ASTM: D6503: Enterococci in Water

Summary
Analytes
Revision
Data and Sites

Official Method Name

Standard Test Method for Enterococci in Water Using Enterolert (TM)

Current Revision

Current edition approved Dec. 10, 1999. Published April 2000.

Media

WATER

Instrumentation

Most Probable Number

Method Subcategory

Microbiological

Method Source

ASTM

Citation

Annual Book of ASTM Standards, Section 11, Water and Environmental Technology, Volume 11.02, Water (I)

Brief Method Summary

This test method is used for the detection of enterococci, such as E. faecium, E. faecalis in drinking water, source water, recreational waters (marine water and fresh), and bottled water. When the reagent is added to the sample and incubated at 41 +/- 0.5^oC for 24 h, Enterolert can detect these bacteria at 1 CFU/100 mL. Fluorescence is produced when enterococci metabolizes the nutrient indicator. Enterolert can be used as a presence-absence test or for quantification (5-tube, 10-tube MPN, 15-tube serial dilution or the Quanti-Tray system).

Scope and Application

This test method describes a simple procedure for the detection of enterococci in water and wastewater. It is based on IDEXX's patented Defined Substrate Technology (DST). This product, Enterolert, utilizes a nutrient indicator that fluoresces when metabolized. It can detect these bacteria at one colony forming unit (CFU)/100 mL within 24 h. The presence of this microorganism in water is an indication of fecal contamination and the possible presence of enteric pathogens. This test method can be used successfully with drinking water, source water, recreational (fresh and marine) water, and bottled water. It is the user's responsibility to ensure the validity of this test method for waters of untested matrices.

Applicable Concentration Range

1 and greater CFU/100 mL

Interferences

The presence of Bacillus spp. can interfere with the testing of marine water samples. To eliminate interference, a 1:10 dilution is required with sterile water (deionized or distilled).

Quality Control Requirements

Check and record temperatures in incubators daily to insure temperature is within stated limits.
Quality control should be conducted on each new lot of Enterolert. See package insert for the recommended quality control procedure, which consists of the following protocol: For each type of the American Type Culture Collection (ATCC) bacterial strain listed below, streak the culture onto labeled TSA or blood agar plates and incubate at 35^C for 18 to 24 h. For each bacterial strain, touch a 1-ml loop to a colony and use it to inoculate a labeled test tube containing 5 mL of sterile deionized water. Close cap and shake thoroughly. For each bacterial strain, take a 1-ml loop from the test tube and use it to inoculate a labeled vessel containing 100 mL. Follow the Enterolert presence/absence steps listed above to test these controls. Compare the test results to the expected results.

Sample Handling

Collect the sample as described in detail in the USEPA microbiological methods manual and in accordance with Practices D 3370.
Sample Storage Temperature and Handling Conditions - Ice or refrigerate water samples at a temperature of 2 to 8^oC during transit to the laboratory. Use insulated containers to ensure proper maintenance of storage temperatures. Take care that sample bottles are not totally immersed in water during transit or storage.

Maximum Holding Time

6 hours

Relative Cost

Unknown

Sample Preparation Methods

This method has 1 analyte associated with it.

Analyte	Detection Level	Bias	Precision	Pct False Positive	Pct False Negative	Spiking Level
Enterococci(E-12282) Enterococci Enterococcus	1.000 CFU/100 mL	N/A	57.60

Precision Descriptor Notes:	A limited collaborative study was conducted. Nine technicians from three laboratories tested three different matrixes at three levels following Practice D 2777. Outliers were rejected in accordance with the statistical tests outlined in Practice D 2777. All data from one technician was rejected for recreational water-marine and single values were rejected for both recreational water-fresh at the low level and for recreational water-marine at the low level. The mean count, the overall standard deviation (St), and the single operator standard deviation (so), are indicated in Table 5 of the Method. NOTE: Bias -- The mean value obtained for the samples (drinking water, recreational water fresh and marine) from the nine technicians for the low-, mid- and high-spiked samples all fall within the 95% confidence interval (Poisson distribution) of the actual values obtained from plating on blood agar.
Detection Level Note:	None.

Precision Descriptor Notes:

A limited collaborative study was conducted. Nine technicians from three laboratories tested three different matrixes at three levels following Practice D 2777. Outliers were rejected in accordance with the statistical tests outlined in Practice D 2777. All data from one technician was rejected for recreational water-marine and single values were rejected for both recreational water-fresh at the low level and for recreational water-marine at the low level. The mean count, the overall standard deviation (St), and the single operator standard deviation (so), are indicated in Table 5 of the Method. NOTE: Bias -- The mean value obtained for the samples (drinking water, recreational water fresh and marine) from the nine technicians for the low-, mid- and high-spiked samples all fall within the 95% confidence interval (Poisson distribution) of the actual values obtained from plating on blood agar.

Detection Level Note:

None.

Revision	PDF/Link
Current edition approved Dec. 10, 1999. Published April 2000.

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ASTM: D6503: Enterococci in Water

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