Evaluation of a most probable number method for the enumeration of Legionella pneumophila from potable and related water samples.

This study compared the performance of a novel MPN method (Legiolert/Quanti-Tray) with the ISO 11731-2 membrane filtration method for the enumeration of Legionella pneumophila from 100 ml potable water and related samples. Data from a multi-laboratory study analysed according to ISO 17994 showed that Legiolert™/Quanti-Tray® yielded on average higher counts of L. pneumophila. The Legiolert medium had a high specificity of 96·4%. The new method represents a significant improvement in the enumeration of L. pneumophila from drinking water-related samples. SIGNIFICANCE AND IMPACT OF THE STUDY: Legionella pneumophila is an opportunistic pathogen of major concern. The current large volume quantitative method employs membrane filtration (MF) and selective culture on GVPC agar followed by confirmation of isolates by serology (ISO 11731-2) We present here the results of a multi-laboratory evaluation of a most probable number (MPN) in-situ confirmed method (Legiolert™/Quanti-Tray®). The results indicate that Legiolert/Quanti-Tray yielded on average higher counts of L. pneumophila than ISO 11731-2. This development significantly improves and simplifies the enumeration of L. pneumophila from potable water samples.

Evaluation of a membrane filtration method for the rapid enumeration of confirmed Clostridium perfringens from water.

UNLABELLED: A modification of the UK reference and ISO 14189 TSCA medium for the enumeration of Clostridium perfringens from water coupled with a membrane filter transfer technique for testing for production of acid phosphatase was evaluated. The new tryptose cycloserine agar (TCA) medium, which lacks sodium metabisulphite but contains sodium pyruvate to improve recovery, allows the isolation and confirmation of Cl. perfringens within 18-24 h of sample processing. Data from a multilaboratory study analysed according to ISO 17994 showed that TCA was equivalent to TSCA for the enumeration of Cl. perfringens. The identification of acid phosphatase-negative isolates revealed a false-negative rate for the TCA method of 0.8%. The TCA membrane filter transfer procedure provides confirmed Cl. perfringens counts in half the time of the TSCA method and is simple to undertake. SIGNIFICANCE AND IMPACT OF THE STUDY: The testing of drinking water for Clostridium perfringens is a regulatory parameter in Europe and the UK. Current UK and ISO methods employ membrane filtration (MF) and TSCA medium followed by subculture and confirmation of isolates by testing for acid phosphatase. This takes 48 h. We present here the results of a multilaboratory evaluation of a MF method that features a simplified isolation medium (TCA) and a membrane transfer procedure for the acid phosphatase test resulting in confirmed results being available in 18-24 h. This development significantly reduces the time to confirmed results for Cl. perfringens from water samples.

Evaluation of acid phosphatase as a confirmation test for Clostridium perfringens isolated from water.

AIMS: To evaluate testing for acid phosphatase as an alternative method for the confirmation of Clostridium perfringens isolated from water. METHODS AND RESULTS: Sixty-two reference strains of Clostridium were tested for their ability to produce acid phosphatase, as well as reduction of sulfite on tryptose sulfite cycloserine agar (TSC) and production of fluorescence in TSC supplemented with 4-methylumbelliferylphosphate (MUP). Additionally 155 environmental presumptive C. perfringens isolates from TSC incubated at 44 degrees C were identified and tested for acid phosphatase production and by the conventional MNLG (testing for motility, nitrate reduction, lactose fermentation and gelatin liquefaction) confirmation procedure. Twenty-seven strains from 15 species of Clostridium-reduced sulfite to some extent on TSC incubated at 44 degrees C, with a significant number of species being able to grow well at this temperature, indicating that a confirmation step is needed for the enumeration of C. perfringens on this medium. All 10 strains of C. perfringens tested, together with one strain each of Clostridium baratii and Clostridium rectum produced acid phosphatase. These also produced fluorescence on MUP supplemented TSC, as did 13 strains of acid phosphatase negative, sulfite-reducing clostridia, representing nine species. Of the environmental isolates, 114 were identified as C. perfringens of which 108 (94.7%) were confirmed by the acid phosphatase test compared with 104 (91.2%) by the MNLG tests. CONCLUSIONS: Testing for acid phosphatase production is at least as reliable, and much simpler to perform, than the current standard confirmation MNLG procedure. Incorporation of MUP into TSC does not reliably improve the identification of presumptive C. perfringens. SIGNIFICANCE AND IMPACT OF THE STUDY: Application of testing for acid phosphatase as a confirmation test for C. perfringens would substantially simplify the analysis for this bacterium from water samples, and reduce the analysis time to confirmed counts.

A simple modified membrane filtration medium for the enumeration of aerobic spore-bearing bacilli in water.

Aerobic spore-bearing bacilli have been proposed as a surrogate indicator for the removal of Cryptosporidium by drinking water treatment processes. Pasteurisation of samples followed by culture on non-selective media is the method of choice. Using white membranes for filtration of water samples makes colony counting difficult. Vital dyes such as neutral red or trypan blue can help when added to the growth medium but these dyes tend to colour the membrane and reduce the contrast between the colonies and the background. The incorporation of bromothymol blue at a concentration of 0.005% (w/v) into nutrient agar facilitated colony counting without inhibiting colony formation compared with unsupplemented nutrient agar. Statistical analysis of the data (ANOVA) confirms this observation. The modified technique was found to be satisfactory with spore suspensions of Bacillus globigii and B. cereus as well as with samples of surface water, settled water and drinking water.

Microbiological safety of water.

Significant advances in water treatment over the last century have resulted in massive improvements in the microbiological safety of public drinking water supplies in the UK and the developed countries. Incidences of illness due to poor treatment or post-treatment contamination are rare, but when they occur tend to attract considerable media attention. A well managed water treatment works and supply system can provide high quality drinking water wherever in the world it is located. As a rule, throughout the world, private supplies tend to be of a poorer quality than public supplies, but poorly managed public supplies have the potential to make a large number of people ill and continued effort is needed to maintain and improve drinking water quality world-wide.

Recovery of Cryptosporidium oocysts from small and large volume water samples using a compressed foam filter system.

A novel filter system comprising open cell reticulated foam rings compressed between retaining plates and fitted into a filtration housing was evaluated for the recovery of oocysts of Cryptosporidium from water. Mean recoveries of 90.2% from seeded small and large volume (100-2000 l) tap water samples, and 88.8% from 10-20 l river water samples, were achieved. Following a simple potassium citrate flotation concentrate clean-up procedure, mean recoveries were 56.7% for the tap water samples and 60.9% for river water samples. This represents a marked improvement in capture and recovery of Cryptosporidium oocysts from water compared with conventional polypropylene wound cartridge filters and membrane filters.

Evaluation of two media for the membrane filtration enumeration of Clostridium perfringens from water.

Two media (mCP medium and Tryptose Sulphite Cycloserine (TSC) agar) were evaluated for recovery of Clostridium perfringens in environmental and part-treated drinking water. For laboratory strains of Clostridium, mCP was more selective and specific for Cl. perfringens than TSC, but was markedly less efficient for the enumeration of both vegetative cells and spores. For samples of river water and part-treated drinking water, TSC recovered significantly greater numbers of Cl. perfringens than mCP. In contrast to previous reports, there was a significant number of false presumptive positive and negative isolates on mCP. TSC is a more suitable medium for the routine monitoring of water supplies for the presence of Cl. perfringens.

Conventional culture for water quality assessment: is there a future?

Conventional culture for the detection, enumeration and identification of micro-organisms has been the traditional tool of the microbiologist. It is, however, time-consuming and labour-intensive and confirmed results often require several days of analysis. Culture may not grow the organisms being sought and for enumeration may only detect a small proportion of the total population. However, it does have the advantage of being simple to use and relatively inexpensive. It is also a direct means of assessing cell viability. Novel fluorogenic dyes and fluorgenic and chromogenic substrates have overcome some of these problems by providing a means of rapid and specific detection and enumeration whilst removing the need for subculture and confirmation tests. Immunological tests such as ELISA have significantly reduced analysis time by providing specific target organism detection. Molecular techniques have removed the need for culture. Improvements in sensitivity, and removal of the inhibitory nature of sample matrices, have allowed analysts to detect low levels of micro-organisms but the questions of viability and comparability with cultural techniques still remain. Are we about to see a change of culture in water quality assessment, or can cultural techniques be developed that reduce analysis time to a few hours and can rapid methods be used for detecting the presence and viability of organisms?

Evaluation of the BBL Crystal Enteric/Nonfermenter kit for the identification of water-derived environmental Enterobacteriaceae.

The Crystal Enteric/Nonfermenter (E/NF) identification kit (Becton Dickinson Microbiology Systems, USA) was evaluated using water-derived bacterial isolates and results compared to those obtained by the API 20E system (BioMérieux, UK). Both the E/NF and 20E systems correctly identified 93% of the Enterobacteriaceae reference cultures. Both systems agreed in the identification of 64.9% of environmental isolates. The E/NF system gave a positive identification to 88.0% of isolates and the 20E to 79.5% of isolates. The principal tests which gave differing reactions between the two systems were arginine dihydrolase, lysine decarboxylase, urease and citrate utilization.