Background - Methods for Detecting and Enumerating Bacterial Indicators in Water
Standard Methods
Classically, the detection and enumeration of bacterial indicators in water relied on the use of the standard methods, Membrane Filtration (MF) and Multiple Tube Fermentation (MTF). In these methods, any microorganisms present in a water sample are grown in solid agar or liquid broth media, which supply the nutritional requirements. Specific compounds are incorporated into the primary media, which allow for selection and differentiation of an organism of interest. Endo agar has been routinely used for the enumeration of coliforms. The presence of lactose in this agar allows for its metabolism with the formation of acid and gas, a hallmark characteristic of the coliform group, which is detected by a colour change (Baker and Herson, 1999). Strain segregating techniques need to be applied as a confirmatory test to verify that faecal coliforms are in fact E. coli. This verification procedure is time consuming, labour intensive and requires a laboratory setting. In addition, studies have shown that the confluent growth of background microorganisms on Endo agar plates prominently used in the Membrane Filtration procedure has the ability to mask the presence of coliforms (Grasso et al., 2000) therefore yielding smaller counts of the targeted coliform organisms. High levels of heterotrophic bacteria typically found in natural surface waters may amplify this masking effect. Incorrect enumeration of other non-target organisms with the membrane filtration method may lead to false-positive readings. Turbidity caused by the presence of algae, particulates, or other interfering material in natural waters may also alter the effectiveness of the Membrane Filtration procedure (American Public Health Association, 1995).
Recognition of some of the inefficiencies of these standard methods has led to the growing use of rapid defined substrate methods, including the Coliplate™ technique (Alonso et al., 1999).
Rapid Defined Substrate Methods - Including the Coliplate™ Technique
The rapid detection of bacterial indicators is extremely important when assessing the microbiological safety of drinking and recreational waters. The introduction of rapid defined substrate techniques, which utilize chromogenic and fluorogenic substrates incorporated into selective media has led not only to the faster detection of bacterial indicators but also the improved accuracy for their enumeration (Manafi, 1996). These methods do not require laboratory conditions, or confirmatory testing, and are a cost effective alternative to the traditional standard methods. The ability to analyze water samples more or less on-site as opposed to transporting samples to a certified laboratory makes this technique an attractive method for regulatory and volunteer water quality monitoring programs. Also, the faster turnaround of results may help ensure that hazardous conditions are detected in a suitable time frame.
Defined substrate methods for the detection and enumeration of coliforms and E. coli in drinking and recreational waters have been widely accepted by government and scientific communities (Baker and Herson, 1999), including the American Public Health Association, the Environmental Protection Agency and Environment Canada.
Comparative Evaluation of Methods for Enumerating Bacterial Indicators
in Recreational Waters and the Potential for Methodological Bias
Bacteriological water quality guidelines provide mandatory limits for Total Coliform and E. coli in recreational waters without further specifying the methods to be used. As a result there is often an inconsistency in methods used to enumerate indicator bacteria in recreational water quality monitoring initiatives. Three methods, membrane filtration (MF), multiple tube fermentation (MTF), and defined substrate technology (DST) are recognized as appropriate procedures to enumerate indicator bacteria for routine surface water quality monitoring (American Public Health Association, 1995).
The three methods are each based upon measuring different products of bacterial growth. MF enumerates bacterial colonies on a specific growth substrate. MTF measures metabolic activity as determined by fermentation and the production of gas. DST methods measure the ability of organisms to metabolize a specific labelled substrate, thereby releasing a chromogen (pigment-producing enzyme). These differences in analytical endpoint provide the potential for differing results among methods (Noble et al. 2003).
Validation Studies - Scientific Literature
Numerous investigators have evaluated the potential methodological bias of using different techniques for the assessment of bacteriological water quality. A general consensus of published studies indicate that DST methods using 4-methylumbelliferyl -D-glucuronide (MUG) substrate (utilized by Coliplate™ ) are adequate for the purpose of enumerating E. coli as an indicator of faecal pollution in regulatory monitoring of recreational waters. Moreover, various investigators recommend DST (MUG supplemented) techniques over the standard methods, indicating that the method provides a greater specificity for the detection of E. coli and is therefore more accurate (Hernandez et al., 1991, Manafi, 1996 and Neidhardt et al., 1995).
Details of precision differences between DST methods and the traditional standard techniques have been reported in the literature, indicating the potential for measurement error using standard methods. Noble et al., (2003) compared a DST method with the membrane filtration method and found that the latter tended to give smaller values than the DST method for both Total Coliforms and Faecal Coliforms. Through discussions with the 22 accredited laboratories involved in their study, it was concluded that filtration was difficult and that patchy colony growth often created clustering of bacteria that made differentiation of colony units difficult, ultimately leading to the underestimation of bacteria counts (Noble et al, 2003).
The American Public Health Association, (1995) concluded that the membrane filtration procedure generally underestimates the number of viable coliform bacteria. DST methods including Coliplate™ have a greater ability to enumerate injured or weakened Coliform and E. coli bacteria. Lifshitz and Joshi (1998) found the Coliplate™ method gave estimates of E. coli that were 47 percent greater than the standard membrane filtration method, likely due to its reliability in detecting weakened cells often encountered in natural waters. However, in comparing the two methods (Coliplate™ and membrane filtration) the investigators observed good correlations for total coliform (r2=0.84) and E.coli (r2=0.95) enumerations (Lifshitz and Joshi, 1998).
A study by Hernandez et al., (1991) reported an 87.4 percent confirmation rate for E. coli with a DST assay compared to a 31.7 percent false-positive rate with membrane filtration (Hernandez et al., 1991), further suggesting that DST methods such as Coliplate™ , have a superior recovery rate and greater specificity for E.coli than membrane filtration methods.
Validation Studies - Relevant Research Studies
Recent research undertaken by Schiefer, 2004 specifically compared the Coliplate™ technique to the standard membrane filtration technique for enumerating Total Coliform and E. coli in a large quantity of surface water samples collected throughout Georgian Bay and from various inland lakes. Similar to other studies in the literature this research project found that the membrane filtration method consistently underestimated the density of E. coli in surface water samples compared to the Coliplate™ method. This was attributed to the fact that other microorganisms present in the water samples (natural surface waters have a wide spectrum and high numbers of microorganisms) out-competed the Coliform group of organisms (including E. coli) on the agar used for the standard membrane filtration method. Consequently, merged growth of unidentifiable colonies on the agar plates made enumeration particularly difficult throughout this study. It was concluded that the DST method Coliplate™ is a more reliable and species-specific method for enumerating E. coli in natural surface waters.
Concluding Remarks
Validation studies in the literature and in recent research projects suggest that DST methods including the Coilpate™ technique are a more reliable technique than the standard methods for enumerating bacteria in natural waters. In addition to it's proven accuracy, it is also a more suitable procedure because of its user-friendliness and for efficiency and logistical reasons. One of the challenges of monitoring bacteriological surface water and drinking water quality in more remote regions is the lack of laboratory services and trained personnel to collect, transport and analyze samples in an effective manner. Unlike the traditional standard methods, the DST method, Coliplate™ can be employed by non-technical individuals in a non-laboratory setting and enables the results to be reported in a timely manner. This improves the feasibility for bacteriological water quality monitoring initiatives to be effectively undertaken regardless of the location.
Major users of the Coliplate™ technology include Environment Canada, Provincial and Municipal government groups, Non-Government Organization's, private environmental agencies and various universities. Consequently, Coliplate™ validation studies have been included in a number of research projects and graduate theses.
List of References
Alonso, J.L., A. Soriano, O. Carbajo, I. Amoros and H. Garelick. 1999. Comparison and recovery of Escherichia coli and theremotolerant coliforms in water with a chromogenic medium incubated at 41 and 44.5 C. Applied and Environmental Microbiology. 65: 3746-3749.
American Public Health Association. Standard methods for the examination of water and wastewater: 1995, 18th ed, Eaton, A.D, L.S. Clesceri and A.E. Greenberg (eds.), Washington, D.C.
Baker K.H. and D.S. Herson. 1999. Detection and occurrence of indicator organisms and pathogens. Water Environment Research. 71(5):530-551.
Grasso, G.M., M.L. Sammarco, G. Ripabelli and I. Fanelli. 2000. Enumeration of Escherichia coli and Coliforms in surface water by multiple tube fermentation and membrane filter methods. Microbes. 103: 119-125.
Hernandez, J.F., J.M. Guibert, J.M. Delattre, C. Oger, C. Charriere, B. Hughes, R. Serceau and F. Sinegre. 1991. Miniaturized Fluorogenic Assays for Enumeration of E. coli and Enterococci in Marine Water. Water Science and Technology. 24: 137-141.
Lifshitz, R. and R. Joshi. 1997. Comparison of a novel Coliplate™™ kit and the standard membrane filter technique for enumerating Total Coliforms and Escherichia coli in water. Environmental Toxicology and Water Quality. 13:157-164.
Manafi, M. 1996. Fluorogenic and chromogenic enzyme substrates in culture media and identification tests. International Journal of Food Microbiology. 31:45-58.
Neidhart, S., G. Havemeister, C. Holler and K.O. Gundermann. 1995. Evaluation of MUG-supplemented media for the detection of E. coli in recreational water surveillance. Zentralblatt fur Hygine und Umweltmedizin. 198:152-164.
Nobel, R.T., S.B. Weisberg, M.K. Leecaster, C.D. McGee, K. Ritter, K.O. Walker and P.M. Vainik. 2003. Comparison of beach bacterial water quality indicator measurement methods. Environmental Monitoring and Assessment. 81: 301-312.
Schiefer, K. 2004. An Analysis of Bacteriological Surface Water Quality in Discrete Nearshore Areas of Southeastern Georgian Bay. Master of Science Thesis. University of Guelph.
