Improved method for coliphage detection based on β-galactosidase induction

Proposed Modifications of Environmental Protection Agency Method 1601 for Detection of Coliphages in Drinking Water, with Same-Day Fluorescence-Based Detection and Evaluation by the Performance-Based Measurement System and Alternative Test Protocol Validation Approaches

“…The ability to reliably detect levels of 1.3 to 1.5 PFU/100 ml in less than 8 h or within 1 working day shift provides better public health protection than do assays that require 24 to 48 h. This speed and sensitivity have not yet been achieved with conventional microbiology methods approved for drinking water quality. Other researchers have shown that accelerated amplification with subsequent coliphage detection techniques can meet detection criteria in a single working day (11,12,14,20). The Fast Phage modifications work within the framework of EPA Method 1601 while incorporating the use of betagalactosidase as an 8-h coliphage prediction method.…”

“…The same-day coliphage detection method utilizes an enrichment medium containing isopropyl-␤-D-1-thiogalactopyranoside (IPTG) to induce transcription of the host E. coli lac operon. Coliphage lysis has been shown to be coupled with lac operon expression; thus, there are a large amplification and a rapid extracellular beta-galactosidase enzyme release during coliphage-induced lysis of the infected host in comparison to the uninfected exponentially growing host (11). A transfer aliquot of the amplified primary enrichment material into a secondary enrichment/detection medium containing the enzyme substrate 4-methylumbelliferyl-␤-D-galactoside (MUG-Gal) will fluoresce under 366-nm UV light when the MUG-Gal is cleaved by the extracellular beta-galactosidase to liberate the fluorescent component MUG and indicate the presence of coliphages.…”

“…The host bacterium specific for male-specific coliphages in the EPA methods is ampicillin/streptomycin-resistant E. coli F amp , and the host specified in European Union (EU) standard methods is Salmonella WG 49 (7,29). Simple and rapid methods for coliphage detection have been reported with preliminary detection in a single working day (11,12,14,20). Qualitative detection methods, including EPA Method 1601, are multiple-step procedures that involve coliphage replication in exponential-growth-phase cells of the host E. coli (enrichment step) followed by a spotting on seeded agar for plaque confirmation.…”

Determining the Minimum Inhibitory Concentration of Bacteriophages: Potential Advantages

“…In 1992, the American Public Health Association (APHA) proposed a single-agar-layer plaque for coliphage enumeration from ground and surface water samples. In general, the limitations of this agar-based method are poor plaque visibility [17], false positive results caused by non-viral toxic material [18], poor adsorption of phages on the bacterial surface [19], superinfection immunity [20] and its restriction modification system [21] in which the success of a phage infection depends on the host's restriction-modification (R-M) system, which depends on issues such as the activities of the host methyltransferase, the restriction endonuclease and the number of susceptible sites in the phage genome.…”

Microbiological monitoring for the U.S. Geological Survey National Water-Quality Assessment Program

“…The single-agar layer (SAL), direct plating method with induction of β-galactosidase (Ijzerman and Hagedorn, 1992) is recommended for detection of somatic and F-specific coliphage in streamwater samples. In this method, 100-mL sample volumes are mixed with an agar medium, E. coli host culture, chemicals that induce the β-galactosidase enzyme, and appropriate antibiotics.…”