# EPA United States Office of Water Environmental Protection W a shington DC September 2002 Agency 20460 EPA 821-R-02-023



# Method 1603: 

# Escherichia coli ( E. coli ) in Water by Membrane Filtration Using Modified membrane-Thermotolerant Escherichia coli Agar (Modified mTEC)

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

EPA-821-R-02-023

## 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 ent com b ines the inform ation previously published in Test Methods for Escherichia coli and Enterococci in Water by the Membrane Filter Procedure (EPA-600/4- 85-076) (Reference 18.6 and Improved Enumeration Methods for the Recreational Water Quality Indicators: Enterococci and Escherichia coli (EPA/821/R-97/004) (Reference 18.4. Mention of trade nam es or com m e rcial products does not cons titute 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)

## 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 ry o f M e t h o d

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

. 0 D e fi n i t i o n s
................................................................... 1
4.0 Interferences and Contam ination

................................................. 1
5

. 0 S a fe t y
...................................................................... 1
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 Re a g e n t s a n d S t a n d a rd s
........................................................ 3
8.0 Samp le Collection, Preservation, and Storage

....................................... 6 9.0 Quality Control

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

. 0 Ca l i b ra t i o n a n d S t a n d a rd i z a t i o n
.................................................. 6
1 1

. 0 P ro c e d u re
................................................................... 6
1 2

. 0 D a t a A n a l y s i s a n d Ca 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
............................................................. 8
1 5

. 0 V e r i f i c a t i o n P r o c e d u r e
......................................................... 8
1 6

. 0 P o l l u t i o n P re v e n t i o n
........................................................... 9
1 7

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

............................................................ 9
1 8

. 0 Re fe re n c e s
................................................................... 9

## Method 1603: Escherichia coli ( E. coli ) in Water by Membrane Filtration Using Modified membrane- Thermotolerant Escherichia coli Agar (modified mTEC) September 2002

1.0 Scope and Application
1.1 This m e thod describes a revised Escherichia coli m e m b rane filter (MF) procedure, a single-step m e thod that uses one m e dium , m odified m TEC Agar, and does not require the transfer of the m e m b rane filter to another m e dium or other s ubstrate. The m odified m e dium contains a chrom ogen (5-brom o -6-chloro-3-indoly l

- $ -D-glucuronide), which is catabolized to glucuronic acid and a red- or m a genta-colored com pound by E. coli that produce the enzy m e $ -Dglucuronidase. The apparatus and equipm ent, and sampling, filtration, and verification procedures for this m odified m TEC m e thod are identical to those of the original m TEC m e thod. Because the bacterium is a natural inhabitant only of the int estinal tract of warm -blooded anim als, its presence in water samples is an indication of fecal pollution and the possible presence of enteric pathogens. 1.2 The E. coli test is used as a m easure of recreational water quality

. Epidem iological studies have led to the developm ent of criteria which can be used to prom ulgate recreational water standards based on established relationships between health effects and water quality

. The significance of finding E. coli in recreational water sam ples is the dir ect relationship between the density of E. coli and the risk of gastrointestinal illness associ ated with swim m i ng in the water (Reference 18.3. 1.3 The test for E. coli can be applied to fresh, estuarine, and m a rine 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 E. coli levels in water can be detected and enum erated. 

## 2.0 Summary of Method
2.1 The MF m e thod provides a direct count of E. coli in water based on the developm ent of colonies that grow on the surface of a m e m b rane filter. A water sam ple is filtered through the m e m b rane, which retains the bacteria. After filtration, th e m e m b rane containing the bacteria is placed on a selective and differential m e dium , m odified m TEC Agar, incubated at 35 ± 0.5°C for 2 h to resuscitate the injured or stressed bacteria, and then incubated at 44.5 ± 0.2°C for 22 h. The m odified m e thod elim inates the transfer of the m e m b rane filter to another substrate. The target colonies on m odified m TEC agar are red or m a genta in color after the incubation period. 

## 3.0 D efinitions
3.1 In this m e thod, E. coli are those bacteria which produce red or m a genta colonies on the m odified m TEC agar. 

## 4.0 Interferences and Contamination
4.1 Water samples containing colloidal or suspended particulate m a terial 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. 

## 5.0 Safety
September 2002Method 1603
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, a nd 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 Filter flask, vacuum , usually 1 L, with appropriate 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; or 15 x 60 m m , glass or plastic, with loose-fitting lids; or 15 x 100 m m
. 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.

September 2002 2Method 1603
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 appli cator sticks or plastic loops are alternatives to inoculation loops.



### Note: A platinum loop is required for the cytochrom e oxidase test in the verification procedure.

6.21 Incubator m a intained at 35 ± 0.5°C, with approxi m a tely 90% hum idity if loose-lidded petri dishes are used. 6.22 Waterbath m a intained at 44.5 ± 0.5°C. 6.23 Waterbath m a intained at 50°C for temp ering agar. 6.24 Test tubes, 20 x 150 m m , borosilicate glass or plastic. 6.25 Test tubes, 10 x 75 m m , borosilicate glass 6.26 Caps, alum inum or autoclavable plastic, for 20 m m diam eter test tubes. 6.27 Test tubes screw-cap, borosilicate glass, 16 x 125 m m or other appropriate size. 6.28 Filter Paper. 6.29 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. 5). 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 Phosphate Buffered Saline 7.4.1 Composition: Sodium Dihy drogen Phosphate 0.58 g Sodium Monohy drogen Phosphate 2.5 g Sodium Chloride 8.5 g Reagent-Grade Distilled Water 1 .0 L 7.4.2 Preparation: Dissolve the ingredients in 1 L of reagent-grade distilled water in a flask , and dispense in appropriate am ounts for diluti ons in screw-cap bottles or culture tubes, and/or into containers for use as rinse wate r. Autoclave 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 (References 18.2)

3 September 2002Method 1603
7.5.1 Composition of Stock Phosphate Buffer Solution: Phosphate Dihy drogen Phosphate 34.0 g Reagent-Grade Distilled Water 500.0 m L 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. 7.5.2 Preparation of Stock Magnesium Chlori de Solution: Add 38 g anhy drous MgCl 2 or 81.1 g MgCl 2 C 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. 7.5.3 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. 7.5.4 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 appropriate am ounts for dilutions in screwcap 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 Modified m TEC Agar (Difco 214884 or 214880) 7.6.1 Composition: Protease Peptone #3 Yeast Extract Lactose NaCl Dipotassium Phosphate Monopotassium Phosphate Sodium Laury l Sulfate Sodium Desoxy cholate Chrom ogen (5-brom o -6-chloro-3-indoly l

- $ -D-glucuronide) Agar Reagent-Grade Distilled Water 5.0 g 3.0 g 10.0 g

7.5 g 3.3 g 1.0 g 0.2 g 0.1 g
0.5 g 15.0 g

1.0 L 7.6.2 Preparation: Add 45.6 g dehy drated m odified m TEC Agar to 1 L of reagent-grade distilled water in a flask, and heat to boiling until the ingredients dissolve. Autoclave at 121°C (15 lb pressure) for 15 m i n, and cool in a 50°C waterbath. Pour the m e dium into each 9 x 50 m m culture dish to a 4-5 m m depth (approxim a tely 4-6 m L ), and allow to solidify

. Final pH should be 7.3 ± 0.2. Store in a refrigerator. 7.7 Nutrient Agar (Difco 0001, BD 4311472) 7.7.1 Composition: Peptone 5.0 g Beef Extract 3.0 g Agar 15.0 g Reagent-Grade Distilled Water 1.0 L

September 2002 4Method 1603
7.7.2 Preparation: Add 23 g of Nutrient Agar to 1 L of reagent-grade distilled water, and m i x well. Heat to boiling to dissolve the agar com p letely

. Dispense in screwcap tubes, and autoclave at 121°C (15 lb pressure) for 15 m i n. Rem ove the tubes and slant. Final pH should be 6.8 ± 0.2. 7.8 Try p tic Soy Broth (Difco 0370); Try p ticase Soy Broth (BD 99071) 7.8.1 Composition: Try p tone or Try p ticase 17.0 g Soy t one or Phy t one 3.0 g Sodium Chloride 5.0 g Dextrose 2.5 g Dipotassium Phosphate 2.5 g Reagent-Grade Distilled Water 1.0 L 7.8.2 Preparation: Add 30 g of dehy drated Try p tic/Try pticase Soy Broth to 1 L of reagentgrade distilled water. Warm the broth, and m i x gently to dissolve the m e dium comp letely

. Dispense in screw-cap tubes, and autoclave at 121°C (15 lb pressure) for 15 m i n. Final pH should be 7.3 ± 0.2. 7.9 Sim m ons Citrate Agar (Difco 0091, BD 4311620) 7.9.1 Composition: Magnesium Sulfate 0.2 g Monoam m onium Phosphate 1.0 g Dipotassium Phosphate 1.0 g Sodium Citrate 2.0 g Sodium Chloride 5.0 g Brom Thy m ol Blue 0.08 g Agar 15.0 g Reagent-Grade Distilled Water 1.0 L 7.9.2 Preparation: Add 24.2 g Sim m ons Citrate Agar to 1 L of reagent-grade distilled water. Heat to boiling to dissolve comp letely

. Di spense into screwcap t ubes, and autoclave at 121°C (15 lb pressure) for 15 m i n. Cool the t ubes and slant. Final pH should be 6.8 ± 0.2. 7.10 Try p tone 1% (Difco 0123); Tr y p tophane Broth (BD 4321717 and 4321718) 7.10.1 Composition: Try p tone or Try p ticase Peptone 10.0 g Reagent-Grade Distilled Water 1.0 L 7.10.2 Preparation: Add 10 g try p tone or try p ticase peptone to 1 L or reagent-grade distilled water, and heat, m i xing until dissolved. Di spense in 5-m L volum es into tubes, and autoclave 121°C (15 lb pressure) for 15 m i n. Final pH should be 7.2 ± 0.2. 7.11 EC Broth (Difco 0314, BD 4311187) 7.11.1 Composition: Try p tose or Try p ticase Peptone 20.0 g Lactose 5.0 g Bile Salts no. 3 or Bile Salts Mixture 1.5 g Dipotassium Phosphate 4.0 g Monopotassium Phosphate 1.5 g

5 September 2002Method 1603
Sodium Chloride 5.0 g Reagent-Grade Distilled Water 1.0 L 7.11.2 Preparation: Add 37 g dehy drated EC m e dium to 1 L of reagent-grade distilled water, and warm to dissolve com p letely

. Dispense into ferm entation tubes (20 x 150 m m tubes containing inverted 10 x 75 m m vials). Autoclave at 121°C (15 lb pressure) for 15 m i n. Final pH should be 6.9 ± 0.2. 7.12 Oxidase Reagent 7.12.1 Com position: N, N, N’ , N’ -tetram e thy l

- D -pheny lenediam ine dihy drochloride, 1% aqueous solution (1 g per 100 m L sterile reagent-grade distilled water). 7.13 Kovacs Indole Reagent 7.13.1 Composition: D -dim ethy lam i nobenzaldehy de 10.0 g Am y l or Isoam y l Alcohol 150.0 m L Concentrated (12 M) Hy drochloric Acid 50.0 m L 7.13.2 Preparation: Dissolve D -dim ethy lam i nobenzaldehy de in al cohol, slowly add hy drochloric acid, and m i x. 

## 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. Samp les not collected according to these rules should not be analy zed. 8.1.1 Storage Tem p erature and Handling Conditions Ice or refrigerate water samples at a temp erature of 1-4°C during transit to the laboratory

. Use insulated containers to assure proper m a in tenance of storage tem p erature. Take care that sample 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 C alibration 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
September 2002 6Method 1603
11.1 Prepare the m odified m TEC Agar as directed in section 7.6. 11.2 Mark the petri dish and report form with the sam ple identification and volum e. 11.3 Place a sterile m e m b rane filter on the filter base, gr id side up, and attach the funnel to the base so that the m e m b rane 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 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 20-80 E. coli colonies on the 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 sizes 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 odified m TEC 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 forceps around the edge of the filter to be sure that the filter is properly seated on the agar. Close the dish, invert, and incubate 35 ± 0.5°C for 2 h. 11.9 After a 2 h incubation at 35 ± 0.5°C, transfer the plate to a Whirl-Pak® bag, seal the bag, place the bag with the plate inverted in a test-tube rack , and put the rack in a 44.5 ± 0.2°C waterbath for 22-24 h. 11.10 After 22-24 h, rem ove the plate from the waterb ath, count and record the num ber of red or m a genta colonies with the aid of an illum i nated lens with a 2-5x m a gnification or a stereoscopic m i croscope (See Photo 1). 

## 12.0 Data Analy s is and Calculations Use the following general rules to calculate the E. coli count per 100 m L of sample: 12.1 Select the m e m b rane filter with an acceptable num ber of m a genta or red colonies (20-80), and calculate the num ber of E. coli per 100 m L according to the following general form ula:
Num b er of E. coli colonies E. coli /100 m L = X 100 Volum e of sample filtered (m L) 12.2 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). 12.3 Reporting Results 12.3.1 There should be at least three volum es filtered per sam ple. Report the results as E. coli per 100 m L of sample. 

## 13.0 Method Performance
13.1 The false-positive and false-negative rates, re ported for various environm ental water samples, were <1% and 4%, respectively

.

7 September 2002Method 1603


## 14.0 R eporting Results 14.1 There should be at least three volum es filtered per sam ple. Report the results as E. coli per 100 m L of sample. 

## 15.0 Verification Procedure
15.1 Red or m a genta colonies can be verified as E. coli

. Verification of colonies m a y be required in evidence gathering and is also recom m e nded as a m eans of quality control for the initial use of the test and for changes in 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.

Photo 1. Escherichia coli colonies on m odified m TEC agar. E. coli colonies are red to m a genta.

15.2 Using a sterile inoculation loop, transfer growth from the centers of at least 10 well-isolated colonies to Nutrient Agar plates or slants and to Try p ticase Soy Broth. Incubate the agar and broth cultures for 24 h at 35 ± 0.5°C. 15.3 After incubation, rem ove a loopful of growth from the Nutrient Agar slant with a platinum loop , and deposit it on the surface of a piece of filter paper that has been saturated with freshly prepared Cy tochrom e Oxidase Reagent. If the spot where the bacteria were deposited turns deep purple within 15 seconds, the test is positive. 15.4 Transfer growth from the Try p ticase Soy Broth tube to Sim m ons Citrate Agar, Try p tone Broth, and an EC Broth ferm entation tube. 15.4.1 Incubate the Sim m ons Citrate Agar and Try p tone Broth for 48 h at 35 ± 0.5°C. 15.4.2 Incubate the EC Broth at 44.5 ± 0.2°C in a waterbath for 24 h. The water level m u st be above the level of the EC Broth in the tube. 15.4.3 Add 0.5 m L of Kovacs Indole Reagent to the 48 h Try p tone Broth culture, and shake the tube gently

. A positive test for indole is indicated by a deep red color which develops in the alcohol lay e r on top of the broth. 15.4.4 E. coli is EC gas-positive, indole-positive, a nd oxidase-negative, and does not utilize citrate ( i.e. , the m e dium rem a ins green).

September 2002 8Method 1603 15.5 Alternately , com m e rcially available m u lti-test identification sy stem s m a y be used to verify colonies. Inoculate the colonies in to an identification sy stem for Enterobacteriaceae that includes lactose ferm entation, F -nitropheny l- $ -D-galactopy ranoside (ONPG), and cy tochrom e oxidase test reactions. 

## 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 E. coli 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. 18.2 Bordner R., J.A. Winter, P.V. Scarpino (eds.). 1978. Microbiological m e thods 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. Am. J. Public Health

. 69: 690-696. 18.4 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. 18.5 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. 18.6 Test m e thods for Escherichia coli and Enterococci in Water by the Mem b rane Filter Procedure. 1985. EPA-600/4-85/076. Environm ental Mon itoring and Support Laboratory , Cincinnati, USEPA.

9 September 2002