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Presented at the WISA 2000 Biennial Conference, Sun City, South Africa, 28 May – 1 June 2000 EVALUATION OF DIFFERENT GROWTH MEDIA FOR THE RECOVERY OF SULPHIDE REDUCING ANAEROBIC Clostridium perfringes FROM THE ENVIRONMENT N. Potgieter, E.Musie, R. Sono, C.L. Obi and P.J. du Toit The Department of Microbiology, University of Venda, Private Bag X 5050, Thohoyandou, 0920, South Africa Abstract Clostridium perfringes is a gram positive, spore forming, anaerobic bacterium, and its spores are present in human and animal faecal matter in water and soil. The use of this organism as an indicator of water quality has not received its full attention, even though the presence of Clostridium perfringes in water can be considered conclusive evidence of faecal pollution. This organism is also extremely resistant to the action of chemical and physical factors which are used for the treatment of water. The objective of this study was to optimize and assess different growth media for the detection of Clostridium perfringes from environmental samples Three agar growth media, SFP (Bacto Perfringes agar base), OPSP (Oxoid perfringes agar) and BA (blood agar), were compared for utilization in the membrane filtration counting of Clostridium perfringes from different water sources in rural settlements. The results indicated that SFP agar recovered significantly greater numbers of C.perfringes than OPSP and blood agar. API and other identification test showed a significant number of false presumptive positive and negative isolates on OPSP and blood agar. 1 Introduction Microbial monitoring of drinking water sources requires the use of microorgnisms as indicators of contamination. The most commonly used indicator of faecal pollution of water sources is Eschericia coli, but the use of Clostridium perfringes as a secondary indicator of contamination of water sources and supplies, has recently begun to receive attention (Burger et al., 1984; Arion, 1994; Satory et al., 1998). Clostridium perfringes occur widely in the environment and in human and animal faeces (Rood, 1991). The organism has been incriminated in food and water borne diseases especial gasgangrene and food poisoning in humans (Rood and Cole, 1991). Occurrence in aquatic environments is attributable to faecal pollution (Cabelli, 1977). C. perfringes is endowed with spores which are resistant to adverse environmental conditions and water treatment processes (Grabow et al., 1978). Burger et al. (1984) reported that counts of the organism in waste and polluted waters are usually greater than those of enteric viruses or pathogenic bacteria and that the spores are highly resistant to disinfection and water treatment processes compared with other organisms. This connotes that the absence of C. perfringes in treated water supplies is a possible index of the absence of enteric viruses and bacterial pathogens (Bisson and Cabelli, 1980). The presence of C. perfringes in water meant for human consumption therefore implies that the water is not safe for consumption because it is an opportunistic pathogen. In spite of the excellent features of C. perfringes as a primary candidate for assessing faecal pollution or efficiency of water treatment procedures, its use as a primary indicator has not been generally adopted because of lack of unity of opinions among researchers on the array of available test methods (Grabow and Isaacson, 1978). However, recently, the European Union (1998), proposed that a new microbiological standard for C. perfringes (0/100 ml) be adopted in their directive on drinking water. Several media have been described and employed for the enumeration of C. perfringes. These include mCP medium (Bisson and Cabelli, 1979; Armon and Paymnet, 1988), Tryptose Sulphite Cycloserine (TSC) agar (Satory et al., 1988), and Wilson and Blair Glucose Sulphite Iron (WB ) medium (Burger et al., 1984). In this study SFP (Bacto Perfringes agar base), OPSP (Oxoid perfringes agar) and BA (blood agar) were compared for use in the membrane filtration 2 enumeration of C. perfringes in water. Results obtained are expected to be of value in determining the best media for the enumeration of C. perfringes in our environment. Materials and methods Growth media SFP was prepared as specified by the manufacturers using Bacto SFP Agar base (Code 0811-018), Bacto egg yolk enrichment 50% (Code 3347-61-2), Bacto antimicrobial vial K (Code 333960-3) and Bacto antimicrobial vial P (Code 3268-60-8). OPSP was prepared from Oxoid Perfringes agar base (Oxoid CM543), OPSP-selective supplement A (Oxoid SR76) and OPSPselective supplement B (Oxoid SR77), according to the manufacturer=s instructions. Blood agar plates were supplied by the Department of Medical Microbiology, University of Pretoria, South Africa. Membrane filtration tests Aliquots (10 ml and 100 ml) from environmental water sources were filtered through 47 mm Gelman GN-6 membrane filters (pore size 0,45 µm). The plates were incubated anaerobically in gas jars using Oxoid anerobic gas generating kits. Incubation was at 37YC for 18 - 24 hours. All tests were done in triplicate and results recorded as average values. All black colonies on SFP and OPSP media and colonies with clear haemolysis on blood agar, were counted as presumptive C. perfringes. Identification of isolates Colonies (50 from each media type) were picked from the membranes after incubation and purified on the same medium. Isolates which were gram-positive rods, catalase positive, obligatory anaerobic, and induced stormy fermentation in milk were confirmed as C. perfringes. API tests were used as a final confirmation test of the isolates. 3 Water samples Environmental samples were collected from rural settlements in the Venda region of the Northern Province over a period of six months. The sources included river water, boreholes and wells used by the local population as a primary water source. Samples were collected and analysed within 24 hours after collection. Results In tests on various environmental samples aimed at evaluating the recovery of naturally occurring Clostridium perfringes, the counts were generally higher on SFP agar than on OPSP agar for vegetative cells and spores (Table 1). Blood agar showed a high count of clear haemolysed colonies for most of the water sources, but the confirmation tests indicated that only a low percentage of these colonies (41%) were identified as Clostridium perfringes. API identification of colonies from OPSP agar revealed that all typically black colonies were Clostridium perfringes (96%), while the remaining 4% (grey colonies and white colonies with a black dot in the middle) were other bacterium species. API identification of colonies from SFP agar revealed that 99.8% of the typically black colonies were Clostridium perfringes, whereas the other 0,2% were identified as other Clostridium bacteria. Table 1. Relative recovery of spores and vegetative cells of C.perfringes by SFP, OPSP and BA media (counts/100 ml) Source Vegetative cells Spores OPSP BA SFP OPSP BA SFP River A 30 5056 443 2 20 0 River B 33 5950 943 10 10 5 River C 152 3382 942 8 16 8 Borehole A 6 502 28 5 45 10 Borehole B 10 910 88 0 55 4 Well A 44 5200 890 10 80 40 Well B 2 1278 82 0 10 2 Well C 120 1485 1158 6 2 18 4 Discussion The main objective of this study was to determine the efficiency of different agar mediums for the selective recovery of C.perfringes from environmental samples. The results indicated that SFP agar is a more specific and selective medium for the general recovery of C.perfringes from water when present in the form of vegetative cells. There was not much difference between OPSP and SFP medium for the selective recovery of spores from the same samples. Blood agar (BA) proved to be inadequate as a selective medium because of the high false positive counts. The high counts of C.perfringes present in different water sources in rural communities, indicates that this organism poses a significant health risk for these communities who use these sources to collect water for domestic purposes. Future work will include a comparison of OPSP and SFP media with mCP and TSC medium. Acknowledgements We would like to thank the post graduate students in the Department of Microbiology for their valuable assistance with this project. References Arion (1994). The microbiology of water 1994: Part 1 B Drinking Water. Reports on Public Health and Medical Subjects No 71. Methods for the Examination of Water and Associated Materials. London, HMSO. Armon R and Payment P (1988). A modification of mCP medium for enumerating C. perfringes from water samples. Canadian Journal of Microbiology 34: 78 B 79. Bisson JW and Cabelli VJ (1979). Membrane filter enumeration methods for C. perfringes. Applied and Environmental Microbiology 37: 55 B 66. Bisson JW and Cabelli VJ (1980). Clostridium perfringes as a water pollution indicator. Journal of Water Pollution and Control Fed. 52: 241 B 248. 5 Burger JS, Nupen EM and Grabow WOK (1984). Evaluation of four growth media for membrane filtration counting of Clostridium perfringes in water. Water SA 10(4): 185 B 188. Cabelli VJ (1977). Clostridium perfringes as a water quality indicator. In Bacterial Indicator/Health Hazards Associated with Water. ASTM STP 635. Hoadley, AW and Dutka BJ (Eds). American Society for Testing and Materials, Philadelphia, Pennsylvania. Rood JI and Cole ST (1991). Molecular genetics and pathogenesis of Clostridium perfringes. Microbiological Reviewa 55: 621 B 648. EU (Europeun Union)(1998). Common position (EC) no 13/98 adopted by the council on 19 December 1977 with a view to the adoption of the Council Directive on the quality of water intended for human consumption. Official Journal of the European Communities 26.3.98.C91/1 - C91/27. Grabow WOK, Bateman BW and Burger JS (1978). Microbiological quality indicators for routine monitoring of waste water reclamation systems. Prog. Wat. Technol. 10: 317 B 327. Grabow WOK and Isaacson M (1978). Microbiological quality and epidemiological aspects of reclaimed water. Prog. Wat. Technol 10: 329 B 335. Satory DP, Field M, Curbishley SM and Pritchard AM (1998). Evaluation of two media for the membrane filtration enumeration of Clostridium perfringes from water. Lett. Appl. Microbiol 27: 323 B 327. 6