The two-step enrichment method is a presence/absence method
for the detection of male-specific (F+) and somatic coliphage.
Coliphage are bacteriophage that infect and replicate in coliform bacteria.
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: 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. Divalent
cations are included in the enrichment solution to enhance phage infectivity.
After 24 hours of incubation at 35°C,
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.
USE: The
two-step enrichment method has been validated for use in ground-water samples.
Large sample volumes, such as 1-L volumes or greater, 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. They
are widely distributed in both fecal-contaminated and uncontaminated waters (Sobsey
and others, 1995). 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.
Thus, male-specific coliphage presumably come from warm-blooded animals
or sewage.
MEDIA: The ODML prepares the medium, reagent solutions, and host-culture strains. See preparation instructions (Appendix Q).
REFERENCES:
Sobsey, M.D., Amanti, A., and Handzel, T., 1995, Detection
and occurrence of coliphage indicator viruses in water: In Proceedings of the Water Quality Technology Conference, American
Water Works Association, November 1995, New Orleans, La., p. 2087-2097.
U.S. Environmental Protection Agency, 2000, Method 1601¾Male-specific (F+) and somatic Coliphage in water by two-step enrichment procedure¾April 2000-Draft: Washington, D.C., EPA-821-R-00-009.
NWIS PARAMETER CODES:
99331 Coliphage, somatic, E. coli CN13-host, 2-step enrichment, presence (1) or absence (2) per 100 mL
99332 Coliphage, somatic, E. coli CN13-host, 2-step enrichment, presence (1) or absence (2) per 1 L
99333 Coliphage, somatic, E. coli CN13-host, 2-step enrichment, presence (1) or absence (2) per 4 L
99334 Coliphage, F-specific, E. coli Famp-host, 2-step enrichment, presence (1) or absence (2) per 100 mL
99335 Coliphage, F-specific, E. coli Famp-host, 2-step enrichment, presence (1) or absence (2) per 1 L
99336 Coliphage, F-specific, E. coli Famp-host, 2-step enrichment, presence (1) or absence (2) per 4 L