# Method 1106.1: Enterococci in Water byMembrane Filtration Using membraneEnterococcus-Esculin Iron Agar (mE-EIA)

December 2009

# U.S. Environmental Protection Agency Office of Water (4303T) 1200 Pennsylvania Avenue, NW Washington, DC 20460

EPA-821-R-09-015

## Acknowledgments The following laboratories are gratefully acknowledged fo r their participation in the validation study for this method in disinfected wastewater effluents which was conducted in 2004: Volunteer Research Laboratories • EPA Office of Research and Development, Nati onal Risk Management Research Lab: Mark C. Meckes • U.S. Army Corps of Engineers, Washington Aqueduct: Elizabeth A. Turner, Michael L. Chicoine, and Lisa Neal Volunteer Verification Laboratory • Orange County Sanitation District, Environmental Sciences Laboratory: Charles McGee, Michael von Winckelmann, Kim Patton, Linda Kirchner, James Campbell, Arturo Diaz, and Lisa McMath Volunteer Participant Laboratories • American Interplex: John Overbey, Steve Bradford, and Jessica Young • County Sanitation Districts of Los Angeles County, Joint Water Pollution Control Plant (JWPCP): Kathy Walker, Michele Padilla, and Albert Soof • East Bay Municipal Utility District: Bill Ellgas and Daniel Mills • Environmental Associates (EA): Susan Boutros and Madelyn Glase • Hampton Roads Sanitation District (HRSD) : Anna Rule, Paula Hogg, and Bob Maunz • Hoosier Microbiological Laboratories (HML): D on Hendrickson, Katy Bilger, Keri Nixon, and Lindsey Shelton • Massachusetts Water Resources Authority (MWRA): Steve Rhode and Mariya Gofshteyn • San Francisco Water: Phil Caskey, Paul McGregor, and Bonnie Bompart • University of Iowa Hygienic La boratory: Nancy Hall and Cathy Lord • Wisconsin State Laboratory of Hygiene (WSLH): Sharon Kluender, Linda Peterson, and Jeremy Olstadtiii 

## Disclaimer Neither the United States Government nor any of its employees, contractors, or their employees make any warranty, expressed or implied, or assumes any legal liability or responsibility for any third party’s use of or the results of such use of any information, apparatus, product, or process discussed in this report, or represents that its use by such party would not in fringe on privately owned rights. Mention of trade names or commercial products does not constitu te endorsement or recommendation for use. Questions concerning this method or its application should be addressed to: Robin K. Oshiro Engineering and Analysis Division (4303T) U.S. EPA Office of Water, Office of Science and Technology 1200 Pennsylvania Avenue, NW Washington, DC 20460 oshiro.robin@epa.gov or OSTCWAMethods@epa.goviv 

## Table of Contents

1.0 Scope and Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 2.0 Summary of Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 3.0 Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 4.0 Interferences and Contamination ................................................. 2 5.0 Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 6.0 Equipment and Supplies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 7.0 Reagents and Standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 8.0 Sample Collection, Preservation, and Storage ....................................... 7 9.0 Quality Control ............................................................... 8 10.0 Calibration and Standardization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 11.0 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 12.0 Verification Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 13.0 Data Analysis and Calculations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 14.0 Sample Spiking Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 15.0 Method Performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 16.0 Pollution Prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 17.0 Waste Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 18.0 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24v 

## List of Appendices Appendices A and B are taken from Microbiological Methods for Monitoring the Environment: Water and Wastes (Reference 18.7). Appendix A: Part II (General Operations), Secti on A (Sample Collection, Preservation, and Storage) Appendix B: Part II (General Operations), Sections C.3.5 (Counting Colonies) and C.3.6 (Calculation of Results)December 2009 1 

## Method 1106.1: Enterococci in Water by Membrane Filtration Using membrane-Enterococcus-Esculin Iron Agar (mE-EIA) December 2009 1.0 Scope and Application 1.1 Method 1106.1 describes a membrane filter (MF) pr ocedure for the detection and enumeration of the enterococci bacteria in ambient water. Th e enterococci bacteria are commonly found in the feces of humans and other warm-blooded animals. Although some strains are ubiquitous and not related to fecal pollution, the presence of enteroco cci in water is an indication of fecal pollution and the possible presence of enteric pathogens. 1.2 The enterococci test is recommended as a measure of ambient fresh and marine recreational water quality. Epidemiological studies have led to the development of criteria which can be used to promulgate recreational water standards based on established relationships between health effects and water quality. The significance of finding entero cocci in recreational fresh or marine water samples is the direct relationship between the density of enterococci and the risk of gastrointestinal illness associat ed with swimming in the water (References 18.1 and 18.2). 1.3 For method application please refer to Title 40 C ode of Federal Regulations Part 136 (40 CFR Part 136). 1.4 Method 1106.1 was submitted to interlaboratory valid ation in disinfected wastewater matrices. Based on the 60.5% false negative confirmation rate for unspiked disinfected wastewater, an estimated 14.1% of total colonies observed during the study would have been incorrectly identified as non-enterococci colonies. As a result, Method 1106.1 is not approved for the analysis of disinfected wastewater. A summary of false positive and negative results are provided in Section 15.4.2. Detailed study results are provided in the validation study report (Reference 18.3). Laboratories wishing to test for enterococci in wastewater using a membrane filtration method are referred to EPA Method 1600. 

## 2.0 Summary of Method 2.1 Method 1106.1 provides a direct count of bacteria in water based on the development of colonies on the surface of the membrane filter (Reference 18.4). A water sample is filtered through the membrane which retains the bacteria. Following filtration, the membrane is placed on a selective medium, mE agar, and incubated at 41°C ± 0.5°C fo r 48 ± 3 hours. The filter is then transferred to a differential medium, EIA, and incubated at 41°C ± 0.5°C for 20-30 minutes. Pink to red enterococci colonies on mE will develop a black or reddish-brown precipitate on the underside of the filter after transfer to EIA. A fluorescent lamp with a magnifying lens is used for counting to give maximum visibility of colonies. 

## 3.0 Definitions 3.1 In Method 1106.1, enterococci are those bacteria which produce pink to red colonies after incubation on mE agar and that form a black or reddish-brown precipitate after subsequent transfer to EIA medium. Enterococci include Enterococcus faecalis (E. faecalis) , E. faecium, E. avium , E. gallinarium and their variants. The genus Enterococcus includes the enterococci formerly assigned to the Group D fecal streptococci.Method 1106.1

December 2009 2 

## 4.0 Interferences and Contamination 4.1 Water samples containing colloidal or suspended particulate materials can clog the membrane filter and prevent filtration, or cause spreading of bacterial colonies which could interfere with enumeration and identification of target colonies. 

## 5.0 Safety 5.1 The analyst must know and observe the normal safety procedures required in a microbiology laboratory while preparing, usi ng and disposing of cultures, reagents, and materials, and while operating sterilization equipment. 5.2 The selective medium (mE) and azide-dextrose broth used in this method contain sodium azide as well as other potentially toxic components. Caution must be exercised during the preparation, use, and disposal of these media to prevent inhalation or contact with the medium or reagents. 5.3 This method does not address all of the safety i ssues associated with its use. It is the responsibility of the laboratory to establish appropriate safety and health practices prior to use of this method. A reference file of material safety data sheets (MSDSs) should be available to all personnel involved in Method 1106.1 analyses. 5.4 Mouth-pipetting is prohibited. 

## 6.0 Equipment and Supplies 6.1 Glass lens with magnification of 2-5X or stereoscopic microscope 6.2 Lamp, with a cool, white fluorescent tube 6.3 Hand tally or electronic counting device 6.4 Pipet container, stainless steel, aluminum or borosilicate glass, for glass pipets 6.5 Pipets, sterile, T.D. bacteriological or M ohr, glass or plastic, of appropriate volume 6.6 Sterile graduated cylinders, 100-1000 mL, covered with aluminum foil or kraft paper 6.7 Sterile membrane filtration units (filter base and funnel), glass, plastic or stainless steel, wrapped with aluminum foil or kraft paper 6.8 Ultraviolet unit for sanitization of the f ilter funnel between filtrations (optional) 6.9 Line vacuum, electric vacuum pump, or aspirator for use as a vacuum source (In an emergency or in the field, a hand pump or a syringe equipped w ith a check valve to prevent the return flow of air, can be used) 6.10 Flask, filter, vacuum, usually 1 L, with appropriate tubing 6.11 Filter manifold to hold a number of filter bases (optional)Method 1106.1

December 2009 3 6.12 Flask for safety trap placed between th e filter flask and the vacuum source 6.13 Forceps, straight or curved, with smooth tips to handle filters without damage 6.14 Ethanol, methanol or isopropanol in a small, wi de-mouth container, for flame-sterilizing forceps 6.15 Burner, Bunsen or Fisher type, or electric in cinerator unit for sterilizing loops and needles 6.16 Thermometer, checked against a National Institute of Standards and Technology (NIST) certified thermometer, or one that meets the requirements of NIST Monograph SP 250-23 6.17 Petri dishes, sterile, plastic, 9 × 50 mm with tight-fitting lids; or 15 × 60 mm with loose fitting lids; and 15 × 100 mm with loose fitting lids 6.18 Bottles, milk dilution, borosilicate glass, screw cap with neoprene liners, 125 mL volume 6.19 Flasks, borosilicate glass, screw cap, 250-2000 mL volume 6.20 Membrane filters, sterile, white, grid marked, 47 mm diameter, with 0.45 μm pore size 6.21 Platinum wire inoculation loops, at least 3 mm diameter in suitable holders; or sterile plastic loops 6.22 Incubator maintained at 41°C ± 0.5°C 6.23 Waterbath maintained at 50°C for tempering agar 6.24 Test tubes, 20 × 150 mm, borosilicate glass or plastic 6.25 Caps, aluminum or autoclavable plastic, for 20 mm diameter test tubes 6.26 Test tubes, screw cap, borosilicate glass, 16 × 125 mm or other appropriate size 6.27 Autoclave or steam sterilizer capable of achievi ng 121°C [15 lb pressure per square inch (PSI)] for 15 minutes 

## 7.0 Reagents and Standards 7.1 Purity of reagents: Reagent-grade chemicals shall be used in all tests. Unless otherwise indicated, reagents shall conform to the specifications of the Committee on Analytical Reagents of the American Chemical Society (Reference 18.5). Th e agar used in preparation of culture media must be of microbiological grade. 7.2 Whenever possible, use commercial cultu re media as a means of quality control. 7.3 Purity of water: Reagent-grade water conforming to specifications in: Standard Methods for the Examination of Water and Wastewater (latest edition approved by EPA in 40 CFR Part 136 or 141, as applicable), Section 9020 (Reference 18.6).Method 1106.1

December 2009 4 7.4 Phosphate buffered saline (PBS) 7.4.1 Composition: Sodium dihydrogen phosphate (NaH 2 PO 4 ) 0.58 g Disodium hydrogen phosphate (Na 2 HPO 4 ) 2.5 g Sodium chloride (NaCl) 8.5 g Reagent-grade water 1.0 L 7.4.2 Dissolve the reagents in 1 L of reagent-gr ade water and dispense in appropriate amounts for dilutions in screw cap bottles or culture tubes, and/or into containers for use as rinse water. Autoclave at 121°C (15 PSI) for 15 minutes. Final pH should be 7.4 ± 0.2. 7.5 mE agar 7.5.1 Composition: Peptone 10.0 g Sodium chloride (NaCl) 15.0 g Yeast extract 30.0 g Esculin 1.0 g Actidione (cycloheximide) 0.05 g Sodium azide 0.15 g Agar 15.0 g Reagent-grade water 1.0 L 7.5.2 Add reagents to 1 L of reagent-grade water, mix thoroughly, and heat to dissolve completely. Autoclave at 121°C (15 PSI) for 15 minutes, and cool in a 50°C waterbath. 7.5.3 After sterilization, add 0.24 g nalidixic acid (sodium salt) and 0.15 g triphenyltetrazolium chloride (TTC) to the basal medium and mix. 7.5.4 Dispense mE agar into 9 × 50 or 15 × 60 mm petri dishes to a 4-5 mm depth (approximately 4-6 mL), and allow to solidify. Final pH of medium should be 7.1 ± 0.2. Store in a refrigerator. 7.6 Esculin iron agar (EIA) 7.61 Composition: Esculin 1.0 g Ferric citrate 0.5 g Agar 15.0 g Reagent-grade water 1.0 L 7.6.2 Add reagents to 1 L of reagent-grade water, mix thoroughly, and heat to dissolve completely. Autoclave the medium at 121°C (15 PSI) for 15 minutes, and cool in a 50°C waterbath. After cooling, pour the medium into 9 × 50 or 15 × 60 mm petri dishes to a depth of 4-5 mm (approximately 4-6 mL), a nd allow to solidify. Final pH should be 7.1 ± 0.2. Store in a refrigerator.Method 1106.1

December 2009 5 7.7 Tryptic soy agar (TSA) 7.7.1 Composition: Pancreatic digest of casein 15.0 g Enzymatic digest of soybean meal 5.0 g Sodium chloride (NaCl) 5.0 g Agar 15.0 g Reagent-grade water 1.0 L 7.7.2 Add reagents to 1 L of reagent-grade water, mix thoroughly, and heat to dissolve completely. Autoclave at 121°C (15 PSI) for 15 minutes and cool in a 50°C waterbath. Pour the medium into each 15 × 60 mm culture dish to a 4-5 mm depth (approximately 4- 6 mL), and allow to solidify. Final pH should be 7.3 ± 0.2. 7.8 Brain heart infusion broth (BHIB) 7.8.1 Composition: Calf brains, infusion from 200.0 g 7.7 g Beef heart, infusion from 250.0 g 9.8 g Proteose peptone 10.0 g Sodium chloride (NaCl) 5.0 g Disodium hydrogen phosphate (Na 2 HPO 4 ) 2.5 g Dextrose 2.0 g Reagent-grade water 1.0 L 7.8.2 Add reagents to 1 L of reagent-grade water, mix thoroughly, and heat to dissolve completely. Dispense in 10-mL volumes in scr ew cap tubes, and autoclave at 121°C (15 PSI) for 15 minutes. Final pH should be 7.4 ± 0.2. 7.9 Brain heart infusion broth (BHIB) with 6.5% NaCl 7.9.1 Composition: BHIB with 6.5% NaCl is the same as BHIB br oth (Section 7.8 above), but with additional NaCl. 7.9.2 Add NaCl to formula provided in Section 7.8 above, such that the final concentration is 6.5 %(65 g NaCl/L). Typically, for commerc ial BHIB media, an additional 60.0 g NaCl per liter of medium will need to be added to the base medium. Prepare as in Section 7.8.2. 7.10 Brain heart infusion agar (BHIA) 7.10.1 Composition: BHIA contains the same components as BHIB (Section 7.8 above) with the addition of 15.0 g agar per liter of BHIB. 7.10.2 Add agar to formula for BHIB provided in Section 7.8 above. Prepare as in Section 7.8.2. After sterilization, slant until solid. Final pH should be 7.4 ± 0.2.Method 1106.1

December 2009 6 7.11 Bile esculin agar (BEA) 7.11.1 Composition: Beef extract 3.0 g Pancreatic digest of gelatin 5.0 g Oxgall 20.0 g Esculin 1.0 g Ferric citrate 0.5 g Agar 14.0 g Reagent-grade water 1.0 L 7.11.2 Add reagents to 1 L reagent-grade water, h eat with frequent mixing, and boil 1 minute to dissolve completely. Dispense 10-mL volumes in tubes for slants or larger volumes into flasks for subsequent plating. Autoclave at 121°C (15 PSI) for 15 minutes. Overheating may cause darkening of the medium. Cool in a 50°C waterbath, and dispense into sterile petri dishes. Final pH should be 6.8 ± 0.2. Store in a refrigerator. 7.12 Azide dextrose broth (ADB) 7.12.1 Composition: Beef extract 4.5 g Pancreatic digest of casein 7.5 g Proteose peptone No. 3 7.5 g Dextrose 7.5 g Sodium chloride (NaCl) 7.5 g Sodium azide 0.2 g Reagent-grade water 1.0 L 7.12.2 Add reagents to 1 L of reagent-grade water and dispense in screw cap bottles. Autoclave at 121°C (15 PSI) for 15 minutes. Final pH should be 7.2 ± 0.2. 7.13 Control Cultures 7.13.1 Positive control and/or spiking organism (either of the following are acceptable): • Stock cultures of Enterococcus faecalis ( E. faecalis ) ATCC #19433 • E. faecalis ATCC #19433 BioBalls (bioMérieux Inc., Durham NC) 7.13.2 Negative control organism (either of the following are acceptable): • Stock cultures of Escherichia coli ( E. coli ) ATCC #11775

• E. coli ATCC #11775 BioBalls (bioMérieux Inc., Durham NC)Method 1106.1

December 2009 7 

## 8.0 Sample Collection, Handling, and Storage 8.1 Sampling procedures are briefly described belo w. Detailed sampling methods can be found in Reference 18.7 (see Appendix A). Adherence to sample preservation procedures and holding time limits is critical to the production of valid data. Samples should not be analyzed if these conditions are not met. 8.1.1 Sampling Techniques Samples are collected by hand or with a sampling device if the sampling site has difficult access such as a dock, bridge, or bank adjacent to a surface water. Composite samples should not be collected, since such samples do not display the range of values found in individual samples. The sampling depth for surface water samples should be 6-12 inches below the water surface. Sample containers should be positioned such that the mouth of the container is pointed away from the sampler or sample point. After removal of the container from the water, a small portion of the sample should be discarded to allow for proper mixing before analyses. 8.1.2 Storage Temperature and Handling Conditions Ice or refrigerate bacteriological samples at a temperature of <10°C during transit to the laboratory. Do not freeze the samples. Use insulated containers to assure proper maintenance of storage temperature. Take care that sample bottles are not totally immersed in water during transit or storage. 8.1.3 Holding Time Limitations Sample analysis should begin immediately, preferably within 2 hours of collection. The maximum transport time to the laboratory is 6 hours, and samples should be processed within 2 hours of receipt at the laboratory. 

## 9.0 Quality Control 9.1 Each laboratory that uses Method 1106.1 is requi red to operate a formal quality assurance (QA) program that addresses and documents instrument and equipment maintenance and performance, reagent quality and performance, analyst training and certification, and records storage and retrieval. General requirements and r ecommendations for QA and quality control (QC) procedures for microbiological laboratories are provided in Reference 18.7 (see Appendix B). 9.2 The minimum analytical QC requirements for the analysis of samples using Method 1106.1 include routine analysis of positive and negativ e controls (Section 9.5), filter sterility checks (Section 9.7), method blanks (Section 9.8), and media sterility checks (Section 9.10). Additional analytical QC for the analysis of samples usi ng Method 1106.1 include an initial demonstration of laboratory capability through performance of the initial precision and recovery (IPR) analyses (Section 9.3), ongoing demonstration of labor atory capability through performance of the ongoing precision and recovery (OPR) analysis (Section 9.4). For the IPR and OPR analyses, it is necessary to spike PBS samples with either laboratory-prepared spiking suspensions or BioBalls as described in Section 14.Method 1106.1

December 2009 8 9.3 Initial precision and recovery (IPR) —The IPR analyses are used to demonstrate acceptable method performance (recovery and precision) by each laboratory before the method is used for monitoring field samples. EPA recommends but does not require that IPR analyses be performed by each analyst. IPR samples should be accompanied by an acceptable method blank (Section 9.8) and appropriate media sterility checks (Section 9.10). The IPR analyses are performed as follows: 9.3.1 Prepare four, 100-mL samples of PBS and spike each sample with E. faecalis ATCC#19433 according to the spiking procedure in Section 14. Spiking with laboratoryprepared suspensions is described in Secti on 14.2 and spiking with BioBalls is described in Section 14.3. Filter and process each IPR sample according to the procedures in Section 11 and calculate the number of ente rococci per 100 mL according to Section 13. 9.3.2 Calculate the percent recovery (R) for each IPR sample using the appropriate equation in Section 14.2.4.2 or 14.3.2 for samples spiked with laboratory-prepared spiking suspensions or BioBalls, respectively. 9.3.3 Using the percent recoveries of the four an alyses, calculate the mean percent recovery and the relative standard deviation (RSD) of the recoveries. The RSD is the standard deviation divided by the mean, multiplied by 100. 9.3.4 Compare the mean recovery and RSD with the corresponding IPR criteria in Table 1 , below. If the mean and RSD for recovery of enterococci meet acceptance criteria, system performance is acceptable and analysis of field samples may begin. If the mean or the RSD fall outside of the required range for recovery, system performance is unacceptable. In this event, identify the problem by ev aluating each step of the analytical process, media, reagents, and controls, correct the problem and repeat IPR analyses. Table 1. Initial and Ongoing Precision and Reco very (IPR and OPR) Acceptance Criteria Performance test Lab-prepared spike acceptance criteria BioBallTM acceptance criteria Initial precision and recovery (IPR) • Mean percent recovery • Precision (as maximum relative standard deviation) 15% - 136% 86% - 102% 21% 12% Ongoing precision and recovery (OPR) as percent recovery 14% - 137% 80% - 108%

9.4 Ongoing precision and recovery (OPR) —To demonstrate ongoing control of the analytical system, the laboratory should routinely process a nd analyze spiked PBS samples. The laboratory should analyze one OPR sample after every 20 field samples or one per week that samples are analyzed, whichever occurs more frequently. OPR samples should be accompanied by an acceptable method blank (Section 9.8) and appropriate media sterility checks (Section 9.10). The OPR analysis is performed as follows: 9.4.1 Spike a 100-mL PBS sample with E. faecalis ATCC #19433 according to the spiking procedure in Section 14. Spiking with labor atory-prepared suspensions is described in Section 14.2 and spiking with BioBalls is d escribed in Section 14.3. Filter and process each OPR sample according to the procedures in Section 11 and calculate the number of enterococci per 100 mL according to Section 13.Method 1106.1

December 2009 9 9.4.2 Calculate the percent recovery (R) for the OPR sample using the appropriate equation in Section 14.2.4.2 or 14.3.2 for samples spiked with BioBalls or laboratory-prepared spiking suspensions, respectively. 9.4.3 Compare the OPR results (percent recovery) with the corresponding OPR recovery criteria in Table 1, above. If the OPR resu lt meets the acceptance criteria for recovery, method performance is acceptable and analysis of field samples may continue. If the OPR result falls outside of the acceptance criteria, system performance is unacceptable. In this event, identify the problem by ev aluating each step of the analytical process, media, reagents, and controls, correct the problem and repeat the OPR analysis. 9.4.4 As part of the laboratory QA program, method recovery results for OPR and IPR samples should be charted and updated records main tained in order to monitor ongoing method performance. The laboratory should also develop a statement of accuracy for Method 1106.1 by calculating the average percent recove ry (R) and the standard deviation of the percent recovery (s r ). Express the accuracy as a recovery interval from R - 2s rto R + 2s r . 9.5 Culture Controls 9.5.1 Negative controls —The laboratory should analyze negative controls to ensure that the mE and EIA media are performing properly. Negative controls should be analyzed whenever a new batch of media or reagents is used. On an ongoing basis, the laboratory should perform a negative control every day that samples are analyzed. 9.5.1.1 Negative controls are conducted by filte ring a dilute suspension of viable E. coli (e.g., ATCC #11775) and analyzing as described in Section 11. Viability of the negative controls should be demonstrated using a non-selective media (e.g., nutrient agar or tryptic soy agar). 9.5.1.2 If the negative control fails to exhibit the appropriate response, check and/or replace the associated media or reagents , and/or the negative control, and reanalyze the appropriate negative control(s). 9.5.2 Positive controls —The laboratory should analyze positive controls to ensure that the mE and EIA media are performing properly. Positive controls should be analyzed whenever a new batch of media or reagents is used. On an ongoing basis, the laboratory should perform a positive control every day that samples are analyzed. An OPR sample (Section 9.4) may take the place of a positive control. 9.5.2.1 Positive controls are conducted by filte ring a dilute suspension of viable E. faecalis (e.g., ATCC #19433) and analyzing as described in Section 11. 9.5.2.2 If the positive control fails to exhibit the appropriate response, check and/or replace the associated media or reagen ts, and/or the positive control, and reanalyze the appropriate positive control(s). 9.5.3 Controls for verification media —All verification media should be tested with appropriate positive and negative controls whenever a new batch of media and/or reagents are used. On an ongoing basis, the laboratory should perform positive and negative controls on the verification medi a with each batch of samples submitted to verification. Examples of appropriate controls for verification media are provided in Table 2

.Method 1106.1

December 2009 10 Table 2. Verification Controls Medium Positive Control Negative Control Bile esculin agar (BEA) E. faecalis E. coli Brain heart infusion broth (BHIB) with 6.5% NaCl E. faecalis E. coli Brain heart infusion broth (BHIB) incubated at 45°C E. faecalis E. coli 9.6 Colony verification —The laboratory should verify 10 typical colonies (positive) and 10 atypical colonies (negative) per month or 1 typical colony and 1 atypical colony from 10% of all positive samples, whichever is greater. Verification procedures are provided in Section 12.0. 9.7 Filter sterility check—Place at least one membrane filter on a TSA plate, and incubate for 24 ± 2 hours at 35°C ± 0.5°C. Absence of growth indicates sterility of the filter. On an ongoing basis, the laboratory should perform a filter sterility check every day that samples are analyzed. 9.8 Method blank —Filter a 50-mL volume of sterile PBS, place the filter on an mE plate, and incubate for 48 ± 3 hours at 41°C ± 0.5°C. Absence of growth indicates freedom of contamination from the target organism. On an ongoing basis, the laboratory should perform a method blank every day that samples are analyzed. 9.9 Filtration blank—Filter a 50-mL volume of sterile PBS before beginning sample filtrations. Place the filter on a TSA plate, and incubate for 24 ± 2 hours at 35°C ± 0.5°C. Absence of growth indicates sterility of the PBS and filtration assembly. 9.10 Media sterility check—The laboratory should test media st erility by incubating one unit (tube or plate) from each batch of medium (TSA, mE, EI A, and verification media) as appropriate and observing for growth. Absence of growth indicates media sterility. On an ongoing basis, the laboratory should perform a media sterility check every day that samples are analyzed. 9.11 Analyst colony counting variability —Laboratories with two or more analysts should compare each analyst's colony counts from one positive field sample per month. Colony counts should be within 10% between analysts. Laboratories with a single analyst should have that analyst perform duplicate colony counts of a single membrane filter each month. Duplicate colony counts should be within 5% for a single analyst. If no positive field samples are available, a OPR sample may be substituted for these determinations. 

## 10.0 Calibration and Standardization 10.1 Check temperatures in incubators twice daily with a minimum of 4 hours between each reading to ensure operation within stated limits. 10.2 Check thermometers at least annually against a NIST certified thermometer or one that meets the requirements of NIST monograph SP 250-23. Check mercury columns for breaks. 10.3 Refrigerators used to store media and reagents should be monitored daily to ensure proper temperature control.Method 1106.1

December 2009 11 

## 11.0 Procedure 11.1 Prepare mE and EIA media as directed in Section 7.5 and 7.6, respectively. 11.2 Mark the petri dishes and report form with the sample identification and volume. 11.3 Place a sterile membrane filter on filter base, grid si de up, and attach the funnel to the base so that the membrane filter is held between the funnel and the base. 11.4 Shake the sample bottle vigorously at least 25 tim es to distribute the bacteria uniformly, and measure the desired volume of sample or dilution into the funnel. 11.5 Select sample volumes based on previous knowledge of enterococci concentration, to produce 20- 60 enterococci colonies on each filter. It is recommended that a minimum of three dilutions be analyzed to ensure that a countable plat e (20-60 enterococci colonies) is obtained. 11.6 Smaller sample volumes or sample dilutions can be used to minimize the interference of turbidity or for high bacterial densities. Multiple volumes of the same sample or sample dilutions may be filtered. Note : When analyzing smaller sample volumes (e.g., <20 mL), 20-30 mL of PBS should be added to the funnel or an aliquot of sample should be dispensed into a 20-30 mL dilution blank prior to filtration. This will allow even distribution of the sample on the membrane. 11.7 Filter the sample, and rinse the sides of the funnel at least twice with 20-30 mL of sterile PBS. Turn off the vacuum, and remove the funnel from the filter base. 11.8 Use sterile forceps to aseptically remove the me mbrane filter from the filter base, and roll it onto the mE plate to avoid the formation of bubbles between the membrane and the agar surface. Reseat the membrane if bubbles occur. Run the forceps around the edge of the filter outside the area of filtration, close to the edge of the dish, to be sure that the filter is properly seated on the agar. Close the dish, invert, and incubate at 41°C ± 0.5°C for 48 ± 3 hours. (See Photo 1 ) Note : If mE is prepared in 15 × 60 mm loose lid petri dishes, they should be incubated in a tight fitting container (e.g., plastic vegetable crisper) containing a moistened paper towel to prevent dehydration of the membrane filter and medium.

Photo 1. Enterococci produces pink to dark red colonies on mE agar.Method 1106.1

December 2009 12 11.9 After incubation, transfer the membranes to EIA plates that have been warmed to room temperature (approximately 15-30 minutes), and in cubate at 41°C ± 0.5°C for 20 to 30 minutes. (See Photo 2 ) 11.10 After EIA incubation, count and record coloni es on those membrane filters containing 20-60 pink to red colonies with black or reddish-brown pr ecipitate on the underside of the membrane. Use magnification for counting and a small fluorescent lamp to give maximum visibility of colonies.

Photo 2. Enterococci colonies are pink to dark red on mE and produce a reddishbrown to black precipitate on the underside of the filter when placed on EIA. 

## 12.0 Verification Procedure 12.1 Pink to red colonies on mE that produce a black or reddish-brown precipitate after incubation on EIA are considered to be “typical” enterococci colonies. Verification of typical and atypical colonies may be required in evidence gathering and it is also recommended as a means of quality control. The verification procedure follows. 12.2 Using a sterile inoculating loop or needle, transfer growth from the centers of at least 10 wellisolated typical and 10 well-isolated atypical colonies into a BHIB tube and onto a BHIA slant. Incubate broth for 24 ± 2 hours and agar slants for 48 ± 3 hours at 35°C ± 0.5°C. 12.3 After a 24 hour incubation, transfer growth from each BHIB tube to BEA, BHIB with 6.5% NaCl, and BHIB. 12.3.1 Incubate BEA and BHIB with 6.5% NaCl for 48 ± 3 hours at 35°C ± 0.5°C. 12.3.2 Incubate BHIB for 48 ± 3 hours at 45°C ± 0.5°C. 12.4 Observe all verification media for growth. 12.5 After 48 hour incubation, perform a Gram stain using growth from each BHIA slant. 12.6 Gram-positive cocci that grow and hydrolyze esculin on BEA (i.e

. , produce a black or brown precipitate), and grow in BHIB with 6.5% NaClat 35°C ± 0.5°C and BHIB at 45°C ± 0.5°C are verified as enterococci.Method 1106.1

December 2009 13 12.7 Alternately, commercially available multi-test id entification systems (e.g., Vitek®) may be used to verify colonies. Such multi-test identification systems should include esculin hydrolysis and growth in 6.5% NaCl test reactions. 

## 13.0 Data Analysis and Calculations Use the following general rules to calculate the enterococci count per 100 ml of sample: 13.1 If possible, select a membrane filter with 20-60 pink to red colonies that form a black or reddishbrown precipitate on the underside of the filter when placed on EIA. Calculate the number of enterococci per 100 mL according to the following general formula:

Enterococci / 100 mL = Number of enterococci colonies × 100 Volume of sample filtered (mL)

13.2 See general counting rules in Reference 18.7 (see Appendix B). 13.3 Report results as enterococci CFU per 100 mL of sample. 

## 14.0 Sample Spiking Procedure 14.1 Method 1106.1 QC requirements (Section 9) include the preparation and analysis of spiked PBS samples in order to monitor initial and ongoing method performance. For the IPR (Section 9.3) and OPR (Section 9.4) analyses, it is necessary to spike samples with either laboratory-prepared spiking suspensions (Section 14.2) or BioBalls (Section 14.3) as described below. 14.2 Laboratory-Prepared Spiking Suspensions 14.2.1 Preparation 14.2.1.1 Stock Culture. Prepare a stock culture by inoculating a TSA slant (or other non-selective media) with E. faecalis ATCC #19433 and incubating at 35°C ± 3°C for 20 ± 4 hours. This stock culture may be stored in the dark at room temperature for up to 30 days. 14.2.1.2 Undiluted Spiking Suspension. Prepare a 1% solution of azide dextrose broth by combining 99 mL of sterilephosphate buffered saline and 1 mL ofsterile single strength azide dextrose broth in a sterile screw cap bottle or resealable dilution water container. From the stock culture of E. faecalis ATCC #19433 in Section 14.2.1.1, transfer a small loopful of growth to the 1 % azide dextrose broth solution and vigorously shake a minimum of 25 times. Incubate at 35°C ± 3°C for 20 ± 4 hours. This culture is referred to as the undiluted spiking suspension and should contain approximately 1.0 × 10 6 - 1.0 × 10 7 E. faecalis colony forming units (CFU) per mL of culture.

.Method 1106.1

December 2009 14 14.2.1.3 Mix the undiluted spiking suspension thoroughly by shaking the bottle a minimum of 25 times and prepare a series of dilutions in the following manner: 14.2.1.3.1 Dilution “A”—Aseptically transfer 1.0 mL of the undiluted spiking suspension to 99 mL of sterile PBS and mix thoroughly by shaking the bottle a minimum of 25 times. This is spiking suspension dilution “A” and 1 mL contains 10 -2 mL of the original undiluted spiking suspension. 14.2.1.3.2 Dilution “B”—Aseptically transfer 1.0 mL of dilution “A” to 99 mL of sterile PBS and mix thoroughly by shaking the bottle a minimum of 25 times. This is spiking suspension dilution “B” and 1 mL contains 10 -4 mL of the original undiluted spiking suspension. 14.2.1.3.3 Dilution “C”—Aseptically transfer 11.0 mL of dilution “B” to 99 mL of sterile PBS and mix thoroughly by shaking the bottle a minimum of 25 times. This is spiking suspension dilution “C” and 1 mL contains 10 -5 mL of the original undiluted spiking suspension. 14.2.1.3.4 Dilution “D”—Aseptically transfer 11.0 mL of dilution “C” to 99 mL of sterile PBS and mix thoroughly by shaking the bottle a minimum of 25 times. This is spiking suspension dilution “D” and 1 mL contains 10 -6 mL of the original undiluted spiking suspension. 14.2.2 Sample spiking 14.2.2.1 Add 3.0 mL of the spiking suspension dilution “D” (Section 14.2.1.3.4) to 100 mL (or appropriate volume) of sample and mix thoroughly by shaking the bottle a minimum of 25 times. The volume of undiluted spiking suspension added to each 100 mL sample is 3.0 x 10 -6 mL, which is referred to as V spiked per 100 mL samplein Section 14.2.4.1 below. Filter the spiked sample and analyze the filter according to the procedures in Section 11. 14.2.3 Enumeration of spiking suspension 14.2.3.1 Prepare TSA spread plates, in triplicat e, for spiking suspension dilutions “B”, “C”, and “D”. Note: Agar plates must be dry and free from condensation prior to use. To ensure that the agar surface is dry, plates should be made several days in advance and stored inverted at room temperature or dried using a laminar-flow hood. 14.2.3.2 Mix dilution “B” by shaking the bottle a minimum of 25 times. Pipet 0.1 mL of dilution “B” onto the surface of each TSA plate in triplicate. 14.2.3.3 Mix dilution “C” by shaking the bottle a minimum of 25 times. Pipet 0.1 mL of dilution “C” onto the surface of each TSA plate in triplicate.Method 1106.1

December 2009 15 14.2.3.4 Mix dilution “D” by shaking the bottle a minimum of 25 times. Pipet 0.1 mL of dilution “D” onto the surface of each TSA plate in triplicate. 14.2.3.5 Use a sterile bent glass rod or spreader to distribute the inoculum over the surface of plates by rotating the dish by hand or on a turntable. Note: Ensure that the inoculum is evenly distributed over the entire surface of the plate. 14.2.3.6 Allow the inoculum to absorb into the medium of each plate completely. Invert plates and incubate at 35°C ± 0.5°C for 20 ± 4 hours. 14.2.3.7 Count and record number of colonies per plate. The number of enterococci (CFU / mL) in the undiluted spiking suspension will be calculated using all TSA plates yielding counts within the countable range of 30 to 300 CFU per plate. 14.2.4 Recovery calculations for samples spik ed with laboratory-prepared spiking suspensions Calculate the concentration of enteroco cci (CFU / mL) in the undiluted spiking suspension (Section 14.2.1.2) according to the following equation. Example calculations are provided in Table 3 , below.



### Enterococci undiluted spike 

### = (CFU 1 

### + CFU 2 

### + ...+ CFU n 

### ) / (V 1 

### + V 2 

### + ... + V n 

### ) Where, Enterococci undiluted spike= Enterococci (CFU / mL) in undiluted spiking suspension CFU = Number of colony forming units from TSA plates yielding counts within the ideal range of 30 to 300 CFU per plate V = Volume of undiluted sample on each TSA plate yielding counts within the countable range of 30 to 300 CFU per plate n = Number of plates with counts within the countable range of 30 to 300 CFU per plate Note: The example calculated numbers provided in the tables below have been rounded at the end of each step for simplification purposes. Generally, rounding should only occur after the final calculation.Method 1106.1

December 2009 16 Table 3. Example Calculations of Laboratory- prepared Enterococci Spiking Concentration

Examples CFU / plate (triplicate analyses) from TSA plates Enterococci CFU / mL in undiluted spiking suspension ( Enterococci undiluted spike ) a 10 -5 mL plates 10 -6 mL plates 10 -7 mL plates

Example 1 94, 106, 89 9, 11, 28 1, 0, 4 (94+106+89) / (10 -5 +10 -5 +10 -5 ) = 289 / (3.0 x 10 -5 ) = 9,633,333 = 9.6 x 10 6 CFU / mL

Example 2 32, 55, 72 8, 5, 3 0, 0, 0 (32+55+72) / (10 -5 +10 -5 +10 -5 ) = 159 / (3.0 x 10 -5 ) = 5,300,000 = 5.3 x 10 6 CFU / mL

a Enterococci undiluted spike is calculated using all plates yielding counts within the ideal range of 30 to 300 CFU per plate

14.2.4.1 Calculate true concentration of spiked enterococci (CFU / 100 mL) according to the following equation. Example calculations are provided in Table 4, below. 

### T spiked Enterococci 

### = ( Enterococci undiluted spike 

### ) x ( V spiked per 100 mL sample 

### ) Where, T spiked Enterococci = Number of spiked Enterococci (CFU / 100 mL) Enterococci undiluted spike = Enterococci (CFU / mL) in undiluted spiking suspension V spiked per 100 mL sample = mL of undiluted spiking suspension per 100 mL sample

Table 4. Example Calculations for Determinati on “True” Spiked Enterococci Concentration

Enterococci undiluted spike V spiked per 100 mL sample T spiked Enterococci

9.6 x 10 6 CFU / mL 3.0 X 10 -6 mL per 100 mL of sample (9.6 x 10 6 CFU / mL) x (3.0 x 10 -6 mL / 100 mL) = 28.8 CFU / 100 mL 5.3 x 10 6 CFU / mL 3.0 X 10 -6 mL per 100 mL of sample (2.8 x 10 6 CFU / mL) x (3.0 x 10 -6 mL / 100 mL) = 8.4 CFU / 100 mLMethod 1106.1

December 2009 17 14.2.4.2 Calculate percent recovery (R) of spiked enterococci (CFU / 100 mL) according to the following equation. Example calculations are provided in Table 5 , below).



## R = 100 x (N s 

## - N u 

## ) T Where, R = Percent recovery N s = Enterococci (CFU / 100 mL) in the spiked sample (Section 13) N u = Enterococci (CFU / 100 mL) in the unspiked sample (Section 13) T = True spiked enterococci (C FU / 100 mL) in spiked sample (Section 14.2.4.1) Table 5. Example Percent Recovery Calculations for Lab-prepared Spiked Samples

N s (CFU / 100 mL) N u (CFU / 100 mL) T spiked Enterococci (CFU / 100 mL) Percent recovery (R)

42 <1 28.8 100 x (42 - 1) / 28.8

= 142%

34 10 28.8 100 x (34 - 10) / 28.8

= 83%

10 <1 8.4 100 x (10 - 1) / 8.4 = 107%

14.3 BioBallTM Spiking procedure 14.3.1 Aseptically add 1 BioBallTM to 100 mL of PBS and mix by vigorously shaking the sample bottle a minimum of 25 times. Anal yze the spiked sample according to the procedures in Section 11. 14.3.2 Recovery calculations for samples spiked withBioBalls—Calculate percent recovery (R) of spiked enterococci (CFU / 100 mL) accord ing to the following equation. Example calculations are provided in Table 6 , below.Method 1106.1

December 2009 18 

## R = 100 x (N s 

## - N u 

## ) T

Where, R = Percent recovery N s = Enterococci (CFU / 100 mL) in the spiked sample (Section 13) N u = Enterococci (CFU / 100 mL) in the unspiked sample (Section 13) T = True spiked enterococci (CFU / 100 mL) in spiked sample based on the lot mean value provided by manufacturer Table 6. Example BioBallTM Percent Recovery Calculations

N s (CFU / 100 mL) N u (CFU / 100 mL) T (CFU / 100 mL) Percent recovery (R) 24 <1 32 100 × (24 - 1) / 32 = 72%

36 10 32 100 × (36 - 10) / 32 = 81%



## 15.0 Method Performance 15.1 The performance characteristics of Method 1106.1 (mE-EIA) for enumerating enterococci in marine waters were initially re ported by Levin, et al. (Reference 18.4) and are summarized in Section 15.2. In a subsequent study, Method 1106.1 precision was evaluated in both fresh and marine recreational waters and in sewage treatment plant effluents. The results of this study are summarized in Section 15.3. Based on these study results, Method 1106.1 was approved for enterococci monitoring of ambient recreati onal waters. More recently, Method 1106.1 was evaluated for use in disinfected wastewater matrices. The results of this interlaboratory validation study (Reference 18.3) are summarized in Section 15.4. 15.2 Performance of mE-EIA Method in Marine Waters 15.2.1 Precision - The degree of agreement of repeated measurements of the same parameter expressed quantitatively as the standard deviation or as the 95% confidence limits of the mean computed from the results of a series of controlled determinations. Precision of the mE method was established by Levin et al. (Reference 18.4) who demonstrated that the method did not exceed the expected limits for counts having the Poisson distribution. 15.2.2 Bias - The persistent positive or negative de viation of the results from the assumed or accepted true value. The bias of the ente rococci MF method with mE agar has been reported to be +2% of the true value (Reference 18.4).Method 1106.1

December 2009 19 15.2.3 Specificity - The ability of a method to select and/or distinguish the target bacteria from other bacteria in the same water sample. The specificity characteristic of a method is usually reported as the percent of false positiv e and false negative results. The specificity for this medium as reported for various environmental water samples was 10% false positive and 11.7% false negative (Reference 18.4). 15.3 Collaborative Study Data for Ambient Waters 15.3.1 A collaborative study was conducted among eleven volunteer laboratories, each with two analysts who independently tested local freshand marine recreational waters and sewage treatment plant effluent samples, in duplicate. The data were reported to the Environmental Monitoring and Support Laborat ory - Cincinnati, U.S. Environmental Protection Agency, for statistical analyses. 15.3.2 The results of the study are shown in Figure 1 where S O equals the pooled standard deviation among replicate counts from a single analyst for three groupings (counts less than 30, counts from 30 to 50, and counts greater than 50) and S B equals the pooled standard deviation between means of duplicat es from analysts in the same laboratory for the same groupings. The precision estimates from this study did not differ with the water types tested. 15.3.3 By linear regression, the precision of the method can be generalized as: S O = 0.013 count/100 mL + 2.42 (dilution factor) and S B = 0.152 count/100 mL + 5.16 (dilution factor)

Where dilution factor = 100 Volume of original sample filtered

15.3.4 Because of the instability of microbial populations in water samples, each laboratory analyzed its own sample series and no full measure of recovery or bias was possible. However, all laboratories analyzed a single surrogate sample prepared from a freeze-dried culture of E. faecalis . The mean count ( 0 ) and the standard deviation of the counts (S y ) (including the variability among laboratories for this standardized enterococci sample) were 32.5 colonies/membrane and 9.42 colonies/membrane, respectively.Method 1106.1

December 2009 20Method 1106.1

December 2009 21 15.4 Interlaboratory validation of Method 1106.1 in disinfected wastewater 15.4.1 Ten volunteer participant laboratories, an en terococci verification laboratory, and two research laboratories participated in the U.S. Environmental Protection Agency’s (EPA’s) interlaboratory validation study of EPA Me thod 1106.1. The purposes of the study were to characterize method performance across multiple laboratories and disinfected wastewater matrices and to develop quantitative quality control (QC) acceptance criteria. A detailed description of the of the study and results are provided in the validation study report (Reference 18.3). The false positive and negative assessments for Method 1106.1 are provided in Tables 7 and 8 , respectively. 15.4.2 Method 1106.1 is not approved for the analysis of enterococci in wastewater because of the high estimated number of false positive and negative colonies observed during the validation study. Users wishing to test for enterococci in wastewater using a membrane filtration method are referred to EPA Method 1600. Table 7. False Positive Assessment of Unspiked Disinfected and Unspiked Secondary Wastewater Effluents

Matrix Total colonies False Positive (FP) Assessment Typical Atypical Typical colonies submitted No. FP colonies FP confirmation

rate (%) a % of total colonies that would have been a FP b
Disinfected 877 267 107 0 0 0

Secondary 689 212 80 15 18.8 14.3

Disinfected + Secondary 1566 479 187 15 8 6.1

a False positive confirmation rate = num ber of false positive colonies / num ber of typical colonies submitted b Calculated as follows: (total typical colonies × FP confi rmation rate) / (total number of typical and atypical colonies observed); e.g., (689×(15/ 80))/(689+212) = 0.1434 = 14.3%Method 1106.1

December 2009 22 Table 8. False Negative Assessment of Unspiked Disinfected and Unspiked Secondary Wastewater Effluents

Matrix Total colonies False Negative (FN) Assessment

Typical Atypical Atypical colonies submitted No. FN colonies FN confirmation

rate (%) a % of total colonies that would have been a FN b
Disinfected 877 267 38 23 60.5 14.1

Secondary 689 212 56 31 55.4 13.0

Disinfected + Secondary 1566 479 94 54 57.4 13.5

a False negative confirmation rate = num ber of false negative colonies / number of atypical colonies submitted b Calculated as follows: (total atypical colonies × FN conf irmation rate) / (total number of typical and atypical colonies observed); e.g., (212×(31/ 56))/(689+212) = 0.1302 = 13.0% 

## 16.0 Pollution Prevention 16.1 The solutions and reagents used in this me thod pose little threat to the environment when recycled and managed properly. 16.2 Solutions and reagents should be prepared in vol umes consistent with laboratory use to minimize the volume of expired materials to be disposed. 

## 17.0 Waste Management 17.1 It is the laboratory’s responsibility to comply with all federal, state, and local regulations governing waste management, particularly the bi ohazard and hazardous waste identification rules and land disposal restrictions, and to protect the air, water, and land by minimizing and controlling all releases from fume hoods and benc h operations. Compliance with all sewage discharge permits and regul ations is also required. 17.2 Samples, reference materials, and equipment known or suspected to have viable enterococci attached or contained must be sterilized prior to disposal. 17.3 For further information on waste management, consult “The Waste Management Manual for Laboratory Personnel” and “Less Is Better: La boratory Chemical Management for Waste Reduction,” both available from the American Chemical Society's Department of Government Relations and Science Policy, 1155 16th Street NW, Washington, DC 20036.Method 1106.1

December 2009 23 

## 18.0 References 18.1 Cabelli, V. J., A. P. Dufour, M. A. Levin, L. J. McCabe, and P. W. Haberman, 1979. Relationship of Microbial Indicators to Health Effects at Marine Bathing Beaches . American Journal of Public Health. 69:690-696. 18.2 Dufour, A.P. 1984. Health Effects Criteria for Fresh Recreational Waters , EPA 600/1-84-004. Office of Research and Development, USEPA. 18.3 USEPA. 2005. Results of the Interlaboratory Validation of EPA Method 1106.1 (mE-EIA) for Enterococci in Wastewater Effluent . December 2004. EPA-821-R-04-018. 18.4 Levin, M. A., J. R. Fischer and V. J. Cabelli. 1975. Membrane Filter Technique for Enumeration of Enterococci in Marine Waters . Applied and Environmental Microbiology 30:66-71. 18.5 Reagent Chemicals, American Chemical Society Specifications , American Chemical Society, Washington, DC. For suggestions of the testing of reagents not listed by the American Chemical Society, see AnalaR Standards for Laboratory Chemicals , BDH Ltd., Poole, Dorset, UK and the United States Pharmacopeia. 18.6 APHA. American Public Health Association. 1998. Standard Methods for the Examination of Water and Wastewater (20th ed.), 1015 Fifteenth Street NW, Washington D.C. 18.7 Bordner, R., J.A. Winter and P.V. Scarpino (eds.). 1978. Microbiological Methods for Monitoring the Environment, Water and Wastes , EPA-600/8-78-017. Office of Research and Development, USEPA

## Appendix A: Part II (General Operations), Section A (Sample Collection, Preservation, and Storage)

### Sample Collection 1 1.0 Sample Containers 1.1 Sample Bottles: bottles must be resistant to sterilizing conditions and the solvent action of water. Wide-m outh borosilicate glass bottles with screw-cap or ground-glass stopper or heat-resistant plastic bottles may be used if they can be sterilized without producing toxic materials (see examples A and C in Figure 1). Screw-caps must not produce bacteriostatic or nutritive compounds upon sterilization.

Figure 1. Suggested sample containers. 1.2 Selection and Cleaning of Bottles: Samples bottles should be at least 125 mL volum e for adequate sampling and for good mixing. Bottles of 250 mL, 500 mL, and 1000 mL volume are often used for multiple analyses. Discard bottles which have chips, cracks, and etched surfaces. Bottle closures must be water-tight. Before use, thoroughly cleanse bottles and closures with detergent and hot water, followed by a hot water rinse to remove all trace of detergent. Then rinse them three times with laboratory-pure water. 1.3 Dechlorinating Agent: The agent m ust be placed in the bottle when water and wastewater sam ples containing residual chlorine are anticipated. Add sodium thiosulfate to the bottle before sterilization at a concentration of 0.1 mL of a 10% solution for each 125 mL sample volume. This concentration will neutralize approximately 15 mg/L of residue chlorine. 1.4 Chelating Agent: A chelating agent should be added to sam ple bottles used to collect samples suspected of containing >0.01 m g /L concentrations of heavy metals such as copper, nickel or zinc, etc. Add 0.3 mL of a 15% solution of ethylenediaminetetraacetic acid (EDTA) tetrasodium salt, for each 125 mL sample volume prior to sterilization.

1 The text is taken from Part II, Section A, of the EPA publication "Microbiological Methods for Monitoring the Environment" EPA-600/8-78-017, December 1978. 

### 11.5 Wrapping Bottles: Protect the tops and necks of glass stoppered bottles from contamination by covering them before sterilization with alum inum foil or kraft paper. 1.6 Sterilization of Bottles: Autoclave glass or heat-resistant plastic bottles at 121°C for 15 m inutes. Alternatively, dry glassware m ay be sterilized in a hot oven at 170°C for not less than two hours. Ethylene oxide gas sterilization is acceptable for plastic containers that are not heat-resistant. Sample bottles sterilized by gas should be stored overnight before being used to allow the last traces of gas to dissipate. 1.7 Plastic Bags: The commercially available bags (Whirl-pak) (see example B in Figure 1) are a practical substitute for plastic or glass sam ples bottles in sampling soil, sedim ent, or biosolids. The bags are sealed in m anufacture and opened only at time of sampling. The manufacturer states that such bags are sterilized. 2.0 Sampling Techniques Samples are collected by hand or with a sampling device if the sampling site has difficult access such as a bridge or bank adjacent to a surface water. 2.1 Chlorinated Samples: When samples such as treated waters, chlorinated wastewaters or recreational waters are collected, the sam ple bottle m u st contain a dechlorinating agent (see section 1.3 above). 2.2 Composite Sampling: In no case should a com posite sample be collected for bacteriologic exam ination. Data from individual samples show a range of values. A composite sample will not display this range. Individual results will give information about industrial process variations in flow and composition. Also, one or more portions that make up a composite sample may contain toxic or nutritive materials and cause erroneous results. 2.3 Surface Sampling by Hand: A grab sam ple is obtained using a sample bottle prepared as described in (1) above. Identify the sampling site on the bottle label and on a field log sheet. Remove the bottle covering and closure and protect from contamination. Grasp the bottle at the base with one hand and plunge the bottle mouth down into the water to avoid introducing surface scum (Figure 2). Position the mouth of the bottle into the current away from the hand of the collector and, if applicable, away from the side of the sampling platform. The sampling depth should be 15-30 cm (6-12 inches) below the water surface. If the water body is static, an artificial current can be created, by moving the bottle horizontally in the direction it is pointed and away from the sampler. Tip the bottle slightly upwards to allow air to exit and the bottle to fill. After removal of the bottle from the stream, pour out a small portion of the sample to allow an air space of 2.5-5 cm (1-2 inches) above each sample for proper mixing of the sample before analyses. Tightly stopper the bottle and place on ice (do not freeze) for transport to the laboratory.



### 2Figure 2. Grab sampling technique for surface waters.

3.0 Selection of Sampling Sites and Frequency These will be described for streams, rivers, estuarine, marine, and recreational waters as well as domestic and industrial wastewaters. 3.1 Stream Sampling: The objectives of the initial survey dictate the location, frequency and number of sam ples to be collected. 3.1.1 Selection of Sampling Sites: A typical stream sam pling program includes sampling locations upstream of the area of concern, upstream and downstream of waste discharges, upstream and downstream from tributary entrances to the river and upstream of the mouth of the tributary. For more complex situations, where several waste discharges are involved, sampling includes sites upstream and downstream from the combined discharge area and samples taken directly from each industrial or municipal waste discharge. Using available bacteriological, chemical and discharge rate data, the contribution of each pollution source can be determined. 3.1.2 Small Streams: Sm all streams should be sam pled at background stations upstream of the pollution sources and at stations downstream from pollution sources. Additional sampling sites should be located downstream to delineate the zones of pollution. Avoid sampling areas where stagnation may occur (e.g., backwater of a tributary) and areas located near the inside bank of a curve in the stream which may not be representative of the main channel.



### 33.1.3 Large Streams and Rivers: Large streams are usually not well mixed laterally for long distances downstream from the pollution sources. Sampling sites below point source pollution should be established to provide desired downstream travel time and dispersal as determined by flow rate measurements. Particular care must be taken to establish the proper sampling points. Occasionally, depth samples are necessary to determine vertical mixing patterns. 3.2 Estuarine and Marine Sampling : Sam pling estuarine and m arine waters requires the consideration of other factors in addition to those usually recognized in fresh water sampling. They include tidal cycles, current patterns, bottom currents and counter-currents, stratification, seasonal fluctuations, dispersion of discharges and multi-depth samplings. The frequency of sampling varies with the objectives. When a sampling program is started, it m ay be necessary to sample every hour around the clock to establish pollution loads and dispersion patterns. The sewage discharges may occur continuously or intermittently. When the sampling strategy for a survey is planned, data may be available from previous hy drological studies done by the Coast Guard, Corps of Engineers, National Oceanic and Atm o spheric Administration (NOAA), U.S. Geological Survey, or university and private research investigations. In a survey, float studies and dye studies are often carried out to determine surface and undercurrents. Initially depth samples are taken on the bottom and at five feet increments between surface and bottom. A random grid pattern for selecting sampling sites is established statistically. 3.2.1 Estuarine Sampling: When a survey is ma de on an estuary , samples are often taken from a boat, usually making an end to end traverse of the estuary. Another method involves taking samples throughout a tidal cycle, every hour or two hours from a bridge or from an anchored boat at a number of fixed points. In a large bay or estuary where many square miles of area are involved, a grid or series of stations m ay be necessary . Two sets of samples are usually taken from an area on a given day, one at ebb or flood slack water, and the other three hours earlier, or later, at the half tidal interval. Sampling is scheduled so that the mid-sampling time of each run coincides with the calculated occurrence of the tidal condition. In location sampling sites, one must consider points at which tributary waters enter the m ain stream or estuary , location of shellfish beds and bathing beaches. The sampling stations can be adjusted as data accumulate. For example, if a series of stations half mile apart consistently show similar values, some of these stations may be dropped and other stations added in areas where data shows more variability. Considerable stratification can occur between the salt water from the sea and the fresh water supplied bya river. It is essential when starting a survey of an unknown estuary to find out whether there is any m arked stratification. This can be done by chloride determinations at different locations and depths. It is possible for stratification to occur in one part of an estuary and not in another.



### 4On a flood tide, the more dense salt water pushing up into the less dense fresh river water will cause an overlapping with the fresh water flowing on top. A phenomenon called a salt water wedge can form. As a result, stratification occurs. If the discharge of pollution is in the salt water layer, the contamination will be concentrated near the bottom at the flood tide. The flow or velocity of the fresh water will influence the degree of stratification which occurs. If one is sampling only at the surface, it is possible that the data will not show the polluted underflowing water which was contaminated at the point below the fresh water river. Therefore, where stratification is suspected, samples at different depths will be needed to measure vertical distribution. 3.2.2 Marine Sampling: In ocean studies, the environm ental conditions are mo st diverse along the coast where shore, atm osphere and the surf are strong influences. The shallow coastal waters are particularly susceptible to daily fluctuations in temperature and seasonal changes. Sampling during the entire tidal cycle or during a half cycle may be required. Many ocean studies such as sam pling over the continental shelf involve huge areas and no two areas of water are the same. Selection of sampling sites and depths are m ost critical in marine waters. In winter, cooling of coastal waters can result in wate r layers which approach 0°C. In summer, the shallow waters warm much faster than the deeper waters. Despite the higher temperature, oxygen concentrations are higher in shallow than in deeper waters due to greater water movement, surf action and photosynthetic activity from macrophytes and the plankton. Moving from the shallow waters to the intermediate depths, one observes a m oderation of these shallow water characteristics. In the deeper waters, there is a marked stabilization of conditions. Water tem peratures are lower and more stable. There is limited turbulence, little penetration of light, sparse vegetation and the ocean floor is covered with a layer of silts and sediments. 3.3 Recreational Waters (Bathing Beaches): Sam pling sites at bathing beaches or other recreational areas should include upstream or peripheral areas and locations adjacent to natural drains that would discharge stormwater, or run-off areas draining septic wastes from restaurants, boat marinas, or garbage collection areas. Samples of bathing beach water should be collected at locations and times of heaviest use. Daily sampling, preferably in the afternoon, is the optimum frequency during the season. Weekends and holidays which are periods of highest use must be included in the sampling program. Samples of estuarine bathing waters should be obtained at high tide, ebb tide and low tide in order to determine the cyclic water quality and deterioration that must be monitored during the swimming season.



### 53.4 Domestic and Industrial Waste Discharges: It is often necessary to sample secondary and tertiary wastes from municipal waste treatment plants and various industrial waste treatment operations. In situations where the plant treatment efficiency varies considerably, grab samples are collected around the clock at selected intervals for a three to five day period. If it is known that the process displays little variation, fewer samples are needed. In no case should a composite sample be collected for bacteriological examination. The National Pollution Discharge Elimination System (NPDES) has established wastewater treatment plant effluent limits for all dischargers. These are often based on maximum and mean values. A sufficient number of samples must be collected to satisfy the permit and/or to provide statistically sound data and give a fair representation of the bacteriological quality of the discharge.



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## Appendix B: Part II (General Operations), Sections C.3.5 (Counting Colonies) and C.3.6 (Calculation of Results)

### Counting Colonies 1 1.0 Counting Colonies Colonies should be counted using a fluorescent lamp with a magnifying lens. The flourescent lamp should be nearly perpendicular to the memb rane filter. Count colonies individually, even if they are in contact with each other. The technician must learn to recognize the difference between two or more colonies which have grown into contact with each other and single, irregularly shaped colonies which sometimes develop on membrane filters. The latter colonies are usually associated with a fiber or particulate material and the colonies conform to the shape and size of the fiber or particulates. Colonies which have grown together almost invariably show a very fine line of contact. 2.0 Calculation of Results 2.1 Select the membrane filter with the number of colonies in the acceptable range and calculate count per 100 m L according to the general formula: Count per 100 mL = (No. of colonies counted/Volume of sam ple filtered, in mL) x 100 2.2 Counts Within the Acceptable Limits The acceptable range of colonies that are countable on a membrane is a function of the method. Different me thods m ay have varying acceptable count ranges. All examples in this appemdix assume that the acceptable range of counts is between 20- 6 0 colonies per membrane. For example, assume that filtration of volumes of 50, 15, 5, 1.5, and 0.5 mL produced colony counts of 200, 110, 40, 10, and 5, respectively. An analyst would not actually count the colonies on all filters. By inspection the analyst would select the membrane filter with the acceptable range of target colonies, as defined by the method, and then limit the actual counting to such membranes. After selecting the best membrane filter for counting, the analyst counts colonies and applies the general form ula as in section 2.1 above to calculate the count/100 mL. 2.3 More Than One Acceptable Count 2.3.1 If there are acceptable counts on replicate plates, carry counts independently to final reporting units, then calculate the arithmetic mean of these counts to obtain the final reporting value.

1 The text is largely taken from Part II, Section C, of the EPA publication "Microbiological Methods for Monitoring the Environment" EPA-600/8-78-017, December 1978. Some examples were kindly provided by Kristen Brenner, US EPA. 

### 1Example, if the counts are 24 and 36 for replicate plates of 100 mL each, then the arithmetic mean is calculated as follows: (24 CFU/100 mL + 36 CFU/100 mL) = 30 CFU/ 100 mL 2 2.3.2 If there is more than one dilution having an acceptable range of counts, independently carry counts to final reporting units, then average for final reported value. For example, if volumes of 100, 10, 1 and 0.1 mL produced colony counts of Too Numerous To Count (TNTC), 5 5, 30, and 1, respectively, then two volumes, 10 mL and 1 mL, produced colonies in the acceptable counting range. Independently carry each MF count to a count per 100 mL:

5 5 × 100 = 5 50 CFU /100 mL 10 and

30 × 100 = 3000 CFU /100 mL 1

Calculate the arithmetic mean as in section 2.3.1 above: ( 5 50 CFU/100 mL + 3000 CFU/100 mL) = 1 7 75 CFU/100 mL 2 Report this as 1 7 75 CFU/100 mL.

2.4 If all MF counts are below the lower acceptable count limit, select the most nearly acceptable count. 2.4.1 For example, sample volumes of 100, 10 and 1 mL produced colony counts of 17, 1 and 0, respectively. Here, no colony count falls within recomm e nded limits. Calculate on the basis of the most nearly acceptable plate count, 17, and report as 17 CFU/100 m L. Notethat in this case, because no calculations were done (i.e. this is the count for 100 m L), the count is reported as 17 CFU/100 mL rather than an “estimated count of 17 CFU/100 m L”



### 22.4.2 As a second example, assume a count in which sample volumes of 10 and 1 mL produced colony counts of 18 and 0, respectively. Here, no colony count falls within recommended limits. Calculate on the basis of the most nearly acceptable plate count, 18, and calculate as in section 2.3.2 above.

18 × 100 = 180 CFU /100 mL 10 Report this as an estimated count of 180 CFU/100 mL. 2.5 If counts from all membranes are zero, calculate using count from largest filtration volume. For example, sample volumes of 25, 10, and 2 mL produced colony counts of 0, 0, and 0, respectively, and no actual calculation is possible, even as an estim ated report. Calculate the number of colonies per 100 mL that would have been reported if there had been one colony on the filter representing the largest filtration volume. In this example, the largest volume filtered was 25 mL and thus the calculation would be:

1 × 100 = 4 CFU /100 mL 25 Report this as < (less than) 4 CFU/100 mL. 2.6 If all membrane counts are above the upper acceptable limit, calculate count using the smallest volume filtered. For exam ple, assum e that the volumes 1, 0.3, and 0.01 mL produced colony counts of TNTC, 150, and 110 colonies, respectively. Since all colony counts are above the acceptable lim it, use the colonycount from the smallest sample volume filtered and estimate the count as:

110 × 100 = 1,100,000 CFU /100 mL 0.01

Report this as estimated count 1.1 × 10 6 CFU/100 mL 2.7 If typical colonies are too numerous to count (TNTC), use upper limit count with smallest filtration volume.



### 3For example, assume that the volumes 1, 0.3, and 0.01 mL all produced too many typical colonies, and that the laboratory bench record indicated TNTC. Use the upper acceptable count for the method ( 6 0 colonies in this example) as the basis of calculation with the smallest filtration volume and estimate the count as:

6 0 × 100 = 6 00,000 CFU /100 mL 0.01 Report this as > (greater than) 6 ×10 5 CFU/100 mL 2.8 If colonies are both above and below the upper and lower acceptable limits (i.e., no counts are within the acceptable limits), select the m o st nearly acceptable count. 2.8.1 For example, sam ple volumes of 100, 10 and 1 mL produced colony counts of 6 4, 6 and 0, respectively. Here, no colony count falls within recommended lim its. Calculate on the basis of the m ost nearly acceptable plate count, 6 4, and report as 6 4 CFU/100 mL. Notethat in this case, because no calculations were done (i.e. this is the count for 100 mL), the count is reported as 6 4 CFU/100 mL rather than an “estimated count of 64 CFU/100 mL” 2.8.2 As a second example, assume a count in which sam ple volum es of 100, 10 and 1 mL produced colony counts of 98, 18, and 0, respectively. Here, no colony count falls within recomme nded limits. Calculate on the basis of the m ost nearly acceptable plate count, 18, and calculate as in section 2.3.2 above.

18 × 100 = 180 CFU /100 mL 10 Report this as estimated count 180 CFU/100 mL. 2.9 If there is no result because of a confluent growth, > 200 atypical colonies (TNTC), lab accident, etc., report as No Data and specify the reason.



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