Monday, 3 March 2014

Assessing water quality: a modified technique for detecting viral pathogens

The microbial quality of water has often been used as a method to assess overall water quality, to ensure it is not harmful to human health. Esherichia coli whilst an efficient indicator of bacterial pathogens it is unable to be used as a direct indicator of viral pathogens. Due to this, and because viral contamination has been attributed to >50% of all waterborne illnesses since 1980’s, coliphages (viruses that infect coliform bacteria) are recommended to be used in conjunction with E. coli as bioindicators of faecal contamination and predictors of water quality. However, the additional testing for coliphages was not considered economically feasible by the 2006 Ground Water Rule (a prequalification of water quality for general human use).

Current methods for coliphage detection and enumeration are multi-step procedures which are costly and require 2-3 days for a result. Thus a quicker and more economically viable alternative viral indicator test is required.

This study attempted to simplify the current methodology by developing a same-day fluorescence assay to detect coliphages which meets the same standard of detection as the current methodology, Method 1601. This modified methodology was termed Fast Phage and used an isopropyl-β-D-1-thiogalactopyranoside (IPTG) enrichment medium to induce transcription of the lac operon in the host coliform bacteria E. coli. On this medium uninfected cells grew exponentially whereas the expression of the lac operon induced lysis of infected cells identified by a rapid release of beta-galactosidase enzyme. When this initial enrichment material was transferred to a secondary medium containing enzyme substrate 4-methylumbelliferyl-β-D-galactosidase (MUG-Gal), MUG-Gal was cleaved from the medium by infected cells; beta-galactosidase formed an enzyme-substrate complex with the MUG-Gal. Cleaved substrate liberated the MUG, a component which fluoresces under UV light. Positive fluorescence indicated coliphage presence, non-fluorescence indicated coliphage absence

20 replicate samples were analysed using Method 1601 and Fast Phage. Fast Phage met the performance criteria defined by Method 1601, and as fluorescence was shown occur within 1h it was able to reliably detect coliphage levels rapidly. This was demonstrated by all 5 laboratories that tested the modified methodology with no significant difference in results between Fast Phage, and the reference method, Method 1601.

Testing for coliphages as an indicator of viral pathogens alongside current bacterial indicators is important to more thoroughly assess water quality and further protect human health. This preliminary study does demonstrate that a quicker, cheaper yet still reliable method can potentially be used to detect coliphages in water. The modified method, Fast Phage, meets the established criteria for detecting coliphages. Using a fluorescence-based predictor is novel but can provide an early warning of viral pathogens, as it has demonstrated to yield results within a day. Though many more stringent studies will have to be conducted prior to this modified method being validated in the protocol for water quality testing, the data from this study can be used to support the concept of the Fast Phage method. With further tweaking of the current methodology, I feel there is definitely potential for fluorescence-based coliphage testing to be used in the future as a measure of viral pathogens.

*** I found this paper quite technical in terms of existing protocol and procedures for accepting a new methodology. For the ease of reading and understanding I have tried to draw the main conclusions from the paper, though the paper does contain a LOT more detailed methodologies, results and applications. ***

Salter R., Durbin G., Conklin E., Rosen J. and Clancy J. (2010) Proposed modification 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. Applied and Environmental Microbiology, 76(23), 7803-7810


  1. This does sound very complex! How is this new method cheaper? Are they expecting to replace Method 1601 with Fast phage or are they going to used for different situations?

  2. Method 1601 requires more resources and equipment to analyse water samples than the Fast Phage method, making it more costly. Additionally, as Method 1601 is a much lengthier process it incurs an associated labour cost. Fast Phage is ultimately a modification of the existing Method 1601 and are both used to test the viral concentration within water samples, however the modified method (fast phage), if implemented, will be much more time and cost efficient.