Quality Assurance Project Plan for an E. coli Monitoring Study of Black River, Pine Creek, and Mill Creek in Berrien and Van Buren Counties, Michigan

Grantee: Southwest Michigan Planning Commission MDEQ Tracking Code: 2011-0502

Version 2 June, 2012

□ Approved □ Returned for Modifications

______Signature: Robert Day, MDEQ NPS Unit Chief Date

______Signature: Joseph Rathbun, MDEQ Quality Assurance Reviewer Date MDEQ use only

QUALITY ASSURANCE PROJECT PLAN FOR AN E. COLI MONITORING STUDY OF BLACK RIVER, PINE CREEK, AND MILL CREEK IN BERRIEN AND VAN BUREN COUNTIES, MICHIGAN

BLACK RIVER AND PAW PAW RIVER WATERSHEDS PATHOGEN MONITORING GRANT PROJECT

MDEQ TRACKING CODE: 2011-0502

APRIL 2012 REVISED JUNE 2012

FTC&H PROJECT NO. G110766Q

Fishbeck, Thompson, Carr & Huber, Inc. Engineers • Scientists • Architects • Constructors 1515 Arboretum Drive, SE, Grand Rapids, MI 49546 Telephone: 616-575-3824 TABLE OF CONTENTS

QUALITY ASSURANCE PROJECT PLAN APPROVAL SHEET ...... 4

QAPP DISTRIBUTION LIST ...... 5

1.0 OVERVIEW ...... 7 1.1 Project Task Organization ...... 7 1.2 Laboratory ...... 8 1.3 Special Training Requirements ...... 9 1.4 Project Organization Chart ...... 9

2.0 PROBLEM DEFINITION/BACKGROUND INFORMATION ...... 7 2.1 Site Location ...... 7 2.2 Project Background/Problem Definition ...... 7

3.0 STUDY DESIGN ...... 9 3.1 Study Objectives ...... 9 3.2 Sampling Locations ...... 9 3.3 Sample Collection And Frequency ...... 10 3.4 Pathogen Monitoring...... 11 3.5 Flow Monitoring ...... 12 3.6 Sample Labeling, Handling, and COC...... 12 3.7 Laboratory Analysis ...... 13 3.8 Data Quality Objectives ...... 15 3.9 Training Requirements/Certification ...... 16

4.0 DATA VALIDATION AND REPORTING ...... 17 4.1 Data Management ...... 17 4.2 Asessment and Oversight ...... 17 4.3 Data Validation And Usability ...... 17 4.4 Data Reporting ...... 18 4.5 Documentation and Records ...... 18 4.6 Revisions to the QAPP ...... 18

5.0 REFERENCES ...... 18

LIST OF FIGURES

Figure 1 Project/Task Organization Figure 2 Black River, Mill Creek, and Pine Creek Watersheds Figure 3 Black River Sample Stations Figure 4 Pine Creek and Mill Creek Sampling Stations

LIST OF TABLES

Table 1 Sample Locations Table 2 Sample Collection and Parameters Table 3 Sampling Frequencies and Analyses

LIST OF APPENDICES

Appendix 1 Standard Operating Procedures for Water Quality Sampling in Streams Appendix 2 Area Velocity Flow Meter Installation and Operation Guide (available in Van Buren County Drain Commissioner’s office) Appendix 3 Sample Collection Documentation Form

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Appendix 4 Examples of Sample Labels and Chain-of-Custody Form Appendix 5 Laboratory SOPs and Instrument Calibration and Maintenance Appendix 6 Standard Methods, 19th Edition, 1995

LIST OF ABBREVIATIONS/ACRONYMS

BMP Best Management Practice COC Chain-of-Custody CSO Combined Sewer Overflow DNA Deoxyribonucleic Acid FTC&H Fishbeck, Thompson, Carr & Huber, Inc. GIS Geographic Information System IDEM Indiana Department of Environmental Management IDEP Illicit Discharge Elimination Plan mL Milliliter MDEQ Michigan Department of Environmental Quality MS4 Municipal Separate Storm Sewer System MST Microbial Source Tracking PLSS Public Land Survey System QA Quality Assurance QA/QC Quality Assurance/Quality Control QAPP Quality Assurance Project Plan QC Quality Control SOP Standard Operating Procedure TMDL Total Maximum Daily Load WQS Water Quality Standards

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QUALITY ASSURANCE PROJECT PLAN APPROVAL SHEET

This Quality Assurance Project Plan (QAPP) was prepared by Fishbeck, Thompson, Carr & Huber, Inc. (FTC&H) on behalf of the Southwest Michigan Planning Commission (SWMPC) to guide field and laboratory activities related to water quality monitoring in the Pine Creek, Mill Creek, and Black River Watersheds for E. coli. The QAPP was developed following guidance presented in The Volunteer Monitor’s Guide to Quality Assurance Project Plans, United States Environmental Protection Agency EPA 841-B-96-003, September 1996. This QAPP also follows the Michigan Department of Environmental Quality (MDEQ) 2006 QAPP Guidance: Environmental Monitoring document.

APPROVALS

MDEQ Project Manager Molly Rippke

SWMPC Project Administrator Marcy Colclough

FTC&H Project Coordinator E. Wendy Ogilvie, LEED-AP

FTC&H Project QC Manager Mary Crosby-Davies

FTC&H Field Leader Todd Campbell

Hope College, Laboratory Director Michael J. Pikaart, PhD

Great Lakes Scientific, Laboratory Director Wayne E. Gleiber, Ph.D.

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QAPP DISTRIBUTION LIST

Ms. Molly Rippke Lake Michigan Unit, Surface Water Assessment Section Water Resources Division Michigan Department of Environmental Quality 525 W. Allegan Street, 2nd Floor P.O. Box 30458 Lansing, MI 48909-7958 Phone: (517) 335-1125 Fax: 269-567-9440 E-mail: [email protected]

Ms. Marcy Colclough, Senior Planner Southwest Michigan Planning Commission 185 East Main Street, Suite 701 Benton Harbor, MI 49022 Phone: 269-925-1137 ext 25 Fax: 269-925-0228 E-mail: [email protected]

Ms. E. Wendy Ogilvie, Environmental Specialist Fishbeck, Thompson, Carr & Huber, Inc. 1515 Arboretum Drive, SE Grand Rapids, MI 49546 Phone: 616-464-3915 Fax: 616-464-3996 E-mail: [email protected]

Ms. Mary Crosby-Davies, Vice President / Senior Chemist Fishbeck, Thompson, Carr & Huber, Inc. 1515 Arboretum Drive, SE Grand Rapids, MI 49546 Phone: 616-575-3842 Fax: 616-464-3996 E-mail: [email protected]

Mr. Todd Campbell, Senior Geologist Fishbeck, Thompson, Carr & Huber, Inc. 4775 Campus Drive Kalamazoo, Michigan 49008 Phone: 269-375-3824 Fax: 616-464-3996 E-mail: [email protected]

Dr. Michael J. Pikaart, Ph.D., Associate Professor of Chemistry Hope College Science Center Rm. 3112 35 E. 12th St. Holland, Michigan 49422-9000 USA Voice: (616) 395-7382 Fax: (616) 395-7118 E-Mail: [email protected]

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Dr. Wayne Gleiber, Ph.D., Lab Director Great Lakes Scientific 2847 Lawrence St. Stevensville, MI 49127 Voice: 269-429-1000 Fax: 269-429-1550 Email: [email protected]

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1.0 OVERVIEW

This QAPP presents the organization, objectives, planned activities, and specific quality assurance/quality control (QA/QC) procedures associated with the water quality monitoring program for E. coli in the Pine Creek, Mill Creek, and Black River Watersheds. Specific protocols for sampling, sample handling and storage, chain-of-custody (COC), and laboratory analysis are presented herein.

1.1 PROJECT TASK ORGANIZATION

This section identifies the key individuals and organizations that will participate in the water quality monitoring program, and provides a general description of their roles and responsibilities.

MDEQ Project Manager – Ms. Molly Rippke

Ms. Rippke will provide review of the sampling program presented and administer grant funding for implementation of the water quality monitoring program presented in the QAPP.

SWMPC Project Administrator - Ms. Marcy Colclough

Ms. Colclough will be responsible for overall project administration. She is the Senior Planner of the organization receiving the grant funds for implementation of the water quality monitoring program. Ms. Colclough is also responsible for the management of the grantee moneys received for the project from MDEQ.

FTC&H Project Manager - Ms. E. Wendy Ogilvie

Ms. Ogilvie will be responsible for ensuring that technical and scheduling objectives for the project are successfully achieved. She will be responsible for primary interface with staff from MDEQ on behalf of the grantee.

FTC&H Project QC Manager – Ms. Mary Crosby-Davies

Ms. Crosby-Davies will be responsible for ensuring that the procedures for accuracy, precision, and completeness described in this QAPP are followed for all field and laboratory activities. Ms. Crosby- Davies will assist the Field Leader in training municipal field staff to complete the tasks related to the water quality monitoring.

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Field Leader – Mr. Todd Campbell

Mr. Campbell will be responsible for training municipal field staff to complete the tasks related to the Water quality monitoring in the Pine Creek, Mill Creek and Black River Watersheds.

Hope College, Department of Chemistry – Dr. Michael J. Pikaart, Ph.D.

Dr. Pikaart will be responsible for the laboratory analysis of the water samples for E. coli and for the microbial source tracking (MST) at Hope College. Dr. Pikaart will also be responsible for ensuring that the technical requirements specified in this QAPP are understood and that all data is reported in accordance with the QA/QC specifications described.

Great Lakes Scientific – Dr. Wayne Gleiber, Ph.D.

Dr. Gleiber will be responsible for the laboratory analysis of the water samples for E. coli at MC-01 on Mill Creek. Dr. Gleiber will also be responsible for ensuring that the technical requirements specified in this QAPP are understood and that all data is reported in accordance with the QA/QC specifications described.

1.2 LABORATORY

Laboratory analysis for E. coli in Black River and Pine Creek and all the analyses for MST will be performed by:

Hope College Chemistry Department Science Center Rm. 3112 35 E. 12th St. Holland, Michigan 49422-9000 USA Voice: (616) 395-7382 Fax: (616) 395-7118

Laboratory analysis for E. coli at MC-01 on Mill Creek will be performed by:

Great Lakes Scientific 2847 Lawrence St. Stevensville, MI 49127 Voice: 269-429-1000 Fax: 269-429-1550

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1.3 SPECIAL TRAINING REQUIREMENTS

All project personnel will be qualified and experienced in the project task for which they are responsible. The FTC&H field leader will conduct a sample collection training session for any selected volunteers prior to the first sampling event. Hope College Chemistry Department and Great Lakes Scientific will be responsible for training and certifying their own personnel. The Van Buren County Drain Commissioner’s office has qualified staff to calibrate the flow meters and run the computer software. All field staff will be trained by the FTC&H Field Leader in sample collection, preservation, chain-of-custody, and note taking.

1.4 PROJECT ORGANIZATION CHART

The organizational structure for water quality monitoring project is shown on Figure 1.

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2.0 PROBLEM DEFINITION/BACKGROUND INFORMATION

2.1 SITE LOCATION

The Black River Watershed encompasses approximately 183,490 acres, or 287 square miles in Allegan and Van Buren Counties in southwestern Michigan. 43.8% of the watershed lies in Allegan County, and 56.2% lies in Van Buren County. The Black River Watershed includes portions of Casco, Cheshire, Clyde, Ganges and Lee Townships in Allegan County; Arlington, Bangor, Bloomingdale, Columbia, Covert, Geneva, South Haven, and Waverly Townships along with the Villages of Breedsville and Bloomingdale and the Cities of Bangor and South Haven in Van Buren County (The Black River Watershed Management Plan (BRWMP)). According to the Michigan Center for Geographic Information 2002, the 1992 land use/land cover in the Black River Watershed is approximately 58% agriculture, 33% forested upland, 8% water and wetlands, and 1% developed.

The project focuses on the Pine and Mill Creeks, both coldwater tributaries to the Paw Paw River, located in Berrien and Van Buren Counties. The Paw Paw River Watershed (PPRW) encompasses approximately 446 square miles in Kalamazoo, Van Buren, Berrien and Kalamazoo Counties with the largest portion in Van Buren County (203,720 acres). In the PPRW, there are 39 governmental units including twenty-five townships, four villages, six cities, and one tribe (Pokagon Band of Potawatomi Indians). The watershed contains mostly agricultural (47%) and natural (45%) land cover, along with approximately 7% urban areas (Paw Paw River Watershed Management Plan).

The Pine Creek watershed includes portions of Hartford and Keeler Townships and the City of Hartford. The Mill Creek watershed includes portions of Hartford and Keeler Townships in Van Buren County and Bainbridge, Coloma, and Watervliet Townships and the City of Watervliet in Berrien County. The headwaters of both creeks have been dredged to facilitate drainage, resulting in flashy flow regimes in these agricultural watersheds (Walterhouse, 2006). United States Geological Survey 2000 land cover data indicate that the Pine Creek watershed is largely cultivated (row) crops (63 percent) while the Mill Creek watershed is approximately 61 percent cultivated (row) crops (USGS, 2001b).

2.2 PROJECT BACKGROUND/PROBLEM DEFINITION

The Pine Creek and Mill Creek have both exceeded the standards established for microorganisms listed in R 323.1062 of the State of Michigan Part 4 Water Quality Standards (WQS) promulgated pursuant to Part 31 of the Natural Resources and Environmental Protection Act, 1994 PA 451 as amended. The Rules require that all waters of the state shall be protected for total body contact recreation and shall not contain more than 130 E. coli per 100 milliliter (mL) as a 30-day geometric mean. In addition, at no time

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shall the waters of the state protected for total body contact contain more than 300 E. coli per 100 mL, as a geometric mean of at least three samples collected during the same sampling event.

Pine Creek and Mill Creek were first placed on the Section 303(d) list in 2006 for the impairment of recreational uses due to exceedances of the E. coli WQS. Data collected by the Michigan Department of Environmental Quality (MDEQ) documented exceedances of the total and partial body contact WQS for E. coli at all sampling locations for both Pine and Mill Creek’s during the months of July through September 2005 (MDEQ, August 2009).

The MDEQ has listed 9.98 miles of Pine Creek and 12.77 miles of Mill Creek as impaired by elevated E. coli concentrations. The location of the Pine Creek TMDL is in two different sections of the creek, 4.17 miles in length which extends from the Paw Paw River confluence upstream to 66th Avenue, and 5.81 miles in length which extends from 66th Avenue upstream to the headwaters (MDEQ, August 2009).

Potential sources of E. coli in Pine Creek and Mill Creek include runoff from pastureland and land application of manure, failing septic systems, illicit connections to storm sewers and drains, and inputs from wildlife (MDEQ, August 2009).

The BRWMP indicates that the designated uses for Partial Body Contact and Total Body Contact are both threatened, however, water quality standards are maintained for skiing, canoeing, wading and swimming. In Section 6.7.4 Bacteria/Pathogens in the BRWMP it was indicated that in the last study of fecal coliform bacteria in the Black River Drain (North Branch of the Black River), two sample locations had fecal coliform in excess of 550. However, the testing may now be outdated (BRWMP).

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3.0 STUDY DESIGN

This section provides the QA measures required to obtain data that will meet the objectives of the project, including sampling design, sample collection and preservation, sample custody, analytical methods, and data management.

3.1 STUDY OBJECTIVES

The primary objective of this water quality monitoring project is to identify possible “problem areas” within the Black River, Pine Creek, and Mill Creek and to attempt to identify the source of pathogen contamination using Molecular Source Tracking (MST) techniques. The specific objectives for each watershed are as follows:

a. Black River: Where are the sites with the highest levels of E. coli and what are the sources?

b. Pine Creek: Is the major source of contamination from the urbanized areas or the agricultural operations?

c. Mill Creek: How does E. coli respond to flows in the system? Is E. coli impairing the City Park where residents have access to the creek? How does the hydrograph correlate to contamination levels?

3.2 SAMPLING LOCATIONS

This E. coli monitoring program will consist of surface water sampling at 12 stations on the Black River, Pine Creek, and Mill Creek (Figure 2) between June 1 to October 31, 2012. Flow monitoring will occur at two stations on Mill Creek. The locations chosen for sampling are generally based on previous sampling conducted by MDEQ.

The following sampling locations are proposed in each waterbody as indicated below and in Table 1:

Black River – A total of seven sites in the Black River and its tributaries (Figure 3) are proposed for sampling during wet weather events: two on the Phoenix Drain, two on tributaries to the Black River within the City of South Haven, one on the North Branch of the Black River, one on the South Branch of the Black River, and one on the confluence of Butternut Creek and Tripp Drain.

Pine Creek – A total of 4 sites in Pine Creek (Figure 4) are proposed for sampling. Both dry and wet weather sampling is proposed for the sites PC-01 and PC-02, which are downstream and upstream of the City of Hartford, respectively. Wet weather sampling is proposed for two sites, one at 66th Avenue (PC- 03), and one at 72nd Avenue (PC-04).

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Mill Creek – One sampling site for E. coli on Mill Creek (Figure 4) is proposed at Flaherty Park in Watervliet (MC-01) for wet weather sampling. Samples for E. coli analysis will be collected through the course of a wet weather event at 12-hour intervals, over a 24-hour period, as the Great Lakes Scientific laboratory schedule allows.

Flow will be monitored at Flaherty Park in Watervliet (MC-01) and also at 70th Street (MC-02) to provide additional information of the watershed’s response to a rain event. Data from the pressure transducers will be uploaded during each sampling event. Rain gauges will be installed adjacent to the sampling sites. The rain level will be recorded during each sampling event and after total rainfall.

3.3 SAMPLE COLLECTION AND FREQUENCY

The intent is to commence the sampling program in June 2012. Wet weather samples will be collected during three significant storm events occurring between June 1, 2012, and October 31, 2012. A significant event is considered to be at least 0.25 inches of rainfall within a 24-hour period. Rainfall events exceeding these criteria would be preferred. Determination of a significant rainfall event will be based on meteorological data recorded at the following sites:

Grandview Orchards - Bainbridge Center / Watervliet http://www.agweather.geo.msu.edu/mawn/station.asp?id=bbc

High Acres Fruit Farm - Hartford http://www.agweather.geo.msu.edu/mawn/station.asp?id=hfd

In addition, the discharge at the USGS Gage Station 04102700 - South Branch Black River near Bangor, MI, Latitude 42°21'15", Longitude 86°11'15" (NAD27, Van Buren County, Michigan, Hydrologic Unit 04050002) will also be noted during wet weather events. http://waterdata.usgs.gov/nwis/inventory?agency_code=USGS&site_no=04102700

Black River - Each wet weather sampling event will consist of collecting triplicate samples at each of the seven stations during the rain event. Specifically, one triplicate sample will be collected as close to the first flush of the wet weather event as possible. No dry weather sampling will be conducted at these seven sites.

Pine Creek – Dry weather sampling for PC-01 (Red Arrow) and PC-02 (access road south of I-94) will be conducted monthly, collecting triplicate samples. Wet weather sampling for all 4 sites will consist of collecting triplicate samples, taken as close as possible to the first flush of the rain event.

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Mill Creek – Wet weather sampling for E. coli will occur at MC-01 at 12-hour intervals during a 24-hour period to capture the samples during the complete hydrograph at the site, including the first flush of a rain event. Samples will be put on ice and transported to the Great Lakes Scientific laboratory within 6 hours of collection to meet the sample hold time. Data from the pressure transducers will be uploaded at the Flaherty Park site (MC-01) and at 70th Street (MC-02). All data will be managed using computer software and Microsoft Excel.

Samples will be collected from each station in accordance with the SOP for surface water sampling provided in Appendix 1. Samples will always be collected from the most upstream location first, and then by working downstream to collect subsequent samples.

Personal safety will be of the greatest concern. Field staff will not enter the creek if conditions appear to be unsafe, especially during high-water conditions. The sampling crews will be given discretion to abandon a sampling run, or to abandon sampling at a particular station, whenever safety is a concern. For example, recent tree falls during a storm may make travel unwise. To extend the reach of the person collecting the sample, the container can be temporarily attached to a pole or a clean bucket attached to a rope can be used. If a bucket is used, care will be taken to avoid contaminating the sample. The bucket will be cleaned prior to initial use by mild scrubbing with a 10% household bleach solution for at least one minute, followed by two rinses with distilled water. The bucket will be rinsed an additional two times with distilled water prior to each sample collection. This wash water will be collected in a sealable container and dumped into a sanitary sewer connection (not storm sewer catch basin). Appropriate personal protective equipment, including gloves, will be worn during sampling. Gloves will be replaced after sample collection at each location.

3.4 PATHOGEN MONITORING

Water quality samples, including duplicates and field blanks, will be collected during dry weather at two locations in Pine Creek (PC-01 and PC-02). Grab water samples will also be collected during three storm events at the Mill Creek station in Flaherty Park (MC-01), all four locations on Pine Creek, and all seven locations on the Black River. The grab water samples will be collected in sterile Nalgene polysulfone dilution 1-Liter containers provided by the Hope College Laboratory, or 250-mL screw cap polycarbonate containers provided by Great Lakes Scientific.

Field duplicates will be collected at a rate of 10 percent and field blanks will be collected at a rate of 5 percent. A minimum of one field blank and one duplicate will be collected per sampling event. All samples will be transferred from the automatic sampler to the storage cooler in a manner that minimizes the chance of contamination during transfer A Sample Collection Documentation Form (Appendix 3) will be completed for each sample collected. All information in the sections of Weather Conditions and General

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Observations will be recorded. Only the water depth will be recorded in the Basic Field Measurements section. At each location, staff will observe the surrounding conditions and document any discoloration, excessive turbidity, foaming, floatables, or the presence of a sheen or scum layer along with a general comment on flow on the sample collection form.

3.5 FLOW MONITORING

Stream hydrographs will be developed for both sample stations on Mill Creek (MC-01 and MC-02) using pressure transducers (Hobo model U20 water level/temperature loggers) and manual measurements of stream stage. During field visits, water depth and velocities will be recorded.

Stream hydrographs will be developed by deploying pressure transducers (Hobo model U20 water level/temperature loggers) at MC-01 and MC-02 to measure stream stage at 10-minute intervals throughout the study period. Transducers will be suspended within 1 cm of the sediment surface inside of stilling wells constructed of 5.2-cm ID perforated polyvinyl chloride pipe. An additional transducer will be suspended at the top of the stilling well at a site to record atmospheric pressure. Pressure data will be uploaded from each transducer and transducer clocks will be synchronized during each site visit.

Stream stage will be measured manually at all sites during each visit using staff gauges attached directly to each stilling well. To convert pressure readings to stream stage, atmospheric pressure will be first subtracted from stream pressure. Next, the atmosphere-corrected stream pressure readings that corresponded with the visual stage measurements will be regressed against the measured stage observations for each site. The resulting linear function will then be applied to the entire record of pressure readings from each site to yield a high-frequency record of stream stage for the study period.

Stream stage will be converted to discharge at each site by first calculating rating curves between stage and discharge and applying these functions to the high-frequency stage records. To measure discharge, transects will be established perpendicular to stream flow. Cross sections will be permanently marked. Water depth and velocity will be measured at equally-spaced points along the transects. Water velocity will be measured according to USGS protocols using a Marsh-McBirney Flow Mate 2000 flow meter attached to a top-setting wading rod during each field visit. When water depth is less than 2.5 ft, velocity will be measured at 0.6 x depth. When depth exceeds 2.5 ft, velocity will be measured at 0.2 x and 0.8 x depth. The Windows-based hydrologic software, HYDROL-INF will be used to calculate stream discharge.

3.6 SAMPLE LABELING, HANDLING, AND COC

A unique number will be assigned to each field sample collected. The following example demonstrates the identification code to be used:

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BR1301-(I) - The first two digits of the code represent the Black River, the second two digits of the code are the Public Land Survey System (PLSS) section number, the final two digits are the numerical station number, and the (I) designates an investigative sample. A (D) in place of the (I) indicates the sample is a field duplicate. A (B) in place of the (I) indicates the sample is a field blank.

Sample labeling and handling will follow the SOP included in Appendix 4. This information, along with collection time, date, sampler’s initials, and analytical parameters will be recorded on an adhesive label affixed to each sample container. An example of a completed label is provided in Appendix 4. A COC record will also be completed for all samples. This form will be used to track the possession of the samples from collection, through delivery to the laboratory. A copy of the COC will accompany the laboratory results. An example of the COC record is in Appendix 4.

The samples will be delivered to Hope College Chemistry Department or Great Lake Scientific by no later than 6 p.m. on the day of collection. Sample holding time is limited to six hours from collection. The samples will be placed in a cooler, on ice, and kept at a temperature <4ºC, after collection, for transport to the laboratories.

Sampling and QC procedures will be evaluated by the project QC Manager and the Field Leader. Discrepancies between field duplicate results and positive results for method blanks will trigger a review of sampling procedures. An RPD of 25 percent will also trigger a review. If the RPD is greater than 25 percent, than the results will be reviewed and if one sample in a duplicate pair exceeds WQS while the other sample meets WQS, the samples will be considered invalid. Corrective action will involve identification of the potential cause of failures where possible. In some cases, a site may have to be re- sampled to achieve project goals.

3.7 LABORATORY ANALYSIS

The intent of the wet and dry weather sampling is to assist in identifying potential sources of E. coli and providing baseline data to track longer-term trends.

Samples will be collected in triplicate for determination of E. coli according to the procedure for the detection and enumeration of Total Coliforms and E. coli using the IDEXX Colilert ® Bacterial Analysis Method or the mTEC Agar Method. An appropriate sample dilution will be selected so that a result of “too numerous to count” is not reported. An actual E. coli count will be provided when results fall between the lower limit of 100 cfu/100 mL and the upper limit of 240,000 cfu/100 mL. Sample collection and parameters are provided in Table 2. Field duplicate samples will be submitted to determine field precision. Copies of the laboratory SOPs are contained in Appendix 5.

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The 1-L samples collected for the IDEXX analysis will yield enough volume of water to also be used for the MST analysis. The 250-mL samples collected at MC-01 on Mill Creek will not yield enough volume of water for MST analysis so separate 1-L samples will be collected. Water not used for the IDEXX will be processed by centrifuging and freezing the residuals to conduct the analysis at a later time. The samples for MST analysis will be selected based on the results of the E.coli analyses. The sites with the highest counts of E. coli in areas where multiple sources could be contributing will be considered for MST analysis. The laboratory at Hope College will detect DNA sequences from Bacteriodes spp. and Enterococcus spp. using qPCR-based methods. This technology will be used to determine the source of fecal contamination as either human or bovine, and possibly pig if that is a potential source for a sampled area. The sampling frequencies and analyses to be conducted for each site are listed in Table 3.

Laboratory QC failures will be evaluated by the laboratory director and by the QC Manager. Laboratory instrument calibration frequency and maintenance for sterilizers, pH meters, thermometers, and incubators is included in Appendix 5. The cause of any failures will be identified when possible. In some cases, a re-sampling effort may be required to achieve project goals.

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3.8 DATA QUALITY OBJECTIVES

This section provides the objectives and criteria for data acceptance. The data will be evaluated to determine whether it can be used to satisfy project objectives.

PRECISION

Precision is a measure of the reproducibility of measurements under given conditions. Field precision will be assessed through the collection of field duplicate samples. Duplicate samples will be collected at a frequency of one duplicate per 10 investigative samples, or a portion thereof.

Laboratory precision will be assessed by replicate analyses of sample duplicate pairs. Precision will be calculated using the procedure outlined in Section 9020B-4b in Standard Methods for the Examination of Water and Wastewater, 19 Edition. A copy of this procedure is included in Appendix 6. This method indicates testing the first 10% samples collected during the first event in duplicate and then calculating the range of logarithms of these duplicate pairs. This data is then used to calculate an average logarithm range. Following the initial duplicate samples, a field duplicate will be collected at a frequency of one per 10 of the identified sampling locations, or portion thereof. The procedure presented in Section 9020B-4b will be used to compare the routine field duplicates to the range established from the initial duplicates. Field duplicate results falling within the guidelines of this comparison will be used in judging the precision of a data set. This procedure was identified as a means to judge overall data quality, however, data reviewer discretion will also be considered. This is required since a broad range of results are anticipated with this type of sampling.

ACCURACY

Accuracy cannot be directly measured for bacterial samples. Accuracy will be assessed through the analysis of quality control (QC) samples as described in the laboratory method contained in Appendix 5.

COMPLETENESS

Completeness is a measure of the amount of valid data obtained compared to the amount that was expected to be obtained under correct normal conditions. Completeness is calculated using the following equation:

Completeness, % = number of valid data point s obtained number of reported measurements

The field completeness objective for each project will be greater than 80%. Laboratory completeness objective for each project will be greater than 80%.

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REPRESENTATIVENESS

Representativeness expresses the degree to which data accurately and precisely represents a characteristic of a population, parameter variations at a sampling point, a process condition, or an environmental condition. Field representativeness will be achieved by following the procedures for sample collection and handling presented in this QAPP and by collecting multiple samples during regular intervals. Laboratory representativeness will best be achieved by following the analytical procedures presented in this QAPP and adherence to sample holding times.

COMPARABILITY

Comparability is an expression of the confidence with which a data set can be compared with another. Comparability is also dependent on similar QA objectives. Field comparability will be satisfied by following the sample collection and handling procedures presented in this QAPP. Laboratory comparability will be satisfied by following the sample preparation and analytical procedures presented in this QAPP.

3.9 TRAINING REQUIREMENTS/CERTIFICATION

The Field Leader will conduct a sample collection training session for the designated Project Administrator and field staff prior to the first sampling event. Laboratory analyses will be performed by the laboratories. No other specialized training or certification is required.

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4.0 DATA VALIDATION AND REPORTING

4.1 DATA MANAGEMENT

Field observations and sample collection information will be recorded on the Sample Collection Documentation Form. A copy of this form is included in Appendix 3. It is the responsibility of the person collecting the sample to complete the Wet Weather Flow Evaluation form, sample labels, and the COC form prior to moving on to the next sampling location.

Laboratory data will be documented on bench sheets and will include analysis date, sample size, raw data, and any other information necessary for analytical calculation. Data will be summarized in a spreadsheet format for each event to include sample collection date, monitoring location, and analytical results.

The original sample collection forms and laboratory data summaries will be stored at the SWMPC Project Administrator’s office. Copies of the sample collection forms, COC forms, laboratory results, and QA/QC documentation will be submitted to FTC&H for review. At the conclusion of the study, an editable EXCEL spreadsheet will be submitted to the MDEQ summarizing site latitudes and longitudes, and site data.

4.2 ASESSMENT AND OVERSIGHT

The FTC&H Project QC Manager will review the field and laboratory records after every event to verify the data quality objectives for the project are being met. If the laboratory data quality objectives are not being consistently met, the deficiencies will be brought to the attention of the laboratory and appropriate corrective measures will be implemented. If deficiencies are noted in the collection or recording of field data, the QC Manager will review sampling and field measurement protocol to ensure that sampling personnel are familiar with the procedures.

4.3 DATA VALIDATION AND USABILITY

The Project QC Manager will review all field and laboratory data, within two weeks of receiving the information, to ensure that it is of usable quality for the project. The data will be judged as usable or unusable based on compliance with laboratory SOPs and the stated data quality objectives.

The Project QC Manager, in conjunction with the FTC&H Project Manager, will be responsible for implementing any required corrective action. Communication between the Field Leader, laboratories, Project Coordinator, and the Project Administrator will be critical. All individuals involved with the program will be required to direct questions regarding sample collection and field and laboratory measurements to the Project QC Manager as soon as they arise.

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4.4 DATA REPORTING

The results of the sampling will be supplied to both the SWMPC Project Administrator and to FTC&H. Hard copies will be maintained at the SWMPC office, and digital copies will be maintained at the FTC&H office. Excel spreadsheets will be used to generate tables and graphs of the field data for review. The Geographic Information System (GIS) mapping will be used for geographic presentation of the data and background information, as may be useful to support project conclusions and any recommendations.

4.5 DOCUMENTATION AND RECORDS

Sampling and laboratory records will be maintained at the SWMPC Project Administrators’ office for a minimum of 5 years. These records will include, but are not limited to, Water Quality Monitoring Field Forms, COC forms, and analytical results. Laboratory bench sheets may be requested by the QC Manager as a means to verify that laboratory procedures, as described in the laboratory SOPs, were followed.

The SWMPC Project Administrator, with assistance from FTC&H, will submit all drafts and final products and deliverables in both hard copy and electronic format according to the DEQ-NPS Providing Electronic Versions of Grant Products Guidance.

4.6 REVISIONS TO THE QAPP

Expedited changes to the QAPP may be necessary to reflect changes in project organization, tasks, schedules, objectives, and methods; to address deficiencies and nonconformance; to improve operational efficiency; and/or to accommodate unique or unanticipated circumstances. Requests for expedited changes are directed from the SWMPC Project Administrator to the MDEQ Project Manager in writing. The changes shall become effective immediately upon approval by the MDEQ Project Manager. Any changes to the QAPP will be documented, and revised pages will be forwarded by the SWMPC Project Administrator to all persons on the QAPP distribution list. Expedited changes shall be reviewed, approved, and incorporated into a revised QAPP within 30 days of approval.

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5.0 REFERENCES

Standard Methods for the Examination of Water and Wastewater, 21st Edition. 2005. American Public Works Association, American Water Works Association, Water Environment Federation, Washington D.C.

Fuller, Erin. 2009. Black River Watershed Management Plan. Van Buren Conservation District

Great Lakes Scientific. 2009. Analysis of Surface Water for E. coli. Stevensville, Michigan.

Hope College Water Quality Lab. 2000. Colilert Quanti-tray 2000 Method Standard Operating Procedure. Hope College, Holland, Michigan.

Hope College Water Quality Lab. 2012. Standard Operating Procedures for Microbial Source Tracking. Hope College, Holland, Michigan.

Paw Paw River Watershed Management Plan. August 2008. Southwestern Michigan Planning Commission (SWMPC).

Michigan Center for Geographic Information. 2002. 1992 NLCD Shapefile by County. Lansing, MI: Michigan Center for Geographic Information.

Alexander, Christine, 2009. Total Maximum Daily Load for E. coli for Pine and Mill Creeks, Berrien and Van Buren Counties. Michigan Department of Environmental Quality, Water Bureau, Surface Water Assessment Section, Lansing, MI. July 27, 2009.

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Table 1 - Sample Locations

Sample ID Water Body Site Description Lat Long

BR-01 Unnamed tributary Kal-Haven Trail 42.41315 -86.25957 State Park BR-02 Phoenix Drain Outlet 42.40856 -86.27235

BR-03 Unnamed drain Outlet near 42.41431 -86.26447 Woodland Marina BR-04 North Branch Baseline Road 42.41905 -86.24891 Black River BR-05 South Branch 71-1/2 Street 42.41699 -86.24003 Black River BR-06 Butternut Creek Downstream of 42.409579 -86.210790 Butternut Creek and Tripp Drain confluence BR-07 Phoenix Drain Upstream 42.39485 -86.2661

PC-01 Pine Creek Red Arrow 42.20580 -86.18204 Highway PC-02 Pine Creek Access Road 42.19152 -86.17091 south of I-94 PC-03 Pine Creek 66th Avenue 42.17877 -86.15768

PC-04 Pine Creek 72nd Avenue 42.15709 -86.17353

MC-01 Mill Creek Flaherty Park 42.19045 -86.25802

MC-02 Mill Creek 70th Street (County 42.14294 -86.22284 Line)

Table 2 - Sample Collection and Parameters Sample Detection Bottle Parameter Method Volume Preservative Hold Time Limit Type (mL) IDEXX Lower: Colilert ® <100/100 mL E. coli Bacterial Upper: 1,000 Plastic None 6 hours Analysis 240,000/100 Method mL Lower: mTEC Agar <100/100 ml E. coli Method Upper: 250 Plastic None 6 hours (9213D) 240,000/100 ml DNA sequences qPCR- Dependent on from Bacteriodes based sample type / 1,000 Plastic None 6 hours spp. and methods volume Enterococcus spp.

Table 3. Sampling Frequencies and Analyses Dry Wet mTEC Agar Watershed Sampling Locations Wet Weather Events IDEXX Weather Weather Method MST* Flow Samples Samples (1-L sample) (250-mL June July August September October TOTAL #1 #2 #3 TOTAL sample) I D B I D B I D B I D B I D B I D B I D B I D B Black River BC-01 (unnamed tributary) 0 3 1 3 3 10 10 BC-02 (Phoenix Drain outlet) 0 3 1 3 3 10 10 1-7 BC-03 (unnamed drain) 0 3 1 1 3 1 3 1 1 14 14 (from BC-04 (North Branch Black River) 0 3 1 3 3 10 10 selected BC-05 (South Branch Black River) 0 3 1 3 1 1 3 1 13 13 sites) BC-06 (Butternut Creek/Tripp Drain) 0 3 1 3 3 10 10 BC-07 (Phoenix Drain headwaters) 0 3 1 3 3 10 10 Total 7 sites 0 21 7 1 21 2 1 21 2 1 77 77 0 7 0

Pine Creek PC-01 (Red Arrow) 3 3 1 1 3 3 1 1 3 19 3 1 1 3 1 3 1 1 14 33 1-4 PC-02 (I-94) 3 1 1 3 3 1 1 3 3 1 1 21 3 1 3 3 10 31 (from PC-03 (66th Avenue) 0 3 1 3 3 10 10 selected PC-04 (72nd Street) 0 3 1 3 1 1 3 1 13 13 sites) Total 4 sites 6 1 1 6 1 1 6 1 1 6 1 1 6 1 1 40 12 4 1 12 2 1 12 2 1 47 87 0 4 0

Mill Creek** MC-01 (Flaherty Park) 0 9 3 1 9 1 1 9 1 1 35 35 3 1 MC-02 (70th Street) 0 1 Total 2 sites 0 9 3 1 9 1 1 9 1 1 35 35 3 2

Total 13 Stations 6 1 1 6 1 1 6 1 1 6 1 1 6 1 1 40 42 14 3 42 5 3 42 5 3 159 164 35 14 2 *The MST analysis does not require a separate grab water sample (except at MC-01). The 1-L water sample collected for the IDEXX analysis will be used also for the MST analysis. For MC-01, a separate 1-L water sample will be collected in addition to the 250-ml water sample for mTEC agar analysis. **Flow will be measured at MC-01 and MC-02 only. E. coli samples will be collected at MC-01 every 12 hours, over a 24-hour time period during each storm event. I - Investigative Sample D - Field Duplicate Sample B - Field Blank Sample

FIGURE 1

Project/Task Organization

Ms. Molly Rippke MDEQ Project Manager MDEQ-Water Resources Division-Lake Michigan Unit

Ms. Marcy Colclough Senior Planner SWMPC Project Administrator Southwest Michigan Planning Commission

Ms. Mary Crosby- Ms. E. Wendy Ogilvie Davies Environmental Specialist Vice FTC&H Project Manager President/Senior Fishbeck, Thompson, Carr & Huber, Inc. Chemist FTC&H Project QC Manager Mr. Todd Campbell Dr. Michael J. Pikaart Fishbeck, Senior Geologist Associate Professor of Thompson, Carr & Field Leader Chemistry Huber, Inc. Fishbeck, Thompson, Carr & Huber, Inc. Lab Manager Hope College

Sampling Team Dr. Wayne Gleiber Members Lab Director Great Lakes Scientific

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MDEQ Tracking Code 2011-0502 s Standard Operating Procedure (SOP) For Surface Water Sample Collection

Scope: This SOP describes the procedure to be followed for collection of surface water grab samples using screw cap bottles.

Equipment: Sample containers, 100 mL to 500 mL polyethylene, sterile COLILERT® sample bottles, , sample collection documentation forms, sample labels, clipboard, black or blue indelible ink pens, sampling location map, insulated cooler with ice, chain-of-custody records

Procedure:

1. Locate the sample point. Carefully wade into the stream or lake, taking care to disturb as little bottom sediment as possible. Stand facing the current near one side of the stream or in an area of the lake that has not been disturbed by any wave action.

2. Uncap the sample bottle, and holding in an inverted position (bottom up), lower the bottle into the water at least 6 inches below the surface unless the stream is shallow and collecting at that depth would risk contaminating the sample with sediment. Sampling will not take place in backwater areas or eddies.

3. Carefully tilt the bottle on its side with the opening facing the current and allow water to fill the container. Take care not to collect water that has sediment from disturbance of the stream bottom.

4. Lift the bottle from the water and cap.

5. For stream samples, move to the center of the stream and repeat steps 2 through 4. Move to the other side of the stream and repeat steps 2 through 4.

6. To extend reach of the person collecting the sample, the container can be temporarily attached to a pole or a clean bucket attached to a rope can be used. If a bucket is used, care will be taken to avoid contaminating the sample. The bucket will be thoroughly rinsed between stations. The bucket will be cleaned prior to initial use by mild scrubbing with a 10% household bleach solution for at least one minute, followed by two rinses with distilled water. The bucket will be rinsed an additional two times with distilled water prior to each sample collection. Appropriate personal protective equipment, including gloves, will be worn during sampling. Gloves will be replaced after sample collection at each location.

7. Place all samples in a cooler with ice to maintain temperatures between 35 and 46 degrees, if possible.

8. Transport samples to the laboratory within 6 hours. Samples must be received by the laboratory on the same day as collection.

9. Record all necessary information on field data sheets and chain-of-custody.

Safety Considerations:

1. Listen to weather reports. If taking wet weather samples, be aware of severe weather events or storms and use precaution before deciding to sample.

2. Do not cross private property without the permission of the landowner.

3. Watch for animals and wildlife. Also watch for poison ivy, poison oak, sumac, or other vegetation in the area that can cause skin irritation.

4. Do not walk on unstable river or stream banks. Do not disturb vegetation on the banks.

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5. Be very careful while walking in the stream itself. Rocky bottom areas can be slippery and soft bottom areas may be dangerous in areas where mud, silt, or sand has accumulated in sinkholes.

6. Never wade in swift or high water. Use an extension pole or clean bucket and rope should be considered under these conditions.

7. If at any time you feel uncomfortable about the condition of the monitoring site or surroundings, leave the area. Personal safety is much more important than data collection.

U:\PROJECTS\110766\G110766Q-QAPP\QAPP\SECOND_MDEQ_SUBMITALL_2012-05\APPENDIX 1_SOP FOR SURFACE WATER SAMPLING.DOCX SAMPLE COLLECTION DOCUMENTATION FORM

MONITORING LOCATION:

SAMPLE ID:

COLLECTION DATE:

COLLECTION TIME:

COC NUMBER:

WEATHER CONDITIONS

CLEAR PARTLY CLOUDY FOG/HAZE SNOW

OVERCAST DRIZZLE DOWNPOUR

AIR TEMPERATURE: ºF

NUMBER OF DAYS WITH SIMILIAR WEATHER:

RAINFALL IN PREVIOUS 24 HOURS (CHECK ONE): NONE HEAVY

LIGHT inches (if known)

WIND:

CALM SLIGHT BREEZE MOD. BREEZE WINDY

WIND DIRECTION:

NORTH EAST SOUTH WEST

NORTHEAST SOUTHEAST SOUTHWEST NORTHWEST

GENERAL OBSERVATIONS:

WATER COLOR:

MED. BROWN DK. BROWN GREEN-BROWN GRAY

GREEN RED-BROWN YELLOW-BROWN OTHER:

WATER SURFACE: AQUATIC PLANTS/ALGAE PRESENT:

CALM RIPPLES CHOP NO YES

FIELD MEASUREMENTS:

DEPTH TO WATER: ft.

NOTES:

SAMPLING PERSONNEL

Name (SIGNATURE):

Name (SIGNATURE):

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Example of Sample Labeling

Client Southwest Michigan Planning Commission Sample ID BC-01 (I) Location Black River Analysis E. coli Preservative Yes Collection Date/Time 07/05/12 10:35 am Collected By Joe Sample

06/12/2012 ● DRAFT 1 U:\PROJECTS\110766\G110766Q-QAPP\QAPP\SECOND_MDEQ_SUBMITALL_2012-05\APPENDIX 4B_EXAMPLE SAMPLE LABEL.DOCX Example of Chain-of-Custody Record

Hope College Water Quality Lab Michael Pikaart, PhD

Colilert Quanti-tray 2000 Method Standard Operating Procedure

1. Principle

The Colilert reagent, based on IDEXX’s Defined Substrate Technology, is used for the detection of coliform and E. coli in water. Colilert uses o-nitrophenyl β-D-galactoside (ONPGal) and 4-methylumbelliferyl-α-D-glucuronate (MUG) as the defined substrate indicators of coliform and E. coli, respectively. ONPGal is hydrolyzed by enzymes common to coliform bacteria and produces a yellow color. MUG, by contrast, is hydrolyzed by an enzyme specific E. coli, to release 4-methylumbelliferone which

exhibits fluorescence under a UV365nm lamp. Thus, a yellow colored culture without fluorescence indicates the presence of coliform, and a yellow colored culture along with fluorescence indicates specific E. coli.

The test will detect the presence of a minimum of one and up to a maximum of 2,419 coliform and/or E. coli cells in 100 mL of water within 24 hours.

2. Equipment 2.1 IDEXX Quanti-Tray Sealer w/ insert 2.2 41 ± 0.5°C incubator 2.3 Long-wave UV Lamp (365-366 nm) 2.4 Nalgene polysulfone dilution bottles marked at 90 and 99 mLs. 3. Supplies

3.1 100 mL sterile bacteriological sample bottles or 100 mL Whirlpak bags. Larger sample bottles may be needed for bacteriological duplicates. 3.2 Colilert reagent 3.3 IDEXX Quanti-Tray 2000 3.4 10 mL pipets - sterile, disposable 3.5 50 mL pipets - sterile, disposable 3.6 90 mL sterile dilution blanks (DDI water) 3.7 50 mL sterile dilution blanks (DDI water)

4. Sample Handling

Enviromental samples must arrive at the laboratory within 6 hours of collection. They should be delivered in a Styrofoam cooler containing ice or ice substitute. Samples that arrive after the 6 hour time limit will not be analyzed. Samples must be processed within 2 hours of arriving at the laboratory. Hope College Water Quality Lab Michael Pikaart, PhD

5. Analytical Methodology for freshwater samples with coliform/E. coli values <2400 MPN/100 mL and relatively free from turbidity, color, and/or solids.

A single standard test volume of 100 mL may be used for water which is determined through experience to be relatively free of bacterial contamination as well as turbidity, color, and solids.

5.1 Turn on Quanti-Tray Sealer 10-15 minutes before starting analysis. The sealer is ready to use when the green light comes on. 5.2 Record time sample is inoculated on the worksheet. 5.3 Shake sample 25 times in 7 seconds in 1 ft. arc. 5.4 Aseptically pour off excess to a volume of 100 mL. 5.5 Add colilert reagent. The reagent has a light yellow color in water. 5.6 Shake well to dissolve reagent. 5.7 Place the rubber insert on the input shelf with the large cutout facing away from the Sealer 5.8 Confirm that reagent has dissolved completely. If not, shake bottle again. 5.9 Carefully pour the entire contents of the sample into a sterile Quanti-Tray 2000. 5.10 Lightly tap the small wells to release trapped air. 5.11 Put the Quanti-Tray 2000 onto the rubber insert, well side down, with the open end facing away from the sealer, making sure that the tray is properly seated in the rubber insert. 5.12 Slide the Quanti-Tray into the sealer until the motor begins to draw it in. 5.13 Remove the Quanti-Tray from the back of the sealer. 5.14 Label the Quanti-Tray with the sample number and dilution factor. 5.15 Place the Quant -Tray in the incubator, well side down, within 30 minutes of adding reagent. 5.16 Incubate for 24 hours 41 ± 0.5°C.* 5.17 Record date and time the sample will be read on the incubator door. 5.18 Turn sealer off when not in use.

6. Analytical Methodology for freshwater samples with coliform/E. coli values <2,400 MPN/100mL which contain interfering amounts of color, turbidity, and/or solids.

A single standard test volume of 50 mL diluted with 50 mL sterile DDI water to a 100 mL total volume is used when samples are relatively free from bacterial contamination but contain interfering substances which could mask fluorescence. Adjust this volume according to the history of the each sample site and information obtained from the sampler about possible sewage contamination. Sample dilutions must be used within 20 minutes.

6.1 Turn on Quanti-Tray Sealer 10-15 minutes before starting analysis. The sealer is ready to use when the green light comes on. 6.2 Record time sample is inoculated on the worksheet. 6.3 Shake sample 25 times in 7 seconds in 1 ft. arc. 6.4 Dilute samples as follows: Hope College Water Quality Lab Michael Pikaart, PhD

1 to 2 dilution Pipet 50 mL of sample into a 50 mL sterile dilution blank for a total volume of 100mL.

6.5 Add colilert reagent. The reagent has a light yellow color in water. 6.6 Shake well to dissolve reagent. 6.7 Place the rubber insert on the input shelf with the large cutout facing away from the Sealer. 6.8 Confirm that reagent has dissolved completely. If not, shake bottle again. 6.9 Carefully pour the entire contents of the dilution blank into a sterile Quanti- Tray 2000. 6.10 Lightly tap the small wells to release trapped air. 6.11 Put the Quanti-Tray 2000 onto the rubber insert, well side down, with the open end facing away from the sealer, making sure that the tray is properly seated in the rubber insert. 6.12 Slide the Quanti-Tray into the sealer until the motor begins to draw it in. 6.13 Remove the Quanti-Tray from the back of the sealer. 6.14 Label the Quanti-Tray with the sample number and dilution factor. 6.15 Place the Quanti-Tray in the incubator, well side down, within 30 minutes of adding reagent. 6.16 Incubate for 24 hours @ 41 ± 0.5°C.* 6.17 Record date and time the sample will be read on the incubator door. 6.18 Turn sealer off when not in use.

7. Analytical Methodology for marine samples and and freshwater samples with coliform/E. coli values between 2,400 and 50,000 MPN/100mL

The single standard test volume is 10 mL diluted with 90 mL sterile DDI water to a 100 mL total volume. Adjust this volume according to the history of the each sample site and information obtained about possible bacterial contamination and other interferences. Sample dilutions must be used within 20 minutes.

7.1 Turn on Quanti-Tray Sealer 10-15 minutes before starting analysis. The sealer is ready to use when the green light comes on. 7.2 Record time sample is inoculated on the worksheet. 7.3 Shake sample 25 times in 7 seconds in 1 ft. arc.

7.4 Dilute samples as follows:

1 to 10 dilution Pipet 10 mL of sample into a 90 ml sterile dilution blank for a total volume of 100mL.

7. 5 Add Enterolert reagent. The reagent has a light yellow color in water 7.6 Shake well to dissolve reagent. 7.7 Place the rubber insert on the input shelf with the large cutout facing away from the Sealer 7.8 Confirm that reagent has dissolved completely. If not, shake bottle again. Hope College Water Quality Lab Michael Pikaart, PhD

7.9 Carefully pour the entire contents of the dilution blank into a sterile Quanti- Tray 2000. 7.10 Lightly tap the small wells to release trapped air. 7.11 Put the Quanti-Tray 2000 onto the rubber insert, well side down, with the open end facing away from the sealer, making sure that the tray is properly seated in the rubber insert. 7.12 Slide the Quanti-Tray into the sealer until the motor begins to draw it in. 7.13 Remove the Quanti-Tray from the back of the sealer. 7.14 Label the Quanti-Tray with the sample number and dilution factor. 7.15 Place the Quanti-Tray in the incubator, well side down, within 30 minutes of adding reagent. 7.16 Incubate for 24 hours @ 41 ± 0.5°C.* 7.17 Record date and time the sample will be read on the incubator door. 7.18 Turn sealer off when not in use.

8. Interpretation

Do not read samples before 24 hours. Samples can be incubated up to 28 hours. After 28hours, lack of fluorescence is a VALID NEGATIVE TEST and can be reported as such. Fluorescence after 28 hours is an INVALID result and should not be reported.

Note: Use a Colillert negative (sterility) control with each sample read. This control tray should be stored in the dark between uses.

8.1 Detect fluorescence using the IDEXX viewing box and UV Lamp. Any fluorescence is positive for E. coli. 8.2 Mark each + well with a black marker. 8.3 Ask a supervisor to check each tray for confirmation of fluorescence, if possible.

9. Recording

Count the number of positive large wells and small wells and record on the worksheet next to the correct dilution.

10. Data Reduction 10.1 Calculate the MPN (for each dilution) using the IDEXX MPN tables or the Quanti-Tray 2000 computer program. 10.2 Multiply the result by the dilution factor. 11. Reporting Rules 11.1 Use a tray that has between 30 and 80 positive (fluorescing) wells (small + large) if available. 11.2 If 2 trays fall in the 30-80 range, use the average of the extended counts. 11.3 If no trays fall in the 30-80 range, use the average of the extended counts. Hope College Water Quality Lab Michael Pikaart, PhD

11.4 If both trays are >2420, use the higher dilution. 11.5 If both trays are <0.99, use the lower dilution. 11.6 If only one tray is < or >, use the other tray. 11.7 Round the result to 2 significant figures. 11.8 All calculations should be audited by a supervisor before distribution of reports. 11.9 Report results as E. coli MPN/100 mL 12. Analytical Quality Control

12.1 For each lot of medium, check productivity and accuracy by testing known positive and negative control cultures.

12.2 Perform bacteriological duplicate analyses on 10% of samples tested and at least once per run.

13. Discard of Positive Samples

All Quanti-Trays that have any positive wells (yellow in color or fluorescing) must be sterilized in a 30 minute autoclave cycle and disposed of in an appropriate manner.

ANALYSIS OF SURFACE WATER FOR E. COLI

GREAT LAKES SCIENTIFIC, INC. 2847 LAWRENCE STREET STEVENSVILLE, MI 49127

UPDATED SEPTEMBER 2009

E.COLI SURFACE WATER - 1

ANALYSIS OF SURFACE WATER FOR E. COLI

Table of Contents

Contents Page #

Rules for Lab Custody Procedure ...... E.Coli Surface Water -3 Water Analysis Request Form...... Collection\Sample H2O-8 Water Analysis Record...... Collection\Sample H2O-11 Laboratory Guidelines Receipt of Water Samples from Customer...... Collection\Sample H2O-12 Transfer of Sample to Lab...... Collection\Sample H2O-12 Water Sample Laboratory Write-up...... Collection\Sample H2O-13 Materials and Equipment...... E.Coli Surface Water-4,5 Test Procedure...... E.Coli Surface Water-5

E. COLI SURFACE WATER -2 SEPT 2009 ANALYSIS OF SURFACE WATER FOR E.COLI

Rules for Lab Custody Procedures

The laboratory shall designate a "sample custodian" and an alternate to act in his absence. In addition, the laboratory shall set aside as a "sample storage area," an isolated space with sufficient refrigerator space, which can contain the samples.

Samples should be handled by the minimum possible number of persons.

Incoming samples shall be received only by a custodian, who will indicate receipt by signing the Chain of Custody Record Sheet accompanying the samples and retaining the sheet as a permanent record. Couriers picking up samples shall sign jointly with the laboratory custodian. Immediately upon receipt, the custodian places samples in the refrigerator, except when samples are removed or replaced by the custodian. To the maximum extent possible, only the custodian should be permitted to handle the samples.

The custodian shall ensure that microbiological and nitrate samples are properly stored and maintained at 1-4C. Nitrate samples will be acidified to

Only the custodian will distribute samples to personnel who are to perform tests.

Samples of surface water for E.Coli will be refrigerated upon receipt and analyzed within two hours.

The analyst records information in his laboratory notebook or analytical worksheet, describing the sample, the procedures performed and the results of the testing. The notes shall be dated and indicate who performed the tests. The notes shall be retained as a permanent record in the laboratory and should include any abnormalities which occurred during the testing procedure.

Once the sample testing is completed, microbiological samples can be discarded but identifying tags and laboratory record should be returned to the custodian. Other documentation of work will also be given to the custodian.

Sample Analysis Request Sheet: A sample Analysis Request Sheet should accompany each sample to the laboratory. Samples collectors and lab personnel will complete the appropriate portions.

E. COLI SURFACE WATER -3 SEPT 2009

Great 2847 Lawrence St P.O. Box 166 Lakes Stevensville, Michigan 49127 (269) 429-1000 (269) 429-1550 (FAX) Scientific, Inc e-mail: [email protected] www.glslab.com

______WATER ANALYSIS REQUEST FORM CUSTOMER: Name______

Address______

City, State, Zip______

Phone # ______Fax # ______

LOCATION OF WATER SOURCE: (if different than above)

Name______

Address______

City, State, Zip______

Date Collected: ______Time Collected: ______am pm

Collected by:______

Type of Sample:______(DW=Drinking Water, SP=Swimming Pool, Lake, Stream, etc.)

Location:______(kitchen, basement, outside tap, etc.)

()Tests to perform: Coliform ___ Lead ______E. coli ___ Copper ______Nitrate ___ Other ______

Were "Sample Collection Instructions" (previous page) followed? __ Yes __ No

Was bottle filled to the "EPA Fill Line"? __ Yes __ No Was sample held refrigerated after collection and transported in an iced cooler? __ Yes __ No If No, how did collection differ? ______

COLLECTION\SAMPLEH2O-8 SEPT 2009 WATER ANALYSIS RECORD Date ______Lab Sample # ______

Customer Name ______

Tests to be Performed: Coliform ______E.coli ______Nitrate ______Other ______

Lead ______Copper ______Other ______

Tests Initials Performed Date by Method Result Coliform E. Coli Coliform/E.coli Colilert Per 100ml Per 100ml Aquacheck C# ppm Nitrate ISE pH = ppm

Lead AA ppb

Copper AA ppb

CHAIN-OF-CUSTODY RECORD

Collector to Laboratory Transfer: Date Time Signature Location (if other than lab)

______

Transport to lab in iced cooler? ______Yes ______No

Receipt of Sample at Laboratory and Transfers: Date Time Signature Seal Reason for Condition Transfer

______

______

______

______

Sample Condition and Temp. C Time am pm

Clear Cloudy Particles Yellow Other ______Location of Sample Storage and Temp: Fridge #4 ______C # of Bottles rec'd: ______Sample Preservation Nitrates: 0.2 ml/100 ml addition of sulfuric acid G# Date Time ______By ______Approved by: Date: ______COLLECTION\SAMPLEH2O-11 SEPT 2009 LABORATORY GUIDELINES

RECEIPT OF WATER SAMPLES FROM CUSTOMER

1.) If customer has a Great Lakes Scientific Water Analysis Request Form: a.) Obtain Chain-of-Custody form and complete section "Collector to Lab Transfer" for Date, Time, Location = Great Lakes Scientific, and have them sign at Collector to Lab Transfer b.) Collect money for testing: $26.00 Coliform/E.coli/Nitrates - 1 week for results. $32.00 Coliform/E.coli/Nitrates - 48 hours for results.

2.) If customer does not have a Great Lakes Scientific Water Analysis Request Form, obtain one and complete with the customer as much information as possible: Obtain Chain-of-Custody form and complete as in 1a and 1b above.

3.) Water Analysis Request form will serve as packing slip for #1 and #2.

4.) Log in samples and assign a lab number as in sample collection p.13. Arrange samples from furthest upstream to downsteam for river and stream samples and from most northerly to most southerly for Lake Michigan beach samples

5.) Store samples in refrigerator and analyze within two hours or receipt.

TRANSFER OF SAMPLE TO LAB

1.) Complete Lab Transfer section if received in office, on Chain-of-Custody form: Date, Time, Signature, Seal Condition, Reason= (Office to Lab) 2.) Give samples to Lab Custodian.

COLLECTION\SAMPLEH2O-12 SEPT 2009

WATER SAMPLE LABORATORY WRITE-UP

1.) Take temperature of Nitrate (N) sample and record on Chain-of-Custody Form at Sample Condition and Temp...Time.

2.) Record on customer sample log and assign a lab # to each sample location. If being tested for Coliform, E.coli, and Nitrates and 2 bottles were received, assign them: #Col and #Nit. (Same number for both). Label bottles and boxes - use tape on bottles & box for #. Place in refrigerator. Do Not let water sit out more than 10 minutes.

3.) Complete the following forms (1 set for each sample): a.) Water Analysis Record Date, Lab # for sample, Customer Name - Tests to Perform - Coliform, E.coli, Nitrate, etc. Remainder of this page is completed by technicians. b.) Chain of Custody Record Collector's Name - If brought in, indicate, or name of collector from Water Analysis Request Form. Collector to Lab Transfer - Complete only if we collect sample. Receipt of Sample at Lab & Transfer - complete date, time, sign, seal condition or "none", Transfer - "Lab Receipt". Sample Condition - temperature of sample, if cloudy or unusual. Location of Storage - Refrig. # ____ Temperature - Temperature of refrigerator as recorded a.m. of that day.

4.) Xerox any forms received with sample and complete as a packing slip with date received, condition, how received, etc. Do not write on original forms.

5.) Staple forms together with Water Analysis Record on top. Place samples and all forms in refrigerator #4.

6.) List Nitrates, Lead, and Copper on chemistry board and Coliform/E.coli on micro board (in Refrigerator section of board). Place packing slip on lab bench to be checked by micro technician.

COLLECTION\SAMPLEH2O-13 SEPT 2009

E. coli in Surface Waters

Materials and Equipment 1.) M-Tec Agar (Difco)

Proteose peptone...... 5.0 g Yeast extract...... 3.0 g Lactose...... 10.0 g Sodium chloride, NaCl...... 7.5 g Dipotassium phosphate, K2HPO4...... 3.3 g Monopotassium phosphate KH2PO4...... 1.0 g Sodium lauryl sulfate...... 0.2 g Sodium desoxycholate...... 0.1 g Bromcresol purple...... 0.08 g Bromphenol red...... 0.08 g Agar...... 15.0 g Reagent-grade water...... 1 L

Sterlize by autoclaving: pH should be 7.3 ± 0.2. Pour 4 to 5 mL liquefied agar into culture dishes (15 mm x 100 mm). Store in refrigerator.

2.) Urea substrate

Urea...... 2.0 g Phenol red...... 10 mg Reagent-grade water...... 100 mL

Adjust pH to between 3 and 4. Store at 2 to 8°C. Use within 1 week.

3.) Membrane Pads - Qualitative 417 paper 5.5 cm VWR 28297-868 or equivalent.

4,) Membrane Filters - Osmonics/MSI Microsep mixed ester cellulose membrane 47mm 0.22µm pore size Fisher Scientific Cat # E02WPO4700.

5.) Filter Holding Assembly - Glass microanalysis vacuum filter holder 47mm, 300 ml funnel Fisher Scientific 09-753-102 or equivalent. Holder assembly will be autoclaved before each day's use. Before reuse assembly may be dipped in boiling water for two minutes and allow to cool before reuse.

6.) Vacuum manifold or 1 liter flask with a side arm for mounting filter holder assembly.

7.) Electric vacuum pump to attach to vacuum manifold or 1 liter flask. Place a trap between pump and manifold or 1 liter flask of 500 ml size or greater.

8.) Sterile pipets, cylinder, and 50 ml tubes.

9.) Forceps - Smooth flat membrane forceps. Sterilize by dipping in 95% ethyl alcohol and flaming.

E.COLI SURFACE WATER - 4 SEPT 2009

Materials and Equipment Con't

10.) Petri dishes - 100mm x 15mm.

11.) Fluorescent lamp with magnifying (10x) lens.

Test Procedure

1.) Set up filter holder assemblies on vacuum manifold attached to vacuum pump with a trap before the pump.

2.) Filter 100ml, 50ml, 10ml, or 1 ml sample through membrane filter. If no recent experience indicates volume to use then follow Table 9222:1 in Standard Methods for Water and Wastewater. Lake Michigan beach samples generally require 50 ml and 10 ml. St.Joseph River samples generally require 50 ml, 10 ml and 1 ml. Other inland lakes generally require 50 ml and 10 ml.

3.) Place membrane on mTec agar plate and incubate at 35 ± 0.5°C for 2 hours to rejuvenate injured or stressed bacteria.

4.) Move to 44.5 ± 0.2°C incubator for the next 22 hours.

5.) After incubation place a filter pad in a petri dish and saturate with urea substrate. Place filters from mTec agar onto urea pad for 15 minutes.

6.) Count under fluorcescent light with magnifier any yellow or yellow brown colonies. Verify as E.coli a portion of the selected colonies.

7.) Report results per 100 ml.

E. COLI SURFACE WATER -5 SEPT 2009

HOPE COLLEGE

Standard Operating Procedures for Microbial Source Tracking Hope College Water Quality Lab

Michael Pikaart, PhD, Associate Professor of Chemistry and Biochemistry, Hope College 5/30/2012

This document presents SOPs for collection of environmental samples, enumeration of enteric bacteria by membrane filtration, isolation of DNA for genomic analysis, and PCR protocols for source tracking.

Hope College Water Quality Lab Michael Pikaart, PhD

Table of contents:

Section Procedure Page I Sample collection for culture and source tracking analysis 1 II Membrane filtration for enumeration of Escherichia coli and 7 enterococci in water samples III Collection of suspended bacteria for DNA isolation and source tracking 13 IV DNA Extraction 17 V General Bacteroides PCR Assay 20 VI HF183 (human specific 16S Bacteroides) PCR Assay 22 VII CF128 (cow specific 16S Bacteroides) PCR Assay 25

1

I. Sample collection for culture and source tracking analysis

Hannah Reynolds, 7/21/2011

1. Purpose and Scope

This Standard Operating Procedure (SOP) is for sampling event preparation. It includes a comprehensive list of the tasks that must be completed before each sampling event, and descriptions explaining how to accomplish these tasks. The purpose of these tasks is to fully prepare for an environmental water sampling event (to analyze the water samples for fecal contamination).

2. Recommended Quality Control Procedures

1. Users must wear gloves and avoid direct hand contact with sterilized equipment, reagents, and materials to avoid contamination. 2. All work surfaces should be sterilized frequently using a spray bottle filled with 70% ethanol.

3. Equipment and Reagents

• Three 1 L plastic autoclavable screw-top sample bottles per sampling site • Autoclave sterilization tape • Autoclave • P 1000 calibrated micro-pipettes • 1000 μL general use micro-pipette tips • Vacuum Pump(s) • Household Bleach • 5% sodium thiosulfate solution • 3 five-gallon buckets • Reverse osmosis or distilled water • Large water bath • Filter Housings • Incubator (37ºC)

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Hope College Water Quality Lab Michael Pikaart, PhD

• Incubator (41ºC) • Incubator/shaker (37ºC) • Food vacuum sealer and vacuum sealer bags • Modified mTEC media (LOT # 1110977) o 1 M NaOH • mEI media (LOT # 1094514) o 2,3,5-Triphenyltetrazolium o 0.48 g/mL Nalidixic acid • Sterile Petri-plates • TSB media • -80ºC freezer stocks of E. coli and Enterococcus fecalis cultures • Aluminum Foil

• Monopotassium phosphate (KH2PO4)

• Anhydrous MgCl2 or magnesium chloride hexahydrate (MgCl2 •6H2O) • 1 L glass screw-top bottles • Forceps • Lighters and or matches • Ethanol burners • 70% ethanol • Pure ethanol • Large spray bottles • 50 mL beakers • 1000 mL waste beakers • Sterile membrane filters w/ absorbent pads, cellulose nitrate, pore size- 0.45 µm, 47 mm. diameter (Whatman- VWR International cat. no.-7141 104) • Nalgene analytical test filter funnels, sterile (Thermo Scientific-VWR International cat. no.- 28198- 861) • Nalgene Wash Bottles, Polypropylene Copolymer, Narrow Mouth, 500 mL (Thermo Scientific- VWR International cat. no. 16651-904) • pH strips • Portable calibrated turbidity meter • Portable calibrated pH and temperature meter • Large coolers and ice packs to keep samples cold • Cold Room (3°C) • Sterile 50 mL screw-top centrifuge tubes • DNA filtration housings • Battery operated rechargeable pipetters • 5 mL, 10 mL, 25 mL, and 50 mL glass pipettes • 1.5 mL pop-top autoclavable centrifuge vials • 2 mL screw-top autoclavable cryogenic vials • Label maker and cryogenic labels • Fine-tip and regular-tip sharpies (black, red, and blue)

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Hope College Water Quality Lab Michael Pikaart, PhD

4. Procedure

Reagent Preparation

To prepare stock monopotassium phosphate (KH2PO4) solution, dissolve 34.0g of monopotassium phosphate (KH2PO4) in 500 mL reagent-grade water. Adjust the pH of the solution to 7.2 with 1 M NaOH, and bring the volume to 1 L with reagent-grade water. Sterilize by autoclave at 121°C (15 PSI) for twenty minutes (liquid 20).

To prepare stock magnesium chloride (MgCl2) solution add 38 g anhydrous MgCl2 or 81.1 g magnesium chloride hexahydrate (MgCl2 •6H2O) to 1 L reagent-grade water. Sterilize by autoclave at 121°C (15 PSI) for twenty minutes (liquid 20).

After sterilization, store the stock solutions in the refrigerator until needed. Handle aseptically. If evidence of mold or other contamination appears, the affected stock solution should be discarded and a fresh solution should be prepared.

PBW Preparation

The appropriate amount of phosphate buffered water (PBW) should be prepared before each sampling event. About 1 L of PBW should be prepared for every three sampling sites.

To prepare PBW 1.25 mL of stock phosphate buffer and 5 mL of MgCl2 should be mixed with each liter of RO water. To mix the solution, place parafilm over the top of the container containing the PBW and invert the container several times.

Continue with autoclave directions for PBW.

Media Preparation

3

Hope College Water Quality Lab Michael Pikaart, PhD

Prepare both mTEC and mEI agar according to the directions on the bottles. Prepare the media in large Erlenmeyer flasks. Allow for 4 mL of media per plate, and prepare the appropriate amount of media according to the desired number of plates. Once prepared, sterilize the media according to the directions in the autoclave section. Once sterilized, keep the flasks of media in the water-bath set at 55°C. Add the additives indicated on the bottles and mix by stirring. Pour 4 mL of media into each sterile plate (make sure bottom of plate is covered). Allow the media to solidify well before packing the plates and storing them in the refrigerator. Note: mTEC media is sensitive to light and should be wrapped in aluminum foil and kept out of sunlight.

Prepare 200 mL TSB media according to package directions. Prepare the media in large flask and autoclave on the liquid 20 setting. This media will last for three months once sterilized and refrigerated. It will be used for positive culture preparation.

Preparation of Positive Cultures

Pipette out 5 mL of TSB into two 15 mL screw-top centrifuge tubes. Take the freezer stocks of E. coli and Enterococcus fecalis cultures from the -80°C freezer and allow them to thaw for about ten minutes. Pipette out 100 µL of each positive stock culture into the appropriately labeled tubes. Allow the tubes to incubate overnight at 37°C.

Sterilization

Three large spray bottles should be filled with 70% ethanol. All work surfaces should be sprayed with the ethanol and wiped down.

Bleach and sodium thiosulfate solutions should be prepared for sterilization of non-autoclavable equipment. A 10% bleach solution should be prepared for sterilization of equipment; to prepare bleach solution mix 1.5 L household bleach with 13.5 L tap water in a large 5 gallon bucket. 0.1% sodium thiosulfate should be prepared to neutralize the bleach on the equipment; 300 mL 5% sodium thiosulfate should be added to 14.7 L RO water in a 5 gallon bucket. Note that

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Hope College Water Quality Lab Michael Pikaart, PhD

prepared bleach and sodium thiosulfate solutions are effective for 24-hours before new solutions must be made.

Autoclave Sterilization

There is an autoclave located in the laboratory preparation room on the third floor next to the advanced biology lab, and another autoclave located in the preparation room adjoined to the microbiology laboratory on the third floor. Open the door to the autoclave when it is not in use or pressurized, and place the tray of materials to be autoclaved (marked with autoclave tape) inside using heat protection gloves. Close the door to the autoclave and make sure the door is sealed tightly. Choose the appropriate autoclave setting, and wait for the autoclave to complete all cycles (the digital readout will say complete when the autoclave is finished). Check to make sure the pressure inside the autoclave is reading zero psi on the digital display. Carefully and slowly open the door to the autoclave (there will be hot steam) using heat protection gloves. Leave these gloves on, and remove the tray from the autoclave; set the tray with autoclaved materials on the bench-top to cool. Note that lids to glass bottles may be tightened immediately after removal from the autoclave, but plastic bottles should be allowed to cool fully before their lids are tightened (all lids should be loose while in the autoclave).

• 3 sample bottles per sampling site should be autoclaved. To autoclave bottles loosen the caps all the way and place autoclave tape on the lid of each bottle. Place the bottles to be autoclaved on an autoclave tray. Autoclave bottles on the preset gravity setting for twenty minutes (nicknamed gravity 20). • To autoclave media, distribute the prepared media into separate glass 1000 mL Erlenmeyer flasks with no more than 400 mL of media per flask to prevent boiling over. Cover the top of each flask with aluminum foil, poke a small hole in the foil, mark with autoclave tape, and label each with sharpie. Place the flasks on an autoclave tray and load into the autoclave. Autoclave the media on the preset liquid setting for twenty minutes (nicknamed liquid 20). • Micro-pipette tips should be autoclaved before use. Fill autoclavable pipette-tip boxes with the appropriate tips (found in the large chemical stock room on the first floor, a CK key will be needed to get in). Close lids of boxes and place strip of autoclave tape from the top of the box to the bottom of the box so the box could not be opened without noticing whether or not the box has been autoclaved. Place the boxes on an autoclave tray and autoclave on the gravity 20 setting. • Glass pipettes should be placed in a metal glass pipette storage tube before autoclaving to avoid recontamination once they are removed from the autoclave. Autoclave tape should be placed on the tube, and the pipettes should be autoclaved on the gravity 20 setting.

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Hope College Water Quality Lab Michael Pikaart, PhD

• Cryogenic vials should be capped and placed into a larger screw-top autoclavable bottle (possibly an extra sampling bottle). The large bottle should be labeled and marked with autoclave tape. The vials should be autoclaved on the gravity 20 setting. • 1.5 mL centrifuge vials should also be placed in a larger bottle. The bottle should be labeled and marked with tape. The vials should be autoclaved on the gravity 20 setting. • Prepared PBW may be distributed into labeled glass bottles (with caps) no more than 2/3 full. The bottles of PBW should have the lids loosened all the way and marked with autoclave tape; they should be autoclaved on the liquid 20 setting. • The DNA filtration equipment must be autoclaved before each sampling event. The two parts of the filter housing should be placed together (frosted glass ends together) and clipped with the metal clamp that goes with the housing. The tapered end of the piece with the stopper should be covered in aluminum foil, and the non-clamped end of the other piece should be covered with foil as well. This helps prevent recontamination once the filter-housing has been removed from the autoclave. The filter-housings should be placed in the autoclave on trays very carefully, and autoclaved on the gravity 20 setting. The filters should be stored in a sterilized area that is not open to the air until use.

Organization and Planning

Plan out sampling sites and come up with labels for the sites.

Pack coolers with labeled sampling bottles (remember to put ice packs in just before leaving to sample). Make sure pH meter and turbidity meter are calibrated. Pack distilled water for rinsing, alcohol for sanitizing, KIM wipes, pH meter, turbidity meter, several 50 mL centrifuge tubes, a sharpie marker, a blue or black pen, a data sheet for measurements, and a sampling stick or device.

Before sampling turn on 37°C incubator, 41°C incubator, and water-bath (set to 45°C). Make sure there is space in the cold room for sample storage after samples have been collected.

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II. Membrane filtration for enumeration of Escherichia coli and enterococci in water samples

07/05/2011

Eric Hydorn

Scope & Application

This method is a revised Escherichia coli and Enterococci membrane filtration procedure based on the EPA methods cited above used for the detection and enumeration of Escherichia coli and enterococci colonies. Enterococci and E. coli are commonly found in the intestinal tract/feces of humans and other warm-blooded animals. Detection of such colonies is an indication of fecal pollution.

Summary of Method

A specified amount of water sample (1 mL, 10 mL) is filtered through a 47 mm diameter (pore size: 0.45 µl) membrane filter, which retains the bacteria from the water sample. The membrane filter is then placed on mTEC and mEI agar plates. mTEC agar is chosen to detect for Echerichia coli colonies and mEI agar is chosen to detect enterococci coloies. The mTEC plates are incubated at 35oC for 2 hours and then incubated in a 44.5oC water bath for 16 - 22 hours. Bacterial colonies are checked for red magenta color in order to be positive for Escherichia coli. The mEI plates are incubated at 41oC for 18 - 24 hours. All colonies (regardless of color) with a blue halo are recorded as enterococci colonies.

Recommended Quality Control

• Always wear gloves when handling any equipment, reagent bottles, Petri dishes, etc.

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Hope College Water Quality Lab Michael Pikaart, PhD

• Make sure all surfaces/instruments are wiped down and sterilized with 70% ethanol between different samples • Never touch membrane filters or inside of filter funnels • Pipette water samples into middle of filter funnels not onto the sides • Keep mTEC plates under aluminum foil as much as possible to avoid exposure from sunlight • Make sure PBW bottles are autoclaved • Shake sample bottles thoroughly between every replicate

Safety

• Normal microbiological lab procedures when handling samples, cultures, reagents, materials, and operation of sterilizing equipment must be observed

• Always use gloves when handling water samples, equipment, and plates

• Mouth pipetting is prohibited

Equipment

• Air compressor, vacuum pump

• 1000 mL Büchner flask

• Rubber tubing

• PVC pipe filter housing holder

• Rubber stopper w/ one hole (to fit 1000 mL Büchner flask)

• VWR® 1000 µL pipette tips (cat. no. 83007-376)

• 1000 µL pipette (pipetman)

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Hope College Water Quality Lab Michael Pikaart, PhD

• Electronic pipette

• 10 mL KIMAX Reusable Measuring (Mohr) Pipets, Class A, Color-Coded, Kimble Chase (cat. no. 89003-450)

• 50 mL beaker

• 1000 mL beaker (for waste)

• Alcohol burner

• Matches

• Tweezers

• Sterile membrane filters w/ absorbent pads, cellulose nitrate, pore size- 0.45 µm, 47 mm. diameter (Whatman- VWR International cat. no.-7141 104)

• Nalgene analytical test filter funnels, sterile (Thermo Scientific-VWR International cat. no.- 28198- 861)

• 60 x 15 mm Petri dishes w/ tight fitted lids (pre-filled with mTEC & mEI media- see media preparation procedure)

• Nalgene Wash Bottles, Polypropylene Copolymer, Narrow Mouth, 500 ml (Thermo Scientific- VWR International cat. no. 16651-904)

• Permanent marker for labeling plates

• 2 x large 27.5 L plastic bucket (for bleach and sodium thiosulfate)

Reagents

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Hope College Water Quality Lab Michael Pikaart, PhD

• Phosphate Buffered Water (PBW) DI H2O 4 L K2HPO4 5 mL MgCl2 20 mL

o Autoclave before use

• Bleach (10%) DI H2O 13 L Standard commercial 1.3 L bleach (~6% sodium hypochlorite) i.e. Clorox

• sodium thiosulfate (10%) DI H2O 13 L sodium thosulfate (5%) 1.3 L

• ethanol (70%)

• 200 proof ethanol

Methods

1. Set up a. Prepare bleach solution in a large 27.5 L bucket b. Prepare sodium thiosulfate solution in a large 27.5 L bucket c. Label 60 x 15 mm Petri dishes- media type (mTEC or mEI), sample site I.D., dilution (negative, 1 mL, or 10 mL) d. Load 1000 µL pipette tips into box and autoclave. e. Apparatus i. Attach one end of rubber tubing to PVC pipe filter housing adapter and the other end to the rubber stopper in the 1000 mL Büchner flask. ii. Attach one end of another piece of rubber tubing to the intake spout of the vacuum pump and the other end to the sidearm of the Büchner flask f. Fill alcohol burner with ethanol and fill a 50 mL beaker with 10 mL of alcohol g. Fill Nalgene wash bottle with PBW and autoclave prior to filtering h. Set out (within an arms reach): alcohol burner, matches, 50 mL beaker containing ethanol (tweezers inside beaker), 1000 mL beaker (for waste), Nalgene wash

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Hope College Water Quality Lab Michael Pikaart, PhD

bottle w/ PBW, individually labeled mTEC and mEI petri dishes, box of sterile membrane filters, 1000 µL pipette tips, 1000 µL pipette, 10 mL glass pipettes, electric pipette, and sterile Nalgene analytical test filter funnels.

2. Procedure a. Bulk samples are kept in a cold room while individual samples are picked out and filtered. b. Open sterile Nalgene analytical test filter funnel package and fix the base onto the PVC pipe adapter. Take clear cylinder off and place top down (end that does not touch blue filter funnel base) on the lab bench. c. Dip tweezers in ethanol and pass through alcohol burner flame, wait until flame extinguishes, keep open flame away from ethanol beaker. d. Open sterile membrane filter, carefully without touching filter, and take filter along with white absorbent pad with tweezers. Place grid side up and absorbent pad side down on the blue filter funnel base. Replace clear cylinder (make sure it clicks into place and is secured). Discard filter packaging in 1000 mL beaker waste. Replace tweezers in 50 mL beaker containing the ethanol. i. Negative Control 1. Perform this first before contamination comes in contact with filter funnel 2. Wash down sides of filter funnel with PBW. Fill bottom of filter funnel with PBW so the entire membrane filter is submerged. 3. Vacuum the PBW out of the filter funnel and submerge membrane filter with PBW for the second time. Vacuum PBW out. ii. 1 mL sample 1. Shake sample bottle containing water sample. 2. Wash down sides of filter funnel with PBW. Fill bottom of filter funnel with PBW so the entire membrane filter is submerged. 3. Use 1000 µL pipette and 1000 µL pipette tips to pipette 1 mL of sample water into filter funnel- make sure sample water gets pipetted into center of filter funnel- replace pipette tip between every replicate. 4. Vacuum out water mixture in filter funnel. Wash down sides of filter funnel with PBW to submerge membrane filter and vacuum out contents of filter funnel. iii. 10 mL sample 1. Shake sample bottle containing water sample. 2. Use 10 mL glass pipettes and electric pipette to pipette 10 mL of sample water into filter funnel - make sure to pipette sample water into center of filter funnel. 3. Vacuum out water mixture in filter funnel. Wash down sides of filter funnel with PBW to submerge membrane filter and vacuum out contents of filter funnel. e. Take off clear filter funnel cylinder top and place, top down, (end that does not touch blue filter funnel base) on lab bench.

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Hope College Water Quality Lab Michael Pikaart, PhD

f. Take tweezers from ethanol and pass over alcohol burner flame, wait until flame extinguishes, allow the tweezers to cool briefly and pick up the membrane filter from the blue filter funnel base - take ONLY the membrane filter, NOT the absorbent pad. g. Place filter in corresponding labeled Petri dish. Set dish aside (place mTEC under aluminum foil cover - light sensitive) h. Discard absorbent pad in 1000 mL waste beaker. i. The above procedure was repeated for: 1 replicate of negative, 3 replicates each for 1 mL and 10 mL, for each media type - mTEC and mEI media (14 plates total for each water sample). During events where high colony counts were expected, 10-1 dilutions were prepared for each sample and the procedure for 1 mL sample was followed as above using this diluted sample. This was also performed in triplicates which led to 20 total plates for each water sample. j. When replicates are finished, remove filter funnel base and cylinder and 10 mL pipette. Place in bleach solution bucket for 30 minutes. After the 30 minutes, switch filter funnel and pipette to sodium thiosulfate bucket for 30 minutes.

References

EPA Method 1600: Enterococci in Water by Membrane Filtration Using membrane- Enterococcus Indoxyl-ß-D-Glucoside Agar (mEI) EPA, Office of Water SOP for filtering and plating Enterococci

EPA Method 1603: Escherichia coli (E. coli) in Water by Membrane Filtration Using Modified membrane-Thermotolerant Escherichia coli Agar (Modified mTEC) EPA Office of Water SOP for filtering and plating Escherichia coli

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III. Collection of suspended bacteria for DNA isolation and source tracking

7/22/2011 Angela Aumaugher and Andrea Houg

1. Purpose and Scope This SOP is for processing of a water sample to achieve a sample concentrate for DNA extraction. The purpose is to obtain the bacteria from a larger volume of water in a concentrated volume, from which the DNA can be extracted. Various types of samples have been processed in different ways as detailed in the following procedures.

2. Recommended Quality Control Procedures 1. Lab workers should wear gloves and goggles and avoid contact with the water as it may contain pathogens from fecal contamination.

2. Cross contamination between samples should be avoided. Only one sample should be taken to the designated work area at a time and the bench top and equipment should be wiped down with 70% alcohol between samples. Pipette tips must be changed between each sample. Filtration housing must be disinfected between each sample.

4. After processing, filtrate water should be disinfected and properly disposed of. Water may be poured into a bucket with bleach and left to set for 30 minutes, then dumped down the sink.

5. Care should be exercised when handling the filtration housing as they are expensive and can be unsteady in this set-up.

3. Equipment and Reagents 1 L Erlenmeyer flask

90 mm Filter Holder with metal clamp, Cole-Parmer item # YO-02923-30

90 mm (40μm pore) filters, Fisher Scientific catalog # HAWP09000

forceps

alcohol burner

50 mL centrifuge tubes, 15 mL centrifuge tubes, or 250 mL centrifuge bottles (depending on sample type)

phosphate buffered water (see preparation…)

vortex

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Hope College Water Quality Lab Michael Pikaart, PhD

centrifuge

vacuum pump with associated hoses

5 mL pipette

1 mL pipette tips

2 mL cryogenic vials

label maker

-80ºC freezer

70% alcohol

10% bleach solution

10% sodium thiosulfate solution

4. Procedure: a. Normal Sampling Event 1. Fill a 50 mL centrifuge tube with 45 mL phosphate buffered water. Repeat for each sample to be filtered and label with the sampling event number and site ID. 2. Remove sample from cold room. Pour a small amount of ethanol into a beaker and light the ethanol burner. Insert the stopper of the first piece of the filter holder into the top of a 1 L Erlenmeyer flask. 3. Sterilize forceps by dipping in alcohol and flaming, then use them to place a filter centered on the top surface of the first piece of the filtration apparatus. Place the second piece of the filter holder directly on top such that the two flat surfaces are against each other and the filter is between. Use a metal clamp to hold the complex in place. 4. Mix the sample well. Pour some of the sample into the filtration apparatus and turn on the vacuum pump. If filtration seems to be proceeding at an acceptable rate, pour in the rest of the sample. If filtration does not seem to be proceeding at an acceptable rate because the sample is very turbid, pour in about half of the sample; you will need to repeat the process using a second filter. Between 800- 1000 mL of each sample should be filtered. 5. Once all the water has been filtered, immediately turn off the pump. Remove the clamp, sterilize the forceps, and use forceps and the upper part of the filter holder to fold the filter lengthwise into fourths. Insert filter into the appropriate 50 mL centrifuge tube. 6. Record the volume of water filtered using the markings on the Erlenmeyer flask. Then, return the filtrate to the original sample bottle for subsequent disinfection. 7. Transfer the filtration apparatus to the 10% bleach solution for 30 minutes, followed by the 10% sodium thiosulfate solution for 5 minutes.

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Hope College Water Quality Lab Michael Pikaart, PhD

8. Wipe down the work surface with 70% alcohol and repeat steps 2-6 until all samples are processed. At this point, tubes may be stored in the refrigerator for several days.

9. Vortex each tube well (approximately 5 minutes) so that bacteria detaches from the membranes. 10. Remove membrane with sterile forceps and discard. Repeat with each sample, sterilizing forceps between each. 11. Centrifuge tubes at 3,500 rpm for 20 minutes. 12. Set up vacuum pump apparatus. Attach tubing from a pump to a side-arm Erlenmeyer flask. Attach tubing from flask to a sterile 5 mL pipette. 13. Put a sterile 1 mL pipette tip onto the tip of the 5 mL pipette. Siphon off most of the supernatant without disturbing the pellet, leaving 5 mL in the bottom of the tube. Repeat for each sample, changing pipette tips between each.

14. Obtain 2 mL cryogenic tubes in triplicates for each sample. Make cryo-dot labels including the sample site ID, the sampling event number, the letters SC for sample concentrate, and the letters A, B, or C for each of the triplicates. Label the tubes. 15. Vortex 50 mL sample tube to break up the pellet. Pipette 1.5 mL into cryogenic tubes A, B and C. Repeat for each sample, changing pipette tips between each. 16. Store cryogenic tubes in -80ºC freezer until ready to use.

b. Sewage Spill Event: 1. Using a graduated cylinder, transfer 200 mL of sample water into a 250 mL centrifuge bottle labeled with sampling event and site ID and cap tightly. 2. Centrifuge at 8000 rpm for 20 minutes. 3. Attach a 5 mL pipette to vacuum hose that is attached to side arm flask and vacuum pump. Place 1 mL pipette tip to the end of the pipette. 4. Using vacuum on the pipette, siphon off supernatant to a volume of approximately 5 mL without disturbing the pellet. 5. Attach a 10 mL pipette to a pipette gun. Take up the remaining supernatant in the pipette and wash the pellet from the sides and bottom of the container. Be sure to reconstitute the entire pellet into solution. 6. Using a 1000 μL pipette and associated pipette tips, aliquot the sample concentrate into three 2 mL cryogenic tubes, each labeled with the sampling event and site ID, “sc” for sample concentrate, and A, B, or C to denote which triplicate. Tubes A & B should contain 1.5 mL each of the sample concentrate. Tube C should contain any remaining sample concentrate. 7. Sample concentrate may be stored in a -80°C freezer until needed.

c. Raw Sewage Sample Processing 1. Add 50 mL of raw sewage sample to each of two 50 mL centrifuge tubes.

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Hope College Water Quality Lab Michael Pikaart, PhD

2. Centrifuge at 3000 rpm for 15 minutes. 3. Pour off the supernatant without disturbing the pellet. Add 1.5 mL PBW to the pellet and reconstitute by vortexing. 4. Transfer into a sterile 1.5 mL micro-centrifuge tube labeled with ‘RS’ to denote raw sewage, an ID for the sampling site, the date, and ‘SC’ for sample concentrate.

d. Effluent (treated) Sewage Sample Processing 1. Add 250 mL of effluent sewage sample into each of two 250 mL centrifuge bottles. 2. Centrifuge at 3000 rpm for 15 minutes. 3. Attach a 5 mL pipette to vacuum hose that is attached to side arm flask and vacuum pump. Place 1 mL pipette tip to the end of the pipette. 4. Using vacuum on the pipette, siphon off supernatant to a volume of approximately 1.5 mL without disturbing the pellet. 5. Attach a 10 mL pipette to a pipette gun. Take up the remaining supernatant in the pipette and wash the pellet from the sides and bottom of the container. Be sure to reconstitute the entire pellet into solution. 6. Transfer solution into a 1.5 mL micro-centrifuge tube labeled ‘EF’ for effluent sewage, an ID for the sampling site, the date, and ‘SC’ for sample concentrate.

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Hope College Water Quality Lab Michael Pikaart, PhD

IV. Standard Operating Procedure (SOP) for DNA Extractions

07/21/2011 Eric Hydorn

Scope & Application

The isolation of genomic DNA from bacteria is performed in order to isolate the DNA strand from the rest of the bacteria. This strand of clean DNA is required for amplification processes such as PCR/Gel or QPCR. With this method we are able to break down the rigid cell walls of the bacteria by a series of filtrations and buffers added to the solution resulting in a pure strand of genomic DNA.

Summary of Method

This procedure described the use of Qiagen QIA-amp DNA mini kit for preparing genomic DNA from bacterial cell pellets obtained from environmental sampling.

Recommended Quality Control

• The filtered sample concentrate are in 1.5 mL microcentrifuge tubes, which must be concentrated to 2 µL prior to beginning the extraction. • Pipette tips must be sterilized before use that will fit micro-pipetters of 1000 µl, 100µl and 10µl • Before beginning the procedure, calculations must be made for aliquoting reagents. Aliquot enough reagents for the total number of samples, plus a DNA extraction negative, plus one extra. • Labels must be created for each of the samples and all of the extractions may be stored in a -20°C freezer afterwards.

DNA Extraction from Water Sample Materials

• QIAamp® DNA Mini Kit (Cat.No.-51306, QIAGEN) • Ethanol (96-100%) • Sterile 1.5 mL microcentrifuge tube • Molecular Grade Water • Centrifuge (NAPCO, 2002) • Water bath (ISOTEMP 102S, Fisher Scientific) or heat block (Digital Heatblock, VWR) • Buffers and QIAamp Mini spin columns are found in the QIAamp DNA mini kit. • Fisherbrand- Culture Test tubes (where buffers are placed) Catalog No. 14-956-6B • Filter Tips- TipOne- Beveled Filter Tips • Various Micropipetters • Latex or Nitrile Gloves

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Hope College Water Quality Lab Michael Pikaart, PhD

• Paper Towels • 70% Ethanol (in spray bottle for sterilization) • Sharpie markers for labeling different buffer tubes and microcentrifuge tubes

Procedure:

1. Turn on a water bath to 37°C and 2 dri-baths, one to 95°C and another to 56°C.

2. Pellet bacteria by centrifugation for 3 minutes at 10,000 rpm (1.5 mL of culture).

3. Remove approximately 1.3 mL of supernatant to reach 200 µL of sample concentrate. Prepare an extraction negative of 200 µL of molecular grade water in a separate microcentrifuge tube.

4. Suspend bacterial pellet in 180 µL of Lysis buffer.

5. Vortex the samples to thoroughly mix.

6. Incubate for 30 minutes at 37ºC.

7. Add 20 µL proteinase K and 200 µL Buffer AL. Mix by vortexing.

8. Incubate at 56ºC for 30 minutes and then for a further 15 minutes at 95ºC. (NOTE: EXTENDED INCUBATION AT 95°C CAN LEAD TO DNA DEGREDATION)

9. Briefly centrifuge the 1.5 mL microcentrifuge tube to remove drops from the inside of the lid.

10. Add 200 µL pure ethanol to the sample, and mix by pulse-vortexing for 15 seconds. After mixing, briefly centrifuge the 1.5 mL microcentrifuge tube to remove drops from the inside of the lid.

11. Carefully apply the mixture from step 10 to the QIAamp Mini Spin Column (in a 2 mL collection tube) without wetting the rim. Close the cap, and centrifuge at 6000 x g (8000 rpm) for 1 minute. Place the QIAamp Mini Spin Column in a clean 2 mL collection tube (provided), and discard the tube containing the filtrate.

12. Carefully open the QIAamp Mini Spin Column and add 500 µL Buffer AW1 without wetting the rim. Close the cap, and centrifuge at 8000 rpm for 1 minute. Place the QIAamp Mini Spin Column in a clean 2 mL collection tube (provided), and discard the collection tube containing the filtrate.

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Hope College Water Quality Lab Michael Pikaart, PhD

13. Carefully open the QIAamp Mini Spin Column and add 500 µL Buffer AW2 without wetting the rim. Close the cap and centrifuge at full speed 14,000 rpm for 3 minutes.

14. Recommended: place the QIAamp Mini Spin Column in a new 2 mL collection tube (not provided) and discard the old collection tube with the filtrate. Centrifuge at full speed for 1 minute.

15. Place the QIAamp Mini Spin Column in a clean 1.5 mL microcentrifuge tube (not provided), and discard the collection tube containing the filtrate. Carefully open the QIAamp Mini Spin Column and add 100 µL Buffer AE or distilled water. Incubate at room temperature for 1 minute, and then centrifuge at 8000 rpm for 1 minute.

16. Repeat step 15.

17. Place in -20°C freezer for storage.

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Hope College Water Quality Lab Michael Pikaart, PhD

Standard Operating Procedure (SOP) for general Bacteroides PCR Assay

7/28/2011 Sangeetha Srinivasan, PhD

References:

Bernhard, A. E., and K. G. Field. 2000. A PCR assay to discriminate human and ruminant feces on the basis of host differences in Bacteroides-Prevotella genes encoding 16S rRNA. Appl. Environ. Microbiol. 66:4571-4574.

CFX96 and CFX 384 Real-Time PCR Detection Sysytems Instrument Operator’s manual.

1. Purpose and Scope

This Standard Operating Procedure (SOP) is for conventional PCR assay targeting general Bacteroides 16S rRNA gene. The total amplicon length for the target is 676bp. This SOP is intended to be used for BioRad CFX96™ Real-Time System. The purpose of this qPCR assay is to determine the presence or absence of contamination from any fecal sources in water or other environmental samples

2. Recommended Quality Control Procedures:

1. Users must wear gloves at all times and avoid direct hand contact with PCR reagents to avoid contamination. 2. Use separate designated spaces for reagent preparation and sample addition. Do not move the pipet tips, microtube racks, microtubes, pipettes and other supplies from one space to another. 3. Routinely wipe the hood/bench surfaces with 70% alcohol and UV-disinfect for 15 minutes before and after use. 4. Store reagents in appropriate conditions as per the manufacturer’s product manual.

3. Equipments and Reagents

1) BioRad CFX96™ Real-Time System 2) Primers (reconstituted in DNase free water to100uM and stored in aliquots of 10uM in the -20°C freezer): a. Forward Primer: Bac32F 5’AACGCTAGCTACAGGCTT3’ b. Reverse Primer: Bac708R 5’ CAA TCG GAG TTC TTC GTG 3’ 3) Qiagen HotStarTaq Mastermix kit-1000 units (A premixed solution containing HotStarTaq DNA Polymerase, PCR Buffer, and dNTPs. The solution provides a final concentration of 1.5 mM MgCl2 and 200 µM each dNTP) 4) DNase-free water 5) TempAssure PCR 0.1 ml 8-strips, flat caps, natural (USA Scientific # 1402-3700) 6) Barrier (filter) pipette tips

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Hope College Water Quality Lab Michael Pikaart, PhD

7) 1.5 ml RNase/DNase-free microcentrifuge tubes 8) Mini centrifuge for PCR tubes

4. Procedure

1) Thaw all the reagents and DNA samples

2) Prepare a super mix for n+4 reactions (n = total samples) in a 1.5 mL centrifuge tube at the PCR Prep Station as given below.

HotStar Taq Master Mix - 10 µL Primer (forward) (10µM) - 1µL Primer (reverse) (10µM) - 1µL Molecular Grade water - 3 µL

3) Mix the reagents well by vortexing gently and centrifuge briefly.

4) Aliquot 15 microlitre of the mastermix into the PCR tubes.

5) Move to the sample addition station and add 5 microlitre of the sample DNA in duplicates, positive control DNA from raw sewage (aliquoted from stock previously amplified and sequenced to confirm the presence of maker) and molecular grade water as negative controls, both in triplicates.

6) Briefly centrifuge the tubes.

7) Set the protocol in the Real-Time System using the cycling program as follows

1 95.0 C for 15:00 2 94.0 C for 0:30 3 53.0 C for 0:30 4 72.0 C for 1:00 5 GOTO 2, 39 more times 6 72.0 C for 8:00 END

8) Check the PCR product by agarose gel (1.2%, w/v) electrophoresis (95 V for ~1 hrs). The product size for the CF128 assay is 676 bp.

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Hope College Water Quality Lab Michael Pikaart, PhD

Standard Operating Procedure (SOP) for HF183 (human specific 16S Bacteroides) PCR Assay

7/28/2011 Sangeetha Srinivasan, PhD

References:

Bernhard, A. E., and K. G. Field. 2000. A PCR assay to discriminate human and ruminant feces on the basis of host differences in Bacteroides-Prevotella genes encoding 16S rRNA. Appl. Environ. Microbiol. 66:4571-4574.

CFX96 and CFX 384 Real-Time PCR Detection Sysytems Instrument Operator’s manual.

1. Purpose and Scope

This Standard Operating Procedure (SOP) is for conventional PCR assay, nicknamed as HF183, targeting human specific Bacteroides 16S rRNA gene. The total amplicon length for the target is 525bp. This SOP is intended to be used for BioRad CFX96™ Real-Time System. The purpose of this qPCR assay is to determine the presence or absence of contamination from human fecal sources in water or other environmental samples

2. Recommended Quality Control Procedures:

1. Users must wear gloves at all times and avoid direct hand contact with PCR reagents to avoid contamination. 2. Use separate designated spaces for reagent preparation and sample addition. Do not move the pipet tips, microtube racks, microtubes, pipettes and other supplies from one space to another. 3. Routinely wipe the hood/bench surfaces with 70% alcohol and UV-disinfect for 15 minutes before and after use. 4. Store reagents in appropriate conditions as per the manufacturer’s product manual.

3. Equipments and Reagents

9) BioRad CFX96™ Real-Time System 10) Primers (reconstituted in DNase free water to100uM and stored in aliquots of 10uM in the -20°C freezer): a. Forward Primer: HF183 ATC ATG AGT TCA CAT GTC CG b. Reverse Primer: Bac708R CAA TCG GAG TTC TTC GTG

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Hope College Water Quality Lab Michael Pikaart, PhD

11) Qiagen HotStarTaq Mastermix kit-1000 units (A premixed solution containing HotStarTaq DNA Polymerase, PCR Buffer, and dNTPs. The solution provides a final concentration of 1.5 mM MgCl2 and 200 µM each dNTP) 12) DNase-free water 13) TempAssure PCR 0.1 ml 8-strips, flat caps, natural (USA Scientific # 1402-3700) 14) Barrier (filter) pipette tips 15) 1.5 ml RNase/DNase-free microcentrifuge tubes 16) Mini centrifuge for PCR tubes

4. Procedure

3) Thaw all the reagents and DNA samples

4) Prepare a super mix for n+4 reactions (n = total samples) in a 1.5 mL centrifuge tube at the PCR Prep Station as given below.

HotStar Taq Master Mix - 10 µL Primer (forward) (10µM) - 1µL Primer (reverse) (10µM) - 1µL Molecular Grade water - 3µL

3) Mix the reagents well by vortexing gently and centrifuge briefly.

4) Aliquot 15 microlitre of the mastermix into the PCR tubes.

5) Move to the sample addition station and add 5 microlitre of the sample DNA in duplicates, positive control DNA from raw sewage (aliquoted from stock previously amplified and sequenced to confirm the presence of maker) and molecular grade water as negative controls, both in triplicates.

6) Briefly centrifuge the tubes.

7) Set the protocol in the Real-Time System using the cycling program as follows

1 95.0 C for 15:00

2 94.0 C for 0:30

3 58.0 C for 0:30

4 72.0 C for 1:00

5 GOTO 2, 40 more times

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Hope College Water Quality Lab Michael Pikaart, PhD

6 72.0 C for 8:00

END

8) Check the PCR product by agarose gel (1.2%, w/v) electrophoresis (95 V for ~1 hrs). The product size for the HF 183 assay is 525 bp.

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Hope College Water Quality Lab Michael Pikaart, PhD

Standard Operating Procedure (SOP) for CF128 (cow specific 16S Bacteroides) PCR Assay

7/28/2011 Sangeetha Srinivasan, PhD

References:

Bernhard, A. E., and K. G. Field. 2000. A PCR assay to discriminate human and ruminant feces on the basis of host differences in Bacteroides-Prevotella genes encoding 16S rRNA. Appl. Environ. Microbiol. 66:4571-4574.

CFX96 and CFX 384 Real-Time PCR Detection Sysytems Instrument Operator’s manual.

1. Purpose and Scope

This Standard Operating Procedure (SOP) is for conventional PCR assay, nicknamed as CF128, targeting cow specific Bacteroides 16S rRNA gene. The total amplicon length for the target is 580bp. This SOP is intended to be used for BioRad CFX96™ Real-Time System. The purpose of

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Hope College Water Quality Lab Michael Pikaart, PhD

this qPCR assay is to determine the presence or absence of contamination from cow fecal sources in water or other environmental samples

2. Recommended Quality Control Procedures:

1. Users must wear gloves at all times and avoid direct hand contact with PCR reagents to avoid contamination.

2. Use separate designated spaces for reagent preparation and sample addition. Do not move the pipet tips, microtube racks, microtubes, pipettes and other supplies from one space to another.

3. Routinely wipe the hood/bench surfaces with 70% alcohol and UV-disinfect for 15 minutes before and after use.

4. Store reagents in appropriate conditions as per the manufacturer’s product manual.

3. Equipments and Reagents

17) BioRad CFX96™ Real-Time System 18) Primers (reconstituted in DNase free water to100uM and stored in aliquots of 10uM in the -20°C freezer): a. Forward Primer: CF128F 5’CCA ACY TTC CCG WTA CTC3’ b. Reverse Primer: Bac708R 5’ CAA TCG GAG TTC TTC GTG 3’ 19) Qiagen HotStarTaq Mastermix kit-1000 units (A premixed solution containing HotStarTaq DNA Polymerase, PCR Buffer, and dNTPs. The solution provides a final concentration of 1.5 mM MgCl2 and 200 µM each dNTP) 20) DNase-free water 21) TempAssure PCR 0.1 ml 8-strips, flat caps, natural (USA Scientific # 1402-3700) 22) Barrier (filter) pipette tips 23) 1.5 ml RNase/DNase-free microcentrifuge tubes 24) Mini centrifuge for PCR tubes

4. Procedure

5) Thaw all the reagents and DNA samples

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Hope College Water Quality Lab Michael Pikaart, PhD

6) Prepare a super mix for n+4 reactions (n = total samples) in a 1.5 mL centrifuge tube at the PCR Prep Station as given below.

HotStar Taq Master Mix - 10 µL

Primer (forward) (10µM) - 1µL

Primer (reverse) (10µM) - 1µL

Molecular Grade water - 3µL

3) Mix the reagents well by vortexing gently and centrifuge briefly.

4) Aliquot 15 microlitre of the mastermix into the PCR tubes.

5) Move to the sample addition station and add 5 microlitre of the sample DNA in duplicates, positive control DNA from raw sewage (aliquoted from stock previously amplified and sequenced to confirm the presence of maker) and molecular grade water as negative controls, both in triplicates.

6) Briefly centrifuge the tubes.

7) Set the protocol in the Real-Time System using the cycling program as follows

1 95.0 C for 15:00

2 94.0 C for 0:30

3 58.0 C for 0:30

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Hope College Water Quality Lab Michael Pikaart, PhD

4 72.0 C for 1:00

5 GOTO 2, 39 more times

6 72.0 C for 8:00

END

8) Check the PCR product by agarose gel (1.2%, w/v) electrophoresis (95 V for ~1 hrs).

The product size for the CF128 assay is 580 bp.

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