MODIFICATION OF FUNG DOUBLE TUBE AND CP ANASELECT OXYPLATE METHODS TO IMPROVE THEIR PERFORMANCE IN ENUMERATING CLOSTRIDIUM PERFRINGENS FROM SEWAGE AND ENVIRONMENTAL WATERS

ABSTRACT

The Fung Double Tube (FDT) and the newly developed CP AnaSelect Oxyplates methods can enumerate concentrations of Clostridium perfringens without need for external anaerobic generating systems. Because Clostridium perfringens is a reliable indicator of fecal contamination and it is one of the fastest growing fecal bacteria, these two methods were evaluated as feasible methods to screen recreational waters for sewage contamination. To increase the sensitivity and selectivity of these methods, three modifications were evaluated. The first modification was to pretreat the water samples using a microwave oven to attain high temperature (70C) for a short time (2.5 min) to reduce the interfering growth of non-C. perfringens colonies on the selective media. The second modification was the addition of phosphatase reaction, thus enumerated colonies could be confirmed as C. perfringens. The third modification was to increase the sample volume for the FDT test from 5 to 10 mL/tube. The data collected showed that these modifications improved the selectivity and sensitivity of these two methods to enumerate C. perfringens from sewage-contaminated water samples as well as environmental water samples such as streams, harbors, canals and coastal swimming beaches. The recovery efficiencies of the FDT and experimental CP AnaSelect Oxyplate for C. perfringens were similar to traditionally used membrane C. perfringens, tryptose sulfite cycloserine and Shahidi Ferguson perfringens agar media by membrane filtration technology, followed by incubation in anaerobic chambers. These results show that the modified FDT and CP AnaSelect Oxyplate methods are feasible and reliable methods to monitor environmental waters for C. perfringens. The FDT method is especially promising because it is simple, inexpensive and can produce results in 5–6 h.

PRACTICAL APPLICATIONS

Clostridium perfringens is currently being evaluated as a reliable indicator of sewage contamination in recreational waters. However, traditional methods to assay for C. perfringens are expensive and cumbersome because of the need for external anaerobic generating systems. In addition, target colonies must be confirmed by a second test. In this study, two methods are described, with capabilities to self-generate anaerobic conditions and to immediately confirm the target colonies as C. perfringens. Moreover, one of the methods meets the difficult criterion of obtaining results in 6 h so decision on closing the beach can be reached on the same day the beach water sample is tested. Since these two methods are feasible and reliable, it will encourage many laboratories to assay their recreational waters for C. perfringens and a national database to determine the quality of recreational waters based on concentrations of C. perfringens can be developed.