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Analytical Methods

Male-specific (F+) and somatic coliphage in water by single-agar layer procedure: U.S. Environmental Protection Agency Method 1602. Updated March 2013

The single agar layer (SAL) procedure is a quantitative method for the detection of male-specific (F+) and somatic coliphage in water. Coliphage are viruses (bacteriophage) that infect coliform bacteria and are indicators of fecal contamination. Antibiotic-resistant host-culture strains E. coli CN-13 (resistant to nalidixic acid) and E. coli F-amp (resistant to streptomycin and ampicillin) are used as hosts for somatic and male-specific coliphage, respectively. This method must be done in the laboratory by a trained microbiologist.

THEORY:
A 100-mL water sample is combined with magnesium chloride, log-phase host bacteria, and double-strength tryptic soy agar.  The sample mixture is poured into plates and incubated overnight. Circular lysis zones (plaques) are counted and summed for all plates from a single sample. The quantity of coliphage in a sample is expressed as plaque forming units (PFU) / 100 mL.  

USE:
Although this method may be used for all water matrices, it has only been validated for use in ground water (U.S. Environmental Protection Agency, 2001).  In reality, the small volume that is practical (100 mL) limits the method’s usefulness in monitoring ground water, where larger sample volumes are needed to detect microorganisms.  The USGS uses the SAL method to enumerate viruses in surface waters, because 100 mL is generally sufficient to detect coliphage in surface water. 

Two main groups of coliphage are used as viral indicators.  Somatic coliphage infect coliform bacteria by attachment to the outer cell membrane or cell wall.  Male-specific coliphage attach only to the F-pilus of coliforms that carry the F+ plasmid: F-pili are made only by bacteria grown at higher temperatures. 

See SAL method directions (Appendix C12).

MEDIA
See preparation instructions for media, reagent solutions, and host-culture strains (Appendix C12).

RECOMMENDED QUALITY-CONTROL
Three QC sample types are recommended for Method 1602 in a surface water monitoring program: field blanks, replicate samples, and matrix spike samples.

A field blank (an aliquot of sterile deionized water transferred to a sterile sample bottle in the field and processed in the same manner as the environmental sample) is used to ensure contamination is not occurring during sample collection and processing. Field blanks should be collected near the beginning of a study and every 20th sample thereafter.

A replicate sample (a second sample collected at the same time as the environmental sample and processed in the same manner as the environmental sample) is used to determine sampling and analytical variability and should be collected for 10-15 percent of the samples.

A matrix spike sample (a second sample collected at the same time as the environmental sample, spiked in the lab with control organisms, and analyzed in the same manner as the environmental sample) is used to identify any matrix interferences from the water source on performance of the analytical method. Matrix spikes should be collected whenever a new water source is sampled and every 20th sample thereafter. Other matrix interferences, such as those resulting from extremes in streamflow, may affect the ability of the method to detect coliphage and matrix spikes should also be included.

REFERENCE
U.S. Environmental Protection Agency, 2001, Method 1602: Male-specific (F+) and somatic coliphage in water by single agar layer (SAL) procedure: Office of Water, Washington D.C., EPA 821-R-01-029.

This document can be obtained at http://www.epa.gov/nerlcwww/microbes/epamicrobiology.html

NWIS PARAMETER CODES:
90903 Coliphage, somatic, E. coli CN-13 host, plaques per 100 mL

90904 Coliphage, F-specific, E. coli F-amp host, plaques per 100 mL

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