EPA-OW: 1601: Coliphage in water by two-step enrichment procedure

Summary
Analytes
Revision
Data and Sites

Official Method Name

Male-specific (F⁺) and somatic coliphage in water by two-step enrichment procedure

Current Revision

April 2001

Media

WATER

Instrumentation

Plate Count

Method Subcategory

Microbiological

Method Source

EPA-OW

Citation

USEPA, 2001, Method 1601: Male-specific (F+) and somatic coliphage in water by two-step enrichment procedure: U.S. Environmental Protection Agency Report 821-R-01-030, 40 p.

Brief Method Summary

Coliphage presence in ground water is an indication of fecal contamination. Method 1601 (two-step enrichment procedure) is a performance-based method for detecting the presence of male-specific (F+) and somatic coliphage in ground water and other waters. Laboratories are permitted to modify or omit any steps or procedure, with the exception of the coliphage stock enumeration procedure (Section 11.3), provided that all performance requirements set forth in the validated method are met. The laboratory may not omit any quality control analyses.

The two-step enrichment procedure requires enrichment of coliphage in a nutrient broth with host bacteria followed by spotting onto a lawn of host bacteria and assessing lysis zone formation in the lawn. This two-step enrichment method was validated as a qualitative, presence-absence method, and Method 1601 was written with this use in mind. Although the method potentially may be used as a quantitative assay of coliphage concentrations in an MPN format, the two-step enrichment method has not been validated this way.

This method is for use in the Environmental Protection Agency?s (EPA?s) data gathering and monitoring programs under the Safe Drinking Water Act and the Clean Water Act.

Scope and Application

The two-step enrichment procedure determines the presence or absence of male-specific (F⁺) and somatic coliphages in groundwater and other waters.

This method is intended to help determine if groundwater is affected by fecal contamination.

Applicable Concentration Range

Interferences

During the enrichment phase of the two-step enrichment procedure, other bacteria in the water sample can grow and interfere with the spot-test confirmation step. Bacteria may grow over the lysis zone and obscure visualization, resulting in a false negative. Generally, when bacteria have overgrown lysis zones, they appear as raised colonies or a confluent growth of raised colonies. As a result, they are distinguishable from the surrounding lawn of host bacteria. If this problem is noted, follow the procedure described in the method report.

Quality Control Requirements

The minimum QA requirements consist of an initial demonstration of laboratory capability through performance of the initial demonstration of capability (IDC) test, ongoing analysis of spiked reagent water and field samples to evaluate and document data quality, and analysis of positive controls and method blanks as tests of continued acceptable performance.

Sample Handling

NOTE: Unless the sample is known or suspected to contain infectious agents (e.g., during an outbreak), samples should be shipped as noninfectious and should not be marked as infectious. U.S. Department of Transportation (DOT) regulations (49 CFR 172) prohibit interstate shipment of more than 4 L of solution known to contain infectious materials. State regulations may contain similar regulations for intrastate commerce. If an outbreak is suspected, ship less than 4 L at a time.
Samples are collected in plastic bottles or carboys and shipped to the laboratory for analysis. Samples must be shipped at 2^oC to 8^oC using wet ice, Blue Ice, or similar products to maintain temperature. Samples must be stored at 4^oC ? 1^oC. Do not freeze.
For the two-step enrichment procedure using 100-mL samples: Collect 250 mL of sample for each of the two coliphage types to allow for sample re-analysis, if necessary.
For the two-step enrichment procedure using 1-L samples: Collect 2.5 L of sample for each of the two coliphage types to allow for sample re-analysis, if necessary.
Dechlorination procedure: Although this method was validated for use with unchlorinated ground water, it potentially can be used with chlorinated waters. If the sample has been chlorinated, add 0.5-mL of 10% sodium thiosulfate per 1-L of sample at time of sample collection.

Maximum Holding Time

Two-step enrichment procedure: 48 hrs from sample collection to beginning of analysis.
Raw sewage sample: 24 hrs from sewage sample collection and analysis, unless re-titered and titer has not decreased by more than 50%. If titer has not decreased by m

Relative Cost

Less than $50

Sample Preparation Methods

This method has 1 analyte associated with it.

Analyte	Detection Level	Bias	Precision	Pct False Positive	Pct False Negative	Spiking Level
Coliphage(E-12215) Coliphage	N/A	N/A	N/A

Precision Descriptor Notes:	Method 1601 is a performance-based method and the laboratory is permitted to modify certain method procedures to improve performance or lower the costs of measurements, provided that all quality control (QC) tests cited in Section 9.2.6 are performed and all QC acceptance criteria are met. The laboratory is not permitted to modify the double agar layer QC spiking suspension enumeration procedure.
Detection Level Note:

Revision	PDF/Link
April 2001

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EPA-OW: 1601: Coliphage in water by two-step enrichment procedure

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