Evaluation of an alternative method for the enumeration and confirmation of Clostridium perfringens from treated and untreated sewages

Abstract

Aims: Clostridium perfringens is recommended as a suitable indicator bacterium for human enteric viruses, Giardia cysts and Cryptosporidium oocysts in finished water and in the assessment and evaluation of water treatment. Several agars and confirmation procedures were evaluated in parallel with the Australian/New Zealand Standard (AS/NZ) Method for the enumeration of Cl. perfringens from treated and untreated sewage samples. Methods and Results: The current AS/NZ method utilizes tryptose sulfite cycloserine agar (TSC), lactose gelatin medium (LG) and nitrate motility medium (NM) at an incubation temperature of 37°C. Sixty treated and untreated sewage samples were used to evaluate TSC agar, membrane Cl. perfringens agar (mCP), Perfringens agar (OPSP) and Perfringens agar with 4‐methylumbelliferyl phosphate (OPSP–MUP) for enumeration of Clostridium. An incubation temperature of 44°C for 24 h was used for comparison. Confirmation procedures were also evaluated using 103 isolates and included LG and NM, ortho‐nitrophenyl‐β‐d‐galactopyranoside (ONPG) with MUP (ONPG–MUP) and phosphatase reagent (PR). OPSP compared favourably with TSC agar. One false negative result was obtained from each of the LG/NM and ONPG–MUP procedures. No false results were obtained using the PR confirmation procedure. Conclusions: OPSP agar and PR were determined as suitable replacements for the AS/NZ Standard procedure with no interference from spreading organisms. Significance and Impact of the Study: This is a simple and rapid method for isolating and enumerating Cl. perfringens from sewage samples and confirmed results can be reported more quickly due to shorter analytical turnaround times.

Implications of faecal indicator bacteria for the microbiological assessment of roof-harvested rainwater quality in southeast Queensland, Australia

“…OPSP agar plates (for Clostridium perfringens) were incubated anaerobically at 44 8C for 24 h. The confirmatory test for Clostridium perfringens was performed according to the method described previously (Wohlsen et al 2006). Sample serial dilutions were made and filtered through nitrocellulose membranes (47 mm diameter) with 0.45 mm pores (Advantec, Tokyo, Japan) and placed on modified mTEC agar (Difco, Detroit, Mich.), membrane-Enterococcus indoxyl-b-D-glucoside (mEI) agar (Difco), and oleandomycin-polymyxin-sulfadiazine perfringens (OPSP) agar with supplement (Oxoid, London, UK) for the isolation of E. coli, enterococci, and spore-forming Clostridium perfringens, respectively.…”

Real-Time PCR Detection of Pathogenic Microorganisms in Roof-Harvested Rainwater in Southeast Queensland, Australia

“…Modified mTEC agar plates were incubated at 35°C for 2 h to recover stressed cells, followed by incubation at 44°C for 22 h (39), and mEI agar plates were incubated at 41°C for 48 h (38). OPSP agar plates (for C. perfringens) were incubated anaerobically at 44°C for 24 h. The confirmatory test for C. perfringens was performed according to the method described previously (43). For bacterial enumeration, all water samples were tested in triplicate.…”

Abilities of the mCP Agar Method and CRENAME Alpha Toxin-Specific Real-Time PCR Assay To Detect Clostridium perfringens Spores in Drinking Water

“…Furthermore, the presence of C. perfringens in water is also associated with fecal contamination, and it has been evaluated and utilized as an alternative indicator of fecal pollution (4-14). Thus, a simple and reliable culture-based method to isolate and enumerate C. perfringens, the membrane filtration method on mCP agar, has been elaborated and evaluated to monitor the presence of C. perfringens in water (15)(16)(17)(18)(19)(20). In the European Union, the mCP agar method is the reference method used to assess the presence of C. perfringens in water intended for human consumption (21).…”