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Analytical Methods

Male-specific (F+) and somatic coliphage in water by a two-step enrichment procedure: U.S. Environmental Protection Agency Method 1601. Updated March 2013

The two-step enrichment method is a presence/absence method for the detection of male-specific (F+) and somatic coliphage. Coliphage are viruses (bacteriophages) 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.

For the enrichment step, large sample volumes are supplemented with magnesium chloride, log-phase host E. coli, and tryptic soy broth and are incubated overnight. After 24 hours of incubation at 35C, samples are spotted onto a lawn of suitable host bacteria and incubated overnight. Positive results for coliphage appear as a clear halo around the spot. Bacteria from the sample grow on the spot, while phage radiate from the spot to lyse the surrounding E. coli lawn. Zones of lysis within the spot are also considered indicative of a positive result.

The two-step enrichment method has been validated for use in ground-water samples. One liter sample volumes are recommended for the detection of coliphage in ground 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 two-step enrichment method directions (Appendix C13).

See preparation instructions for media, reagent solutions, and host-culture strains (Appendix C13).

In a groundwater sampling program, one type of QC sample is recommended for Method 1601—matrix spike 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.

Note: Replicate samples are not recommended for groundwater samples being analyzed by Method 1601. Due to the decreased chance of detecting coliphage in groundwater, replicate samples will not appropriately characterize sampling and analytical variability. Field blanks are also not recommended for this method for groundwater samples. However, if greater than 15 percent of samples are positive for coliphage during any study, field blanks should be collected to ensure contamination is not occurring.


U.S. Environmental Protection Agency, 2001, Method 1601: Male-specific (F+) and somatic coliphage in water by two-step enrichment procedure: Office of Water, Washington, D.C., EPA-821-R-01-030. 

This document can be obtained at

99332 Coliphage, somatic, E. coli CN13-host, 2-step enrichment, presence or absence per 1 L

99335 Coliphage, F-specific, E. coli Famp-host, 2-step enrichment, presence or absence per 1 L

Coding results for PA tests are as follows:

  Value Remark
Present # M
Absent # U