Evaluation of microbiological methods for water quality analysis and detection of Cryptosporidium in surface waters

Tracey D Wohlsen

doi:10.25907/00211

Current methods for enumerating faecal indicator bacteria and water quality analysis lack precision in enumerating bacterial content of water samples due to a lack of a standard quality control. This is in part, responsible for inter-laboratory variations in water quality tests. The objectives of this thesis were to assess the effectiveness of existing methods for the detection and enumeration of faecal indicator bacteria as well as improving existing methods for detection of Cryptosporidium in surface water samples. A number of methods were assessed for their efficacy to detect faecal indicator organisms using a precise quality control tool, i.e. BioBall vials containing freeze-dried E. coli or E. aerogenes cultures. These methods included membrane filtration (MF) using mFC and mLS agars, standard plate count (pour and spread plates), defined substrate technology (DST) using Colilert and Colisure, MPN and Petrifilm. From the methods designed to analyse 100 mL water samples (i.e. MF, DST and MPN), Colilert produced the most consistent results with the lowest coefficient of variation. Similar assessment was performed to evaluate the recovery efficiency of five membrane filtration techniques for the recovery of Cryptosporidium from water samples. Using EasySeed and ColorSeed kits to provide standard inocula, accurately dispensed oocysts (n = 100) and cysts (n = 100) were seeded into distilled and surface water samples. Of the five membrane filters analyzed, the Envirochek HV filters, yielded a higher recovery for both oocysts and cysts in distilled and surface waters than the other filtration methods, when subjected to a 5-sec back-wash. This modified method was also less labour intensive and as such was used in a 12-month survey of Cryptosporidium contamination in the Lake Baroon Catchment in the Sunshine Coast region of Queensland, Australia. This survey indicated there was no relationship between rainfall, indicator bacteria and the detection of oocysts/cysts in surface water at most sites sampled. Increased counts of C. parvum, coinciding with rainfall, were observed at only three sites. During the course of this investigation, an outbreak of cryptosporidiosis occurred in the region caused by the consumption of unpasteurised milk, and milk samples were also analysed. A high number of Cryptosporidium oocysts were observed in the raw effluent of the main sewage treatment plant within the catchment, and this coincided with the outbreak of Cryptosporidium. However, the number of oocysts in surface water during the course of this outbreak did not increase. Sites identified as being contaminated with Cryptosporidium during the initial 12-month survey were revisited after 9-months and a level of risk was assigned to each site. This risk assignment could be used as a model in microbiological studies investigating the occurrence of Cryptosporidium in surface waters. As the water treatment plant receiving water from the Lake Baroon catchment provides drinking water to the region, an evaluation of the ozone disinfection process utilised at the local water treatment plant was assessed at winter and summer temperatures. Containers seeded with 1000 oocysts/L were filled with ozonated water and the level of ozone decay, viability of oocysts, as well the Cryptosporidium inactivation, was measured over 10 minutes. This study showed that ozone inactivated 92% of oocysts in summer and 92.8% in winter. As spore forming C. perfringens has been suggested as a surrogate indicator for the presence of Cryptosporidium, it was necessary to develop an improved method for the isolation and identification of C. perfringens from surface water samples. Several membrane filtration and confirmation methods were evaluated in parallel with the Australian/New Zealand (AS/NZ) Standard method, including membrane Clostridium perfringens (mCP) agar, oleandomycin phosphate/polymyxin B sulphate Perfringens agar (OPSP) and OPSP agar combined with 4-methylumbelliferyl phosphate (MUP) (OPSP-MUP) for enumeration by membrane filtration. For confirmation procedures, Ortho-nitrophenyl-D-galactopyranoside (ONPG) with MUP (ONPG-MUP), plus phosphatase reagent (PR) and lactose gelatin and nitrate motility (LG/NM) agars were evaluated. Treated and untreated sewage water samples were used for filtration and confirmation procedures in this evaluation. OPSP agar, blood agar containing 0.5% neomycin (BANeo) and PR were shown to be a suitable substitute to the AS/NZ Standard method for surface and sewage effluent samples with the selectivity and sensitivity of the PR procedure at 100%. This improved method is suitable for detection of spore forming C. perfringens as an indicator organism for evaluating the inactivation of Cryptosporidium in water and waste-water samples.

Evaluation of microbiological methods for water quality analysis and detection of Cryptosporidium in surface waters

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