EPA-OW: 1602: Coliphage in water by single agar layer (SAL)

Summary
Analytes
Revision
Data and Sites

Official Method Name

Male-specific (F⁺) and somatic coliphage in water by single agar layer (SAL) procedure

Current Revision

April 2001

Media

WATER

Instrumentation

Plate Count

Method Subcategory

Microbiological

Method Source

EPA-OW

Citation

USEPA, 2001, Method 1602: Male-specific (F+) and somatic coliphage in water by single agar layer (SAL) procedure: U.S. Environmental Protection Agency Report 821-R-01-029, 38 p.

Brief Method Summary

Coliphage presence in ground water is an indication of fecal contamination. Method 1602 is a performance-based method for enumerating 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.

This single agar layer procedure requires the addition of host bacteria, magnesium chloride, and double-strength molten agar medium to the sample, followed by pouring the total volume of the mixture into plates. All plates from a single sample are examined for plaque formation (zones of bacterial host lawn clearing). The quantity of coliphage in a sample is expressed as plaque forming units (PFU) / 100 mL.

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 single agar layer (SAL) procedure detects and enumerates 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 single agar layer procedure the sample and host bacteria should not remain in contact with each other for more than 10 minutes prior to plating and after plating the agar must harden within 10 minutes. Increased contact time or agar hardening time may result in replication of phages such that the initial phage concentration is overestimated. The entire plating procedure from combining sample with host to hardening of single-agar layer plates should not exceed 20 minutes.

Quality Control Requirements

The minimum QA requirements consist of an initial demonstration of laboratory capability through performance of the initial precision and recovery (IPR) test, analysis of spiked samples to evaluate and document data quality, and analysis of standards and blanks as tests of continued acceptable performance. Laboratory performance is compared to established performance criteria to determine if the results of analyses meet the performance criteria of the method.

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.
Collect 250 mL 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 groundwater, it potentially can be used with chlorinated ground waters. If the sample has been chlorinated, add 0.5-mL 10% sodium thiosulfate per 1-L of sample at time of sample.

Maximum Holding Time

SAL 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 more than 50%, ca

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	57.00 % RSD (ML)

Precision Descriptor Notes:	Method 1602 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 9.2.5 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 (Section 11.3).
Detection Level Note:

Precision Descriptor Notes:

Method 1602 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 9.2.5 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 (Section 11.3).

Detection Level Note:

Revision	PDF/Link
April 2001

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EPA-OW: 1602: Coliphage in water by single agar layer (SAL)

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