# EPA United States Office of Water Environmental Protection W a shington DC September 2002 Agency 20460 EPA 821-R-02-022



# Method 1600: Enterococci in Water by Membrane Filtration Using membrane-Enterococcus Indoxyl- 

# $ -D-Glucoside Agar (mEI)

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

EPA-821-R-02-022

## Acknowledgments

This m e thod was developed under the direction of Jam es W. Messer and Alfred P. Dufour of the U.S. Environm ental Protection Agency ' s (EPA) Hum a n Exposure Research Division, National Exposure Research Laboratory , Cincinnati, Ohio. 

## Disclaimer

The Engineering and Analy s is Division, of the Office of Science and Technology , has reviewed and approved this report for publication. The Office of Science and Technology directed, m a naged, and reviewed the work of Dy nCorp in preparing this re port. Neither the United States Governm e nt nor any of its em ploy ees, contractors, or their employ ees m a ke any warranty , expressed or implied, or assum es any legal liability or responsibility for any third party ’ s use of or the results of such use of any inform ation, apparatus, product, or process discussed in this report, or represents that its use by such party would not infringe on privately owned rights. This docum en t com b ines the inform ation previously published in Method 1600: Membrane Filter Test Method for Enterococci in Water (EPA-821-R-97-004) (Reference 18.8) and Improved Enumeration Methods for the Recreati onal Water Quality Indicators: Enterococci and Escherichia coli (EPA/821/R-97/004) (Reference 18.7). Mention of trade nam es or com m e rcial products does not constitute endorsem ent or recom m e ndation for use.

Questions concerning this m e thod or its application should be addressed to: Robin K. Oshiro Engineering and Analy s is Division (4303T) U.S. EPA Office of Water, Office of Science and Technology 1200 Pennsy l vania Avenue, NW Washington, DC 20460 oshiro.robin@epa.gov 202-566-1075 202-566-1053 (facsim ile)

## Introduction

EPA has been increasingly concerned with the pub lic health risks of infectious diseases caused by m i crobial organism s in our nation' s beaches. In response to this problem , EPA has established the Beaches Environm ental Assessm ent Closure and Health (BEACH) Program

. This analy tical m e thod is published for use in the BEACH Program

. In 1986, EPA issued a revision to its bacteriological am bient water quality criteria recom m e ndations to include new indicator bacteria, E. coli and enterococci, which provide a better correlation with swim m i ng-associated gastrointes tinal illness than the previous criteria recom m e ndations for fecal coliform bacteria. These revised criteria are useful to public health offi cials because they enable quantitative estim ates of illness rates associat ed with swim m i ng in polluted water. This m e thod is a revision of EPA' s previous enterococci m e thod, used since 1985 in am bient water quality m onitoring. It reduces analy s is tim e to 24 hours and im proves analy tical quality

. The m e thod has been validated in single- and m u lti-laborat ory studies and has undergone peer review.

## Table of Contents

1

. 0 S c o p e a n d A p p l i c a t i o n ......................................................... 1 2

. 0 S u m m a r y o f M e t h o d

........................................................... 1 3

. 0 D e f i n i t i o n s ................................................................... 1 4

. 0 I n t e r f e r e n c e s ................................................................. 1 5

. 0 S a f e t y ...................................................................... 2 6

. 0 E q u i p m e n t a n d S u p p l i e s ........................................................ 2 7

. 0 R e a g e n t s a n d S t a n d a r d s ........................................................ 3 8.0 Samp le Collection, Preservation, and Storage

....................................... 5 9.0 Quality Control

............................................................... 6 1 0

. 0 C a l i b r a t i o n a n d S t a n d a r d i z a t i o n .................................................. 6 1 1

. 0 P r o c e d u r e ................................................................... 6 1 2

. 0 D a t a A n a l y s i s a n d C a l c u l a t i o n s .................................................. 7 13.0 Method Perform ance ........................................................... 7 1 4

. 0 R e p o r t i n g R e s u l t s ............................................................. 7 1 5

. 0 V e r i f i c a t i o n P r o c e d u r e ......................................................... 7 1 6

. 0 P o l l u t i o n P r e v e n t i o n ........................................................... 8 1 7

. 0 W a s t e M a n a g e m e n t

............................................................ 8 1 8

. 0 R e f e r e n c e s ................................................................... 8

## Method 1600: Enterococci in Water by Membrane Filtration Using membrane-Enterococcus Indoxy l - 

## $ -D-Glucoside Agar (mEI) September 2002

1.0 Scope and Application 1.1 This m e thod describes a m e m b rane filter (MF) pro cedure for the detection and enum eration of the enterococci bacteria in water. Enterococci are com m only found in the feces of hum ans and other warm -blooded anim als. Although som e strains ar e ubiquitous and not related to fecal pollution, the presence of enterococci in water is an indi cation of fecal pollution and the possible presence of enteric pathogens. 1.2 The enterococci test m easures th e bacteriological quality of recreational waters. Epidem iological studies have led to the developm ent of criteria which can be used to prom ulgate recreational water standards based on the established relations hip between health effects and water quality

. The significance of finding enterococci in recrea tional water samples is the direct relationship between the density of enterococci in the wate r and swim m i ng-associated gastroenteritis studies of m a rine and fresh water bathing beaches (Reference 18.3, Reference 18.4). 1.3 The test for enterococci can be applied to potab le, fresh, estuarine, m a rine, and shellfish growing waters. 1.4 Since a wide range of sample volum es or dilutions can be analy zed by the MF technique, a wide range of enterococci levels in wate r can be detected and enum erated. 

## 2.0 Summary of Method 2.1 The MF m e thod provides a direct count of bacteria in water based on the developm ent of colonies on the surface of the m e m b rane filter (Reference 18.5). A water sam ple is filtered through the m e m b rane which retains the bacteria. Following f iltration, the m e m b rane containing the bacterial cells is placed on a selective m e dium , m E I agar, and incubated for 24 h at 41°C. All colonies (regardless of color) with a blue halo are record ed as enterococci colonies. Magnification and a sm all fluorescent lam p are used for counting to give m a xim u m visibility of colonies. 

## 3.0 D efinitions 3.1 In this m e thod, enterococci are those bacteria which produce colonies with a blue halo after incubation on m E I agar. Enterococci include Streptococcus faecalis , Streptococcus faecium , Streptococcus avium , and their variants. 

## 4.0 Interferences 4.1 Water samples containing colloidal or suspended particulate m a terials can clog the m e m b rane filter and prevent filtration, or cause spreading of bacterial colonies which could interfere with identification of target colonies.

1 September 2002Method 1600 

## 5.0 Safety 5.1 The analy s t/technician m u st know and observe the norm a l safety procedures required in a m i crobiology laboratory while preparing, using, and disposing of cultures, reagents, and m a terials, and while operating sterilization equipm ent. 5.2 Mouth-pipetting is prohibited. 

## 6.0 E quipment and Supplies 6.1 Glass lens with m a gnification of 2-5x or stereoscopic m i croscope. 6.2 Lamp , with a cool, white fluorescent tube. 6.3 Hand tally or electronic counting device. 6.4 Pipet container, stainless steel, alum inum or borosilicate glass, for glass pipets. 6.5 Pipets, sterile, T.D. bacteriological or Mohr, glass or plastic, of appropriate volum e. 6.6 Graduated cy linders, 100-1000 m L , covered with alum inum foil or kraft paper and sterile. 6.7 Mem b rane filtration units (filter base and funnel) , glass, plastic or stainless steel, wrapped with alum inum foil or kraft paper and sterilized. 6.8 Ultraviolet unit for sanitization of the filte r funnel between filtrations (optional). 6.9 Line vacuum , electric vacuum pum p, or aspirator for use as a vacuum source. In an em ergency or in the field, a hand pum p or a sy ringe 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 appropria te tubing. A filter m a nifold to hold a num ber of filter bases is optional. 6.11 Flask for safety trap placed between th e filter flask and the vacuum source. 6.12 Forceps, straight or curved, with sm ooth tips to handle filters without dam a ge. 6.13 Ethanol, m e thanol or isopropanol in a sm all, wi de-m outh container, for flam e-sterilizing forceps. 6.14 Burner, Bunsen or Fisher ty pe, or electric in cinerator unit for sterilizing loops and needles. 6.15 Therm o m e ter, checked against a National Institute of Standards and Technology (NIST) certified therm o m e ter, or one that m eets the requirem e nts of NIST Monograph SP 250-23. 6.16 Petri dishes, sterile, plastic, 9 x 50 m m , with tight-fitting lids. 6.17 Bottles, m ilk dilution, borosilicate glass, screw-cap with neoprene liners, m a rked at 99 m L for 1:100 dilutions. Dilution bottles m a rked at 90 m L or tubes m a rked at 9 m L m a y be used for 1:10 dilutions. 6.18 Flasks, borosilicate glass, screw-cap, 250-2000 m L volum e. 6.19 Mem b rane filters, sterile, white, grid m a rked, 47 m m diam eter, with 0.45 ± 0.02 μm pore size. 6.20 Inoculation loops, at least 3 m m diam eter, and needles, nichrom e or platinum wire, 26 B & S gauge, in suitable holders. Sterile disposable app licator sticks or plastic loops are alternatives to inoculation loops. 6.21 Incubator m a intained at 41 ± 0.5°C. 6.22 Waterbath m a intained at 50°C for temp ering agar. September 2002 2Method 1600 6.23 Test tubes, 20 x 150 m m , borosilicate glass or plastic. 6.24 Caps, alum inum or autoclavable plastic, for 20 m m diam eter test tubes. 6.25 Test tubes, screw-cap, borosilicate glass, 16 x 125 m m or other appropriate size. 6.26 Whirl-Pak® bags. 

## 7.0 Reagents and Standards 7.1 Purity of Reagents: Reagent grade chem icals sh all be used in all tests. Unless otherwise indicated, reagents shall conform to the specifi cations of the Com m ittee on Analy tical Reagents of the Am erican Chem ical Society (Reference 18. 9). The agar used in preparation of culture m e dia m u st be of m i crobiological grade. 7.2 Whenever possible, use com m e rcial cultu re m e dia as a m eans of quality control. 7.3 Purity of Water: Reagent water conform i ng to Specification D1193, reagent water conform i ng Ty pe II, Annual Book of ASTM Standards (Reference 18.1). 7.4 Buffered Dilution Water 7.4.1 Composition: Sodium Dihy drogen Phosphate 0.58 g Sodium Monohy drogen Phosphate 2.50 g Sodium Chloride 8.50 g Reagent-Grade Distilled Water 1.0 L 7.4.2 Preparation: Dissolve the ingredients in 1 L of reagent water in a flask and dispense in appropriate am ounts for dilutions in screw-cap bottles or culture tubes, and/or into containers for use as rinse water. Autoclav e after preparation at 121°C (15 lb pressure) for 15 m i n. Final pH should be 7.4 ± 0.2. 7.5 Phosphate Buffered Dilution Water (Reference 18.2) 7.5.1 Composition of Stock Phosphate Buffer Solution: Phosphate dihy droge n phosphate 34.0 g Reagent-Grade distilled water 500.0 m L 7.5.2 Preparation: Adjust the pH of the solution to 7.2 with 1 N NaOH, and bring the volum e to 1 L with reagent-grade distilled water. Sterilize by filtration or autoclave at 121°C (15 lb pressure) for 15 m i n. 7.5.3 Preparation of Stock Magnesium Chlori de Solution: Add 38 g anhy drous MgCl 2 or 81.1 g MgCl 2 A 6H 2 O to 1 L reagent-grade distilled water. Sterilize by filtration or autoclave at 121°C (15 lb pressure) for 15 m i n. 7.5.4 Storage of Stock Solutions: After sterilization, store the stock solutions in the refrigerator until used. Handle aseptically

. If evidence of m o ld or other contam ination appears, the affected stock solution should be discarded and a fresh solution should be prepared. 7.5.5 Working Phosphate Buffered Dilution Water: Mix 1.25 m L of the stock phosphate buffer and 5 m L of the MgCl 2 stock per liter of reagent-grade distilled water. Dispense in

3 September 2002Method 1600 appropriate am ounts for dilutions in screw-cap bottles or culture tubes, and/or into containers for use as rinse water. Autoclav e at 121°C (15 lb pressure) for 15 m i n. Final pH should be 7.0 ± 0.2. 7.6 mE I A g a r 7.6.1 Composition of Basal Medium (m E Agar, Difco 0333) Peptone Sodium Chloride Yeast Extract Esculin Actidione (Cy c lohexim i de) Sodium Azide Agar Reagent-Grade Distilled Water 10.0 g 15.0 g 30.0 g

1.0 g 0.05 g 0.15 g 15.0 g

1.0 L 7.6.2 Preparation of m E I Medium : Add 71.2 g of dehy drated basal m e dium plus 0.75 gram s of indoxy l $ -D glucoside to 1 L of reagent grade water in a flask and heat to boiling until ingredients dissolve. Autoclave at 121°C (15 lb pressure) for 15 m i n and cool in a 50°C water bath. 7.6.3 Reagents Added After Sterilization: Mix 0. 24 g nalidixic acid in 5 m L reagent-grade sterile distilled water, add a few drops of 0.1N NaOH to dissolve; add to the m E I m e dium

. Add 0.02 g tripheny ltetrazolium chloride separately to the m E I m e dium and mi x . 7.6.4 Alternately , the following solutions m a y be used: 7.6.4.1 Nalidixic acid: Add 0.48 g of nalidixic acid and 0.4 m L 10 N NaOH to 10 m L of reagent-grade distilled water and m i x. Filter-sterilize the solution, and add 5.2 m L per liter of m e dium

. 7.6.4.2 Tripheny ltetrazolium chloride (TTC): Add 0.1 g of TTC to 10 m L of reagentgrade distilled water, and warm to dissolve. Filter-sterilize the solution, and add 2 m L per liter of m e dium

. 7.6.5 Preparation of m E I Agar Plates: Pour the m E I agar into 9x50 m m petri dishes to a 4-5 m m depth (approxim a tely 4-6 m L ), and allow to solidify

. Final pH of m e dium should be 7.1 ± 0.2. Store in a refrigerator. 7.7 Brain Heart Infusion Broth (BHIB) (Difco 0037, BD 4311059) 7.7.1 Com position: Calf Brain Infusion Beef Heart Infusion Peptone Sodium Chloride Disodium Phosphate Dextrose Reagent-Grade Distilled Water 200.0 g 250.0 g

10.0 g

5.0 g 2.5 g 2.0 g 1.0 L 7.7.2 Preparation: Dissolve 37 g of dehy drated brain heart infusion in 1 L of reagent grade water. Dispense in 8-10 m L volum es in screw-cap tubes and autoclave at 121°C (15 lb pressure) for 15 m i n. If the m e dium is not used the sam e day as prepared and sterilized, September 2002 4Method 1600 heat in boiling water bath for several m i n to rem ove absorbed oxy gen, and cool quickly without agitation, just prior to inocula tion. The final pH should be 7.4 ± 0.2. 7.8 Brain Heart Infusion Broth (BHIB) with 6.5% NaC1 7.8.1 Com position: BHIB with 6.5% NaC1 is the sam e as BHIB above, but with additional NaC1. 7.8.2 Preparation: Add 60.0 g NaC1 per liter of m e dium

. Since m o st com m e rcially available dehy drated m e dia contain sodium chloride, this am ount is subtracted from the 65 g per liter required to m a ke a final concentration of 6.5% NaCl. 7.9 Brain Heart Infusion Agar (BHIA) (Difco 0418, BD4311065) 7.9.1 Composition: BHIA contains the sam e components as BHIB above with the addition of 15.0 g of agar per L of BHIB. 7.9.2 Preparation: Suspend 52 g dehy drated BHIA in 1 L of reagent-grade distilled water. Heat to boiling until the ingredients are dissolved. Dispense 10 m L of m e dium in screwcap test tubes, and sterilize for 15 m i n at 121°C (15 lb pressure). After sterilization, slant until solid. Final pH should be 7.4 ± 0.2. 7.10 Bile Esculin Agar (BEA) (Difco 0879) 7.10.1 Composition: Bacto Beef Extract Bacto Peptone Bacto Oxgall Bacto Esculin Ferric Citrate Bacto Agar Reagent-Grade Distilled Water 3.0 g 5.0 g 40.0 g

1.0 g 0 .5 g 15.0 g

1 .0 L

7.10.2 Preparation: Add 64.0 g dehy drated BEA to 1 L reagent-grade distilled water, and heat to boiling to dissolve comp letely

. Dispense 10-m L volum es in tubes for slants or larger volum es into flasks for subsequent plating. Autoclave at 121°C (15 lb pressure) for 15 m i n. Overheating m a y cause darkening of the m e dium

. Cool in a 50°C waterbath, and dispense into sterile petri dishes. Final pH should be 6.6 ± 0.2. Store in a refrigerator. 

## 8.0 Sample Collection, Preservation, and Storage 8.1 Samp ling procedures are described in detail in the USEPA m i crobiology m e thods m a nual, Section II, A (Reference 18.2). Adherence to sample preservation procedures and holding tim e lim its is critical to the production of valid data. Sam p les shall not be analy zed if these conditions are not m e t. 8.1.1 Storage Tem p erature and Handling Conditions Ice or refrigerate bacteriological sam ples at a temp erature of 1-4°C during transit to the laboratory

. Use insulated containers to assure proper m a intenance of storage 5 September 2002Method 1600 temp erature. Take care that sam ple bottles are not totally im m e rsed in water during transit or storage. 8.1.2 Holding Tim e Lim itations Exam ine samples as soon as possible after collection. Do not hold samples longer than 6 h between collection and initiation of analy ses. 

## 9.0 Quality Control 9.1 See recom m e ndations on quality control for m i crobiological analy ses in the USEPA m i crobiology m e thods m a nual, Part IV, C (Reference 18.2). 

## 10.0 Calibration and Standardization 10.1 Check temp eratures in incubators daily to ensure operation within stated lim its. 10.2 Check therm o m e ters at least annually against a NIST certified therm o m e ter or one that m eets the requirem e nts of NIST Monograph SP 250-23. Check m e rcury colum n s for breaks. 

## 11.0 Procedure 11.1 Prepare the m E I agar as directed in 7.6. 11.2 Mark the petri dishes and report form s with sample identification and sample volum es. 11.3 Place a sterile m e m b rane filter on the filter base, grid-side up and attach the funnel to the base so that the m e m b rane filter is now held between the funnel and the base. 11.4 Shake the sample bottle vigorously about 25 tim es to distribute the bacteria uniform ly , and m easure the desired volum e of sample or dilution into the funnel. 11.5 Select sample volum es based on previous knowle dge of the pollution level, to produce 20-60 enterococci colonies on m e m b ranes. Samp le volum es of 1-100 m L are norm a lly tested at half log intervals (e.g., 100, 30, 10, 3 m L ). 11.6 Sm aller sample size or sample dilutions can be u sed to m i nim i ze the interference of turbidity or for high bacterial densities. Multiple volum es of the sam e sample or sample dilutions m a y be filtered, and the results m a y be com b ined. 11.7 Filter the sample, and rinse the sides of the funnel at least twice with 20-30 m L of sterile buffered rinse water. Turn off the vacuum a nd rem ove the funnel from the filter base. 11.8 Use sterile forceps to aseptically rem ove the m e m b rane filter from the filter base, and roll it onto the m E I agar to avoid the form ation of bubbles between the m e m b rane and the agar surface. Reseat the m e m b rane if bubbles occur. Run the fo rceps around the edge of the filter to be sure that the filter is properly seated on the agar. Clo se the dish, invert, and incubate at 41 ± 0.5°C for 24 h. 11.9 After incubation, count and record colonies on t hose m e m b rane filters containing, if practical, 20-60 colonies with a blue halo regardless of co lony color as an enterococci. Use m a gnification for counting and a sm all fluorescent lam p to give m a xim u m visibility of colonies.

September 2002 6Method 1600

Figure 1. Enterococci on m E I Agar. Colonies with a blue halo are considered to be enterococci. 

## 12.0 Data Analy s is and Calculations 12.1 Use the following general rules to calculate the enterococci count per 100 m l of sample: 12.1.1 Select the m e m b rane filter with an acceptable num ber of colonies (regardless of colony color) with a blue halo (20-60). Calculate the num ber of enterococci per 100 m L according to the following general form ula: Num b er of enterococci colonies Enterococci/100 m L = X 100 Volum e of sample filtered (m L) 12.1.2 See the USEPA m i crobiology m e thods m a nual, Part II, Section C, 3.5, for general counting rules (Reference 18.2). 

## 13.0 Method Performance 13.1 Specificity - The specificity of the m e dium used in this m e thod is 6.0% false positive and 6.5% false negative for various environm ental water sam ples (Reference 18.6). The false positive rate was calculated as the percent of colonies which reacted ty pically , but did not verify as m e m b ers of the enterococcus group. The false negative ra te was calculated as the percent of all verified enterococcus colonies not reacting ty pically

. 13.2 Bias - The persistent positive or negative devia tion of the results from the assum e d or accepted true value is not significant (Reference 18.6). 13.3 Precision - The precision am ong laboratories for m a rine water and surface water was 2.2% and 18.9% (Reference 18.6). 

## 14.0 R eporting Results 14.1 There should be at least three volum es tested pe r sample. Report the results as enterococci per 100 m L of sample. 

## 15.0 V erification Procedure 15.1 Colonies of any color having a blue halo after incubation on m E I agar can be verified as enterococci. Verification of colonies m a y be required in evidence gathering and it is also recom m e nded as a m eans of quality control for th e initial use of the test and for changes in

7 September 2002Method 1600

sample sites, lots of com m e rcial m e dia, or m a jor ingredients in m e dia compounded in the laboratory

. The verification procedure follows. 15.2 Using a sterile inoculating needle, transfer cells from the centers of at least 10 well-isolated ty pical colonies into a BHIB tube and onto a BHIA slant. Incubate broth tubes for 24 h and slants for 48 h at 35 ± 0.5°C. 15.3 After a 24 h incubation, transfer a loopful of m a terial from each BHIB tube to BEA, BHIB and BHIB with 6.5% NaCl. 15.3.1 Incubate the BEA and BHIB with 6.5% NaCl at 35 ± 0.5°C for 48 h. 15.3.2 Incubate the BHIB at 45 ± 0.5°C for 48 h. 15.4 Observe for growth. 15.5 After 48 h incubation, apply a Gram stain to growth from each BHIA slant. 15.6 Gram -positive cocci that grow and hy droly ze esculin on BEA ( i.e. , produce a black or brown precipitate), and grow in BHIB at 45 ± 0.5°C and BHIB with 6.5% NaCl at 35 ± 0.5°C are verified as enterococci. 

## 16.0 Pollution Prevention 16.1 The solutions and reagents used in this m e thod pose little threat to the environm ent when recy cled and m a naged properly

. 16.2 Solutions and reagents should be prepared in vol um es consistent with laboratory use to m i nim i ze the volum e of expired m a terials to be disposed. 

## 17.0 Waste Management 17.1 It is the laboratory ' s responsibility to comp ly with all federal, state, and local regulations governing waste m a nagem e nt, particularly the bi ohazard and hazardous waste identification rules and land disposal restrictions, and to protect the air, water, and land by m i nim i zing and controlling all releases from fum e hoods and benc h operations. Comp liance with all sewage discharge perm its and regul ations is also required. 17.2 Samp les, reference m a terials, and equipm ent known or suspected to have viable enterococci attached or contained m u st be sterilized prior to disposal. 17.3 Samp les preserved with HCl to pH <2 are hazar dous and m u st be neutralized before being disposed, or m u st be handled as hazardous waste. 17.4 For further inform ation on waste m a nagem e nt, consult “The Waste Managem e nt Manual for Laboratory Personnel” and “Less Is Better: La boratory Chem ical Managem e nt for Waste Reduction,” both available from the Am erican Chem ical Society ' s Departm e nt of Governm e nt Relations and Science Policy , 1155 16t h Street NW, Washington, DC 20036. 

## 18.0 R eferences 18.1 Annual Book of ASTM Standards, Vol. 11.01, Am erican Society for Testing and Materials, Philadelphia, PA 19103.

September 2002 8Method 1600 18.2 Bordner, R., J.A. Winter and P.V. Scarpino (e ds.), Microbiological Methods for Monitoring the Environm ent, Water and Wastes, EPA-600/8-78- 017. Office of Research and Developm ent, USEPA. 18.3 Cabelli, V. J., A. P. Dufour, M. A. Levin, L. J. McCabe, and P. W. Haberm an, 1979. Relationship of Microbial Indicators to Health Effects at Marine Bathing Beaches. Amer. Jour. Public Health

. 69:690-696. 18.4 Dufour, A.P. 1984. Health Effects Criteria for Fresh Recreational Waters, EPA-600/1-84-004. Office of Research and Developm ent, USEPA. 18.5 Levin, M.A., J.R. Fischer and V.J. Cabelli. 1975. Mem b rane Filter Technique for Enum eration of Enterococci in Marine Waters. Appl. M i crobiol. 30:66-71. 18.6 Messer, J.W. and A.P. Dufour. 1998. A Rapid, Specific Mem b rane Filtration Procedure for Enum eration of Enterococci in Recreational Water. Appl. Environ. M i crobiol. 64:678-680. 18.7 Im proved Enum eration Methods for the Recreationa l Water Quality Indicators: Enterococci and Escherichia coli

. 2000. EPA/821/R-97/004. Office of Science and Technology , Washington D.C. 18.8 Method 1600: Mem b rane Filter Test Method for Enterococci in Water. 1997. EPA-821-R-97- 004. Office of Water, Washington D.C. 18.9 Reagent Chem icals, Am erican Chem ical Society Specifications , Am erican Chem ical Society , Washington, DC. For suggestions of the testing of reagents not listed by the Am erican Chem ical Society , see Analar Standards for Laboratory Ch em icals, BDH Ltd., Poole, Dorset, UK and the United States Pharm acopeia.

9 September 2002