Method EPA 1600 – NPDES Approved for Enterococcus


1.0         Scope and Application

1.1         This method describes a membrane filter (MF) procedure for the detection and enumeration of the enterococci bacteria in water. Enterococci 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 enterococci in water is an indication of fecal pollution and the possible presence of enteric pathogens.

1.2         The enterococci test measures the bacteriological quality of recreational waters. Epidemiological studies have led to the development of criteria which can be used to promulgate recreational water standards based on the established relationship between health effects and water quality. The significance of finding enterococci in recreational water samples is the direct relationship between the density of enterococci in the water and swimming-associated gastroenteritis studies of marine and fresh water bathing beaches (Reference 18.3, Reference 18.4).

1.3         The test for enterococci can be applied to potable, fresh, estuarine, marine, and shellfish growing waters.

1.4         Since a wide range of sample volumes or dilutions can be analyzed by the MF technique, a wide range of enterococci levels in water can be detected and enumerated.

2.0         Summary of Method

2.1         The MF method provides a direct count of bacteria in water based on the development of colonies on the surface of the membrane filter (Reference 18.5). A water sample is filtered through the membrane which retains the bacteria. Following filtration, the membrane containing the bacterial cells is placed on a selective medium, mEI agar, and incubated for 24 h at 41°C. All colonies (regardless of color) with a blue halo are recorded as enterococci colonies. Magnification and a small fluorescent lamp are used for counting to give maximum visibility of colonies.

3.0         Definitions

3.1         In this method, enterococci are those bacteria which produce colonies with a blue halo after incubation on mEI agar. Enterococci include Streptococcus faecalis, Streptococcus faecium, Streptococcus avium, and their variants.

4.0         Interferences

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 identification of target colonies.

5.0         Safety

5.1         The analyst/technician must know and observe the normal safety procedures required in a microbiology laboratory while preparing, using, and disposing of cultures, reagents, and materials, and while operating sterilization equipment.

5.2         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 Mohr, glass or plastic, of appropriate volume.

6.6         Graduated cylinders, 100-1000 mL, covered with aluminum foil or kraft paper and sterile.

6.7         Membrane filtration units (filter base and funnel), glass, plastic or stainless steel, wrapped with aluminum foil or kraft paper and sterilized.

6.8         Ultraviolet unit for sanitization of the filter 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 with a check valve to prevent the return flow of air, can be used.

6.10       Flask, filter, vacuum, usually 1 L, with appropriate tubing. A filter manifold to hold a number of filter bases is optional.

6.11       Flask for safety trap placed between the filter flask and the vacuum source.

6.12       Forceps, straight or curved, with smooth tips to handle filters without damage.

6.13       Ethanol, methanol or isopropanol in a small, wide-mouth container, for flame-sterilizing forceps.

6.14       Burner, Bunsen or Fisher type, or electric incinerator unit for sterilizing loops and needles.

6.15       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.

6.16       Petri dishes, sterile, plastic, 9 x 50 mm, with tight-fitting lids.

6.17       Bottles, milk dilution, borosilicate glass, screw-cap with neoprene liners, marked at 99 mL for 1:100 dilutions. Dilution bottles marked at 90 mL or tubes marked at 9 mL may be used for 1:10 dilutions.

6.18       Flasks, borosilicate glass, screw-cap, 250-2000 mL volume.

6.19       Membrane filters, sterile, white, grid marked, 47 mm diameter, with 0.45 ± 0.02 µm pore size.

6.20       Inoculation loops, at least 3 mm diameter, and needles, nichrome or platinum wire, 26 B & S gauge, in suitable holders. Sterile disposable applicator sticks or plastic loops are alternatives to inoculation loops.

6.21       Incubator maintained at 41 ± 0.5°C.

6.22       Waterbath maintained at 50°C for tempering agar.

6.23       Test tubes, 20 x 150 mm, borosilicate glass or plastic.

6.24       Caps, aluminum or autoclavable plastic, for 20 mm diameter test tubes.

6.25       Test tubes, screw-cap, borosilicate glass, 16 x 125 mm or other appropriate size.

6.26       Whirl-Pak® bags.

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 18.9). The agar used in preparation of culture media must be of microbiological grade.

7.2         Whenever possible, use commercial culture media as a means of quality control.

7.3         Purity of Water: Reagent water conforming to Specification D1193, reagent water conforming Type II, Annual Book of ASTM Standards (Reference 18.1).

7.4         Buffered Dilution Water

7.4.1     Composition:

Sodium Dihydrogen Phosphate                            0.58 g

Sodium Monohydrogen 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 amounts for dilutions in screw-cap bottles or culture tubes,   and/or into containers for use as rinse water. Autoclave after preparation at 121°C (15 lb pressure) for 15 min. 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 dihydrogen phosphate          34.0 g

Reagent-Grade distilled water               500.0 mL

7.5.2     Preparation: Adjust the pH of the solution to 7.2 with 1 N NaOH, and bring the volume to 1 L with reagent-grade distilled water. Sterilize by filtration or autoclave at 121°C (15 lb pressure) for 15 min.

7.5.3     Preparation of Stock Magnesium Chloride Solution: Add 38 g anhydrous MgCl2 or 81.1 g MgCl2A6H2O to 1 L reagent-grade distilled water. Sterilize by filtration or autoclave at 121°C (15 lb pressure) for 15 min.

7.5.4     Storage of Stock Solutions: After sterilization, store the stock solutions in the refrigerator until used. Handle aseptically. If evidence of mold or other contamination 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 mL of the stock phosphate buffer and 5 mL of the MgCl2 stock per liter of reagent-grade distilled water. 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 lb pressure) for 15 min. Final pH should be 7.0 ± 0.2.

7.6         mEI Agar

7.6.1     Composition of Basal Medium (m-EI Agar, Difco 0333)

Peptone   10.0g

Sodium Chloride  15.0g

Yeast Extract  30.0g

Esculin   1.0g

Actidione (Cycloheximide)  0.05g

Sodium Azide   0.15g

Agar  15.0g

Reagent-Grade Distilled Water  1L

7.6.2     Preparation of mEI Medium: Add 71.2 g of dehydrated basal medium plus 0.75 grams of indoxyl B-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 min and cool in a 50°C water bath.

7.6.3     Reagents Added After Sterilization: Mix 0.24 g nalidixic acid in 5 mL reagent-grade sterile distilled water, add a few drops of 0.1N NaOH to dissolve; add to the mEI medium. Add 0.02 g triphenyltetrazolium chloride separately to the mEI medium and mix.

7.6.4     Alternately, the following solutions may be used: Nalidixic acid: Add 0.48 g of nalidixic acid and 0.4 mL 10 N NaOH to 10 mL of reagent-grade distilled water and mix. Filter-sterilize the solution, and add 5.2 mL per liter of medium. Triphenyltetrazolium chloride (TTC): Add 0.1 g of TTC to 10 mL of reagent- grade distilled water, and warm to dissolve. Filter-sterilize the solution, and add 2 mL per liter of medium.

7.6.5     Preparation of mEI Agar Plates: Pour the mEI agar into 9×50 mm petri dishes to a 4-5 mm depth (approximately 4-6 mL), and allow to solidify. Final pH of medium should be7.1 ± 0.2. Store in a refrigerator.