U.S. Environmental Protection Agency Method 1602:
Male-specific (F+) and somatic coliphage in water by single agar
layer (SAL) procedure was developed to enumerate coliphage in ground water and
other waters. 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: This
method is a plaque assay method. A
100-mL sample is combined with magnesium chloride, log-phase host bacteria, and
tryptic soy agar. The sample
mixture is poured into plates and incubated overnight.
If phage particle were present in the sample, they infect an E.
coli cell and reproduce, causing death of the cell and cell lysis.
This continues until a plaque is visible; a plaque is a circular clearing
in the bacterial lawn. The plaques are counted and summed for all plates from a
single sample and expressed as plaques per 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 has been using the SAL method to enumerate viruses in surface waters,
because 100 mL is generally sufficient to detect coliphage in surface water.
Coliphage are recognized to be representative of the
transport and survival of viruses in the environment. They are found in high
numbers in sewage and are thought to be reliable indicators of sewage
contamination of waters. 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:
See SAL method directions (Appendix O).
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, 2001, Method 1602:
Male-specific (F+) and somatic coliphage in water by single agar layer (SAL)
procedure: Washington D.C., EPA 821-R-01-029, 30 p.
NWIS PARAMETER CODES:
90903 coliphage, E. coli CN-13 host, plaques per 100 mL
90904 coliphage, E. coli F-amp host, plaques per 100 mL