County s Stormwater Management Department will be responsible for sample collection, analysis and reporting to Georgia EPD. Georgia EPD will be responsible for providing technical support and necessary guidance documents required for completing the SQAP project successfully. Table 1: Contact Information Atlanta Regional Commission Henry County Stormwater SQAP Review Corey D. Babb Senior Environmental Planner Atlanta Regional Commission 40 Courtland St., NE Atlanta, GA 30303 Ph: 404-463-3323 Fax; 404-463-3254 Email: cbabb@atlantaregional.com James Moore Chief Environmental Specialist Henry Co. Stormwater Management 347 Phillips Dr. McDonough, GA 30253 Ph: 770-288-7246 Fax: 770-288-7257 Email: jamesmoore@co.henry.ga.us Susan Salter Environmental Specialist Watershed Planning and Monitoring Program Georgia EPD 4220 International Parkway, Suite 101 Atlanta, GA 30354 Ph: 404-675-1658 Fax:404-675-6247 Email:susan_salter@dnr.state.ga.us 1.2 Background Information Georgia EPD submits a Statewide Water Quality Report to the United States Environmental Protection Agency (US EPA) on a biennial basis. This report includes reporting requirements for both Section 305(b) and Section 303(d) of the Clean Water Act (CWA), as amended. In fulfillment of a specific requirement of this report, Georgia EPD has chosen to submit an integrated list composed of both 305(b) and 303(d) waterbodies. The 305(b) list is composed of waterbodies that are either supporting their designated uses or are not supporting their designated uses and have had a Total Maximum Daily Load (TMDL) completed for the parameter(s) causing the impairment. Those listed on the 303(d) list are waterbodies not currently supporting their designated use(s) and have yet to have a TMDL established for one or more parameters i. GA EPD received an approval letter from i Georgia Department of Natural Resources, Environmental Protection Division. 2008. Water Quality in Georgia 2006 2007. Watershed Protection Branch, Atlanta, Georgia. 1
the United States Environmental Protection Agency (US EPA) on November 10, 2010, approving the State of Georgia s decision to include each of the waters designated by the state in its 303(d) list. The approved list contains several stream segments located within the County that are currently either not supporting at least one designated use but have had a TMDL completed for the parameter(s) causing the water not to meet its use (Category 4a), or are not supporting at least one designated use and TMDL(s) need to be completed for one or more pollutants (Category 5) ii. A list of the stream segments currently not meeting at least one designated use in the County may be found in Appendix A. 1.3 Project Objective The primary objective of this project is to provide the County with the ability to monitor local impaired stream segments and submit their monitoring results to Georgia EPD for use in 305(b)/303(d) listing purposes. A secondary objective is to ensure that timely water quality data is collected for listing purposes and periodic data analysis is conducted for determining the trends in water quality of streams within Henry County that have already had TMDLs and TMDL implementation plans implemented. The final result of this project will be a monitoring report prepared by the County and submitted to Georgia EPD. PART TWO: SAMPLING PLAN 2.1 Study Area The areas of study for this project are impaired stream segments located in the Ocmulgee River Basin and wholly contained within the jurisdictional boundaries of Henry County, Georgia. The stream segments and respective monitoring locations are given in Table 2: Henry County, Georgia Sampling Locations. The sampling locations will include existing Georgia EPD sampling locations previously used in the determination of 303(d) listing status, with the exception of the Little Cotton Indian Creek sampling site. This site was moved approximately 1.3 miles upstream due to the fact that Hooper Reservoir impounds Little Cotton Indian Creek at the previous sample site. The selected site is the nearest access point to the original sample site that has consistent flow. GPS coordinates for the sample location are listed below in Table 2. Maps of the sampling locations may be found in Appendix B. ii Ibid 2
Table 2: Henry County, Georgia Sampling Locations Impaired Segments Segment Location GPS Coordinates (decimal degrees) Reeves Creek Minska Pinska Dam to Little Cotton Indian Creek Reeves Creek @ Tye St Lat: 33 31 51.70 N Long: 84 14 07.56 W Rum Creek Lake Spivey to Little Cotton Indian Creek Little Cotton Indian Creek Confluence of Reeves and Rum Creeks to Clayton Co. Hooper Reservoir Rum Creek @ Rock Quarry Rd Little Cotton Indian Creek @ Rum Creek Parkway Lat: 33 31 10.36 N Long: 84 13 48.96 W Lat: : 33 31 25.58 N Long: 84 13 02.94 W 2.2 Sampling Parameters and Schedule The parameter of concern for this project is fecal coliform. Four geometric means will be collected for FC between September 1, 2011 and August 31, 2012. The geometric mean samples collected for fecal coliform will consist of four samples collected over a 30 day period not less than 24 hours apart. Four samples will be collected during each of the four sampling events. The sampling periods have been assigned as following: First Sampling Event (FC) September 5-12, 2011 Second Sampling Event (FC) January 9-20, 2012 Third Sampling Event (FC) May 7-18, 2012 Fourth Sampling Event (FC) September 3-14, 2012 2.3 Personnel and Resources Staff from Henry County Stormwater Management Department will be responsible for sample collection. Subsequent analysis will be conducted by City of Stockbridge Wastewater treatment plant personnel. The County is staffed with qualified individuals that are able to perform the sampling and routinely conduct NPDES and stream monitoring for regulatory requirements.. The City has personnel qualified to conduct laboratory analysis employed at their NPDES permitted wastewater treatment plant. Staff certification for laboratory analysis of fecal coliform samples has been included in Appendix B. 3
PART THREE: QUALITY ASSURANCE PLAN 3.1 Regulatory Compliance The methodology employed in the collection of water quality samples and the data reported in the final Monitoring Report in fulfillment of this project shall adhere to the requirements of the Watershed Protection Branch Quality Assurance Manual (June 1999) and any applicable Georgia EPD standard operating procedures. The laboratory analyses conducted in fulfillment of this project shall comply with Title 40 of the Code of Federal Regulations, Part 136. 3.2 Field Quality Assurance Georgia EPD has developed standard operating procedures (SOP) establishing uniform methods for the field collection of data, document control, quality assurance, laboratory safety, as well as other activities. These guidance documents were developed to document, and ensure, the validity of measurements, analyses, and the representativeness of samples collected. This project will comply with Georgia EPD s standard operating procedure for Surface Water Sampling (EPD-WPMP-2). iii 3.2 a. Sample Collection Technique (By Parameter) and Sample Representativeness: Field samples will be collected in accordance with Georgia EPD s SOP # EPD-WPMP-2. The sample collection technique utilized will be determined by field personnel on site. Safety is of the utmost concern when choosing a sample collection technique and will often be the determining factor. If waters are safe for wading, the grab sampling while wading technique would be appropriate for fecal coliform. However, if the sampler determines the waters appear to be unsafe for wading, than the fecal coliform sample should be collected by lowering a sampling bucket from a bridge or culvert, or the grab sampling technique should be employed from the safety of the stream bank. The grab sampling technique has been excerpted from the Georgia EPD SOP to include the information relevant to the collection of fecal coliform for this project and is included as follows: Grab Sampling While Wading When an instantaneous sample is required, but water levels prohibit the use of a water sampler lowered from a bridge or culvert, a grab sample is taken from the surface water s midstream point using the actual sample container. iii Georgia Department of Natural Resources, Environmental Protection Division. 2008. SOP# EPD-WPMP-2: Surface Water Sampling. Watershed Protection Branch, Atlanta, Georgia. 4
Procedure Upon arrival at the sampling location, staff members should first don their high-visibility safety vests before proceeding with work. Proceed to water s edge on the upstream side of the bridge/water crossing by safest route possible. Then, an instantaneous grab sample should be taken from the surface water s mid-channel point being careful not to disturb the downstream side. The grab sample should be collected in an area with cross-sectional homogeneity where the water is well mixed. Since turbulence and water velocity principally govern mixing, the selection of a site immediately downstream of a riffle area (in high gradient streams) will ensure good vertical mixing. Horizontal mixing occurs in constrictions in the channel. To collect the sample, first don nitrile, or latex, gloves and move to mid-channel of the stream. While facing upstream, the bottle is submerged with the cap on. Samples or the sample should be taken well below the surface to eliminate chance of collecting surface film. The bottle is positioned so the opening is facing upstream and the lid is removed under water allowing it to fill. Then, once the bottle is filled, the lid is replaced. Once the bottle lid has been replaced, the bottle is removed from the water. Once the sample has been collected, the staff member attaches a label with the time, date, and staff member making the collection marked on the label. The label is sealed with clear tape, and the sample is placed in a cooler of ice for transportation to the laboratory. Prior to transportation, Laboratory Source Documents (chain of custody) containing all information recorded on the sample bottles, are completed, placed inside a plastic bag, and taped to the underside of the cooler lid. iv Samples are transported to the laboratory. 3.2 b. Sample Containers, Preservatives, Refrigeration and Holding Time Limitations: Holding times, sample containers, and preservatives for collected samples will comply with 40 CFR 136 and are listed in Table 3. Fecal coliform tests will be refrigerated upon arrival at the laboratory and will be prepared and analyzed as soon as staff schedules allow (holding time not to exceed 24 hours). All sample containers are pre-sterilized by the Stockbridge Waste Water Treatment Plant Water Quality Control Laboratory prior to field collection. Sample collection devices, if not directly using sample containers and the grab method, should be pre-rinsed between sample collections using water from the next sample site as the rinsing agent. iv Georgia Department of Natural Resources, Environmental Protection Division. 2008. SOP# EPD-WPMP-2: Surface Water Sampling. Watershed Protection Branch, Atlanta, Georgia. 5
Table 3: Sample Container, Preservation Method, and Holding Times Parameter Sample Container Preservation Method Holding Time Fecal Coliform Plastic 250 ml Cool, < 6 o C 6 24 hours 3.2 c. Field Instrument Calibration, Quality Assurance Measures: A field instrument will not be used for this project. The only parameter to be collected in fulfillment of this project will be FC. 3.2 d. Sampling Personnel Training: Sampling personnel agree to follow the standard collection methodology as outlined in this SQAP document under 3.2a.. Any field training which may be deemed necessary will be arranged through, and provided by Henry County Stormwater Management Department. 3.3 Laboratory Analyst Certification/Laboratory Accreditation All laboratory analyses performed in fulfillment of the requirements of this project will be performed by certified laboratory analysts utilizing EPA approved methods. 3.3 a. Certified Laboratory Analyst(s): Robinson, Wayne (14237) 3.3 b. Laboratory: Stockbridge Waste Water Treatment Plant Water Quality Control Laboratory 3.3 c. Laboratory Quality Assurance/Quality Control: All laboratories utilized in fulfilling the requirements of this project will conform to the QA/QC measures required by the specific methods in 40 CFR Part 136. Adequate records concerning all laboratory analyses performed and their respective QA/QC measures will be retained by the laboratory performing the analyses for a minimum of three years and will be available for review. All samples analyzed in fulfillment of this project will conform to the standard protocols as outlined in US EPA s Standard Methods (latest edition), and 40 CFR Part 136. Specific methods to be utilized in this project are listed below in Table 4. The County s SOP for fecal coliform analysis is available in Appendix A. Table 4: Laboratory Analytical Methods Parameter Units Method Fecal Coliform colonies/100 ml SM9222D 6
REFERENCES: City of LaGrange. 2008. Sampling and Quality Assurance Program Plan for West Point Lake Monitoring Program. Water Pollution Control Division. City of LaGrange, Georgia. Clayton County Water Authority. 2002. Sampling and Quality Assurance Plan. Watershed Management Program. Clayton County, Georgia. Stockbridge, Water Quality Control Laboratory. 2008. Fecal Coliform Testing SOP. Bacteriology Laboratory Section. Stockbridge, Georgia. Georgia Department of Natural Resources, Environmental Protection Division. 2002. Guidance On Submitting Water Quality Data for Use by the Georgia Environmental Protection Division in 305(b)/303(d) Listing Assessments. Watershed Protection Branch, Atlanta, Georgia. Georgia Department of Natural Resources, Environmental Protection Division. 2008. SOP# EPD- WPMP-2: Surface Water Sampling. Watershed Protection Branch, Atlanta, Georgia. Georgia Department of Natural Resources, Environmental Protection Division. 2010. Water Quality in Georgia 2008 2009. Watershed Protection Branch, Atlanta, Georgia. 7
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APPENDIX A 9
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2010 List of Non-Supporting Waters in Henry County, GA Stream Segment Basin Use 2010 Criterion Violated/ Category TMDL Completed Big Cotton Indian Creek Panther Creek to Brush Creek Big Cotton Indian Creek Tar Creek to Panther Creek Reeves Creek Minska Pinska Dam to Little Cotton Indian Creek Rum Creek Lake Spivey to Little Cotton Indian Creek Brown Branch Headwaters (Locust Grove) to Wolf Creek Little Cotton Indian Creek Confluence of Reeves and Rum Creeks to Clayton Co. Hooper Reservoir Island Shoal Creek Headwaters to Mackey Creek Panther Creek Headwaters to Big Cotton Indian Creek Ocmulgee Fishing FC 4a TMDL FC 2002 Ocmulgee Fishing FC 5 2016 Priority Ocmulgee Fishing FC 5 2016 Priority Ocmulgee Fishing FC 5 2016 Priority Ocmulgee Fishing Bio F 4a TMDL Bio(F) 2002 & 2007 Ocmulgee Fishing FC 5 2016 Priority Ocmulgee Fishing Bio F 4a TMDL Bio(F) 2007 Ocmulgee Fishing FC - 5 2016 Priority South River Hwy 20 to Snapping Shoals Creek South River Pole Bridge Creek to Hwy 20 Ocmulgee Fishing FC, FCG(PCBs) 4a Ocmulgee Fishing FC, FCG(PCBs) 4a TMDL FC (2007) TMDL FCG(PCBs) (2002) TMDL FC (2002 & 2007) TMDL FCG(PCBs) (2002) 11
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Stockbridge Water Quality Control Laboratory Bacteriology Laboratory Section: Fecal Coliform Testing SOP Effective Date: 1/1/2008 TABLE OF CONTENTS 1. SCOPE AND APPLICATION 2. SUMMARY OF METHOD 2.2 INTERFERENCES 2.3 DEFFINITIONS 3. EQUIPMENT 4. REGENTS AND STANDARDS 5. SAMPLE COLLECTIONS, PRESERVATION AND HANDLING, SUMMARY 6. SAMPLE PREPARATION 7. SAMPLE ANALYSIS 8. DATA COLLECTION 9. QUALITY CONTROL 10. SAFETY/HAZARD MANAGEMENT SECTION 1: SCOPE AND APPLICATION 1.1 THIS METHOD IS USED TO DETECT AND COUNT FECAL COLIFORM BACTERIS IN NON-POTABLE WATER AND IS BASED ON STANDARD METHOSD SM9222D. FECAL COLIFORMS ARE COMMONLY FOUND IN THE FECES OF HUMANS AND WARM- BLOODED ANIMALS, AND THE PRESENCE OF THESE INDICATOR ORGANISMS CAN INDICATE THE POTENTIAL PRESENCE OF BACTERIA OF VIRAL PATHOGENS. SECTION 2: SUMMARY OF METHOD 2.1 A WATER SAMPLE IS FILTERED THROUGH A MEMBRANE THAT HAS A 0.45 µm PORE SIZE TO CAPTURE BACTERIA. THE MEMBRANE FILTER IS PLACED ON AN mfc PLATE, WHICH IS A SLECTIVE MEDIA FOR FECAL COLIFORMS. THE PLATE IS INCUBATED FOR 24 HOURS AT 44.5 DEGREES-C. POSITIVE COLONIES, WHICH HAVE A BLUE COLOR ARE COUNTED AND RECORDED. 2.2 INTERFERENCES 2.2.1 SUSPENDED PARTICULATE MATERIALS IN SAMPLE WATER CAN CLOG THE MEMBRANE FILTER AND CAUSE SPREADING OF BACTERIAL COLONIES. 2.3 DEFINITIONS 2.3.1 FECAL COLIFORM: GRAM-NEGATIVE, NON-SPORE FORMING, ROD- SHAPEBACTERIA, CAPEABLE OF FERMENTION LACTOSE IN 24 HOURS AT 44.5 DEGREES-C AND PRODUCES BLUE COLONIES ON mfc MEDIA. 13
THE FECAL COLIFORMS ARE A SUBGROUP OF THE TOTAL COLIFORMS. THE PREDOMINANT SPECIES IS E. COLI. 2.3.2 POSITIVE GROWTH-BACTERIAL GROWTH THAT RESEMBLES A TYPICAL POSITIVE COLONY WHICH IS A BLUE COLORED COLONY. 2.3.3 NEGATIVE GROWTH-BACTERIAL GROWTH THAT DOES NOT HAVE THE CHARACTERISTICS OF A TYPICAL POSITIVE COLONY AND IS CONSIDERED A NON-COLIFORM. A CREAM COLOR IS TYPICAL OF NEGATIVE GROWTH. SECTION 3: EQUIPMENT AND SUPPLIES 3.1 STERILE GLOVES 3.1.1 LATEX 3.1.2 VINYL 3.2 GLASSWARE: FLASKS, GRADUATED CYLINDERS, BEAKERS 3.3 WEIGHING DISH AND MEDIUM SIZE WEIGHING PAPER 3.4 MAGNETIC STIR BAR: SMALL, MEDIUM AND LARGE 3.5 BALANCE: DENVER INSTRUMENTS PI-G02 3.6 HOTPLATE WITH STIRRER 3.7 AUTOCLAVE: MARKET FORGE STERILMATIC 3.8 ph METER: ORION 420A 3.9 PETRI DISHES, 47MM 3.10 PIPETTES: 2ML, 10ML 3.11 REFRIGERATOR, MAINTAINED AT 1-4 DEGREES-C 3.12 SAMPLE BOTTLES: 250 ML 3.13 VACUUM FILTRATION SYSTEM 3.14 UV STERILISER BOX 3.15 MEMBRANE FILTRATION UNITS 3.16 FORCEPS, STERILE, STRAIGHT AND CURVED 3.17 GLASS SEROLOGICAL DISPOSABLE PIPETS(2,5,10 ML) STERILE AND INDIVIDUALLY WRAPPED. 3.18 MEMBRANE FILTERS, STERILE, WHITE GRIDDED, 47MM DIAMETER, 0.45 UM PORE SIZE 3.19 INOCULATING LOOPS 3.20 TEST-TUBES 16X125 MM 3.21 INCUBATOR, OVEN BLOCK AT 44.5 +/-0.2 DEGREES-C 3.22 WATER BATH, 44.5 DEGREESE-C; LINDBERG/BLUE M 3.23 STEREO MICROSCOPE 3.24 DURHAM TUBES 3.25 FISHER 304R ISOTEMP INCUBATOR 35-DEGREES-C; 3.26 PARTICULATE RESPIRATOR SECTION 4: SECTION 5: REAGENTS AND STANDARDS 4.1 DEIONIZED WATER SYSTEM 4.2 ETHANOL, DILUTED TO 80% 4.3 BUFFERED DILUTION WATER 4.4 mfc BROTH BASE (mfc) 4.5 LAURYL TRYPTOSE BROTH (LTB) 4.6 EC MEDIUM (EC) 4.7 SODIUM THIOSULFATE SAMPLE COLLECTION, PRESERVATION AND HANDLEING: SUMMARY NOTE: TRAINED AND EXPERIENCED MONITORING PERSONNEL WILL COLLECT SAMPLES 5.1 BACTERIOLOGY LABORATORY SECTION WILL PROVIDE AUTOCLAVED SAMPLE BOTTLES. 14
SAMPLE BOTTLES HAVE 0.1 ML OF SODIUM THIOSULFATE ADDED. 5.2 CHAIN OF CUSTODY CARDS ARE FILLED OUT PRIOR TO GOING INTO THE FIELD. 5.3 GRAB SAMPLES WILL BE COLLECTED AT EACH STATION USING BOTTLES PROVIDED BY THE LABORATORY SECTION. 5.4 SAMPLES ARE COLLECTED BY HAND OR WITH A SAMPLING DEVICE. 5.5 BOTTLE COVERINGS IS REMOVED. AVOID CONTAMINATION. 5.6 POSITION THE BOTTLE INTO THE CURRENT UPSTREAM OF THE SAMPLER. 5.7 FILL THE BOTTLE COMPLETELY. 5.8 AFTER REMOVAL OF THE BOTTLE FROM THE STREAM, POUR OUT A SMALL VOLUME TO ALLOW AN AIR SPACE FO ABOUT ONE INCH, AND REPLACE THE CONTAINER LID. 5.9 AFTER SAMPLE COLLECTION, PERFORM FIELD ANALYSIS FOR PH, TEMPATURE, AND DISSOLVED OXYGEN. 5.10 PLACE SAMPLE IN COOLER WITH ICE TO BE DELIVERED TO EHT LABORATORY. 5.11 RECORD FIELD INFORMATION IN STREAM LOG NOTEBOOK. 5.12 ALL SAMPLES ARE DELIVERED TI THE LABORATORY TO ALLOW SAMPLE SETUP WITHIN SIX HOURS FO SAMPLE COLLECTION. SECTION 6: SECTION 7: SECTION 8: SAMPLE PREPARATION 6.1 TURN ON UV STERLIZER AND LET WARM-UP FOR 30 MINUTES. 6.2 REMOVE STERILE FUNNEL FROM UV BOX. 6.3 PLACE FUNNEL BASE IN VACCUM MANIFOLD. 6.4 SELECT SAMPLE DILUTION THAT WILL PRODUCE 20-60 COLONIES. 6.5 MIX SAMPLES COMPLETELY. PERFORM AT A MINIMUM 3 DILUTIONS (10,1,-1,-2), USING STERILE DILUTION WATER (REFER TO STANDARD METHODS TABLE 9222:III). 6.6 PLACE 2MLS OF mfc BROTH BASE TO THE ABSORBENT PAD IN EACH DISH. 6.7 FILTER SAMPLE WITHIN 15 MINUTES OF SAMPLE DILUTION. SAMPLE ANALYSIS 7.1 PLACE A STERILE MEMBRANE FILTER, GRID-SIDE UP, ON FILTER BASE. 7.2 ATTACH FUNNEL TO BASE. 7.3 FIRST, POUR THE CONTENTS OF LOWEST DILUTION ONTO FILTER. 7.4 APPLY VACUUM. 7.5 RINSE WITH 10-20 MLS OF DILUTION WATER. 7.6 TURN OFF VACUUM AND ASEPTICALLY REMOVE FILTER WITH STERILE FORCEPS. 7.7 KEEP FORCEPS IN 80% ALCOHOL WHEN NOT IN USE. 7.8 PLACE FILTER, GRID SIDE UP, ONTO PETRI DISH BY ROLLING IT ONTO mfc BROTH SOAKED PAD. 7.9 REPEAT STEPS 7.1 7.6 FOR EACH REMAINING DILUTION GOING FROM LOWEST DILUTION TO HIGHEST. 7.10 USE A NEW, UV STERILIZED FUNNEL FOR EACH SAMPLE. 7.11 PLACE LABELED INVERTED DISHES IN 44.5 DEGREE INCUBATOR FOR 24 HOURS+/-2 HOURS. 7.12 AFTER INCUBATION, COUNT AND RECORD ALL DISH COLONIES THAT ARE VARIOUS SHADES OF BLUE. 7.13 NOTE: CREAM COLORED COLONIES ARE NEGATIVE. 7.14 IF COLONIES ARE IN QUESTION, USE A TRANSFER LOOP TO ISOLATE COLONIES TO LTB TUBES AND EC BROTH TUBES. OBSERVE FOR GROWTH TURBIDITY AND GAS FORMATION IN THE INVERTED TUBE. DATA COLLECTION 8.1 RECORD THE NUMBER OF COLONIES FOR EACH DILUTION ON BENCH SHEET. 8.2 CALCULATE # OF COLONIES FOR EACH DILUTION. 15
8.3 IF AT LEAST ONE DILUTION IS WITHIN THE IDEAL COLONY COUNT RANGE 20-60 COLONIES, USE ONLY THAT DILUTION TO CALCULATE THE FINAL RESULTS USING THE FORMULA CFU/100ML = COLONIES COUNTED X 100 / DILUTION VOLUME. REPORT RESULTS AS CFU/100MLS. 8.3.1 IF NO DILUTIONS FALL WITHIN THE IDEAL COUNT RANGE, USE ALL DILUTIONS TO CALCULATE THE FINAL RESULTS AND DENOTE RESULTS AS CALCULATED SEE STANDARD METHODS 9222B.6. FOR FURTHER GUIDANCE. 8.3.2 LOG DATA RESULTS IN TO THE LABORATORY INFORMATION MANAGEMENT SYSTEM(LMS). SECTION 9: QUALITY CONTROL 9.1 SAMPLE REPLICATE 9.1.1 ANALYZE A LABORATORY REPLICATE FOR 10% OF SAMPLES WITH AT LEAST ONE REPLICATE PER FILTRATION BATCH. 9.1.2 CONVERT RESULTS TO LOGARITHMS AND CALCULATE THE RANGE (DIFFERENCE IF LOGARITHMS OF A REPLICATE SET). THE RANGE SHOULD BE <3.27 TIMES THE MEAN RANGE OF AT THE LAST 15 REPLICATE SETS. 9.2 METHOD BLANKKS 9.2.1 FILTER A 100 ML VOLUME OF BUFFERED DILUTION WATER FOR EACH FILTRATION BATCH. PLACE THE FILTER ON AN mfc DISH AND LABEL AS WATER BLANK. INCUBATE BLANK WITH SAMPLES AT 44.5+/-0.2 FOR 24 HOURS =/- 2 HOURS. 9.2.2 ABSENCE OF GROWTH IN THE METHOD BLANK DEMONSTRATES THAT MEDIA, FILTRATION EQUIPMENT, FILTERS, AND DILUTION WATER ARE FREE OF CONTAMINATION OF THE TARGET ORGANISM FROM IMPROPER HANDLING, INADEQUATE STERLIZATION OF ENVIRONMENTAL EXPOSURE. 9.2.3 IF METHOD BLANKS HAVE GROWTH, DETERMINE THE CAUSE OF CONTAMINATION. IF THE CAUSE OF CONTAMINATION HAS ALSO AFFECTED SAMPLES, REPORT ALL SAMPLES IN THE FILTRATION BATCH WITH A QUALIFIER STATEMENT. 9.3 MEDIA STERILITY CHECK 9.3.1 PLACE A MEMBRANE FILTER DIRECTLY ON mfc MEDIA BROTH. AFTER 24 INCUBATION, NO GROWTH OF COLONIES SHOULD BE OBSERVIED. PRESENCE OF COLONIES INDICATES METHOD CONTAMINATION. 9.4 POSITIVE CONTROL 9.4.1 DILUTE PLANT INFLUENT 1/100. AT THE END OF THE RUN FOR THAT DAY, FILTER THE PLANT INFLUENT AND INCUBATE SAMPLE AT 44.5 DEGREES-C. GOOD COLONY FORMATION INDICATES GOOD MEDIA. 9.4.2 PERFORM AT LEAST ONE POSITIVE CONTROL FOR EACH NEW BATCH OF PREPARE MEDIA. SECTION 10: SAFETY 10.1 MEDIA AND CULTURE DISPOSAL ALL MEDIA AND BACTERIA MUST BE AUTOCLAVED FOR 45 MINUTES BEFORE DISPOSAL. 10.2 READ MSDS INFORMATION FOR REAGENT HANDLING AND DISPOSAL. 10.3 UV STERILIZATION CABINET ALTHOUGH UNIT IS LIGHT-TIGHT, EYE PROTECTION IS RECOMMENDED. 10.4 PERSONAL PROTECTIVE EQUIPMENT (PPE) EYE PROTECTION EQUIPMENT AND GLOVES ARE TO BE WORN AT ALL 16
TIMES WHILE WORKING IN THE BACTERIOLOGY LABORATORY SECTION. LABORATORY COATS ARE TO BE WORN AT ALL TIMES. NO OPEN TOE SHOES ARE ALLOWED. NO FOOD ALLOWED AT ANY TIME. 10.5 AUTOCLAVE AUTOCLAVE PRESSURE MUST BE AT ZERO POUNDS PER SQUARE INCH BEFORE DOOR IS OPENED. 10.6 REPORT ANY ACCIDENTS TO YOUR IMMEDIATE SUPERVISOR. 10.7 ANY BROKEN GLASSWARE MUST BE DISCARDED. SECTION 11: REFERENCES 11.1 STANDARD METHODS 20 TH, EDITION; SM9222D 11.2 EPA MICROBIOLOGICAL METHODS FOR MONITORING THE ENVIROMENT WATER AND WASTES (EPA-600/878017). 17
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APPENDIX B 21
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