EPA Method 1601

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EPA Method 1601:
Coliphage in water by two-step enrichment procedure. Official Name: Male-specific (F+) and somatic coliphage in water by two-step enrichment procedure

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.

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.

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.

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.

QC 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.

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



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.


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