The
The detection and enumeration of
Although the method included in the directive has been withdrawn, it is used by, among others, water suppliers for water control tests due to the short time it takes to obtain results. In this case, it is worth noting what differences in the quantification of
Nineteen surface water samples were collected to detect and enumerate
The performance characteristics of the method using the TSC and mCP media were determined according to PN-EN ISO 13843:2017 (2017). The characteristics of the methods included the following parameters: sensitivity – the fraction of the total positives correctly assigned in the presumptive counts; specificity – the fraction of the total negatives correctly assigned in the presumptive counts; selectivity – the ratio of the target colonies number to the total number of colonies in the sample volume; false-positive rate – the fraction of positive results (e.g., typical colonies) that are subsequently shown to be due to non-target organisms; false-negative rate – the fraction of negative results (e.g., atypical colonies) shown to be target organisms; efficiency – the fraction of total colonies correctly assigned in the presumptive counts. The tests included 847 isolated and confirmed bacterial strains, including 466 isolates from the TSC medium and 381 isolates from the mCP medium.
A comparison of relative bacterial recovery (including statistical analysis) was carried out according to PN-EN ISO 17994:2014 (2014) using parallel tests of water samples according to the procedure ISO/CD 6461-2:2002 (2002) and according to the procedure described in Council Directive 98/83/EC (1998).
Method performance characteristics on the substrates compared were determined using natural surface water samples. The water samples and isolated bacterial strains were tested according to appropriate procedures, including confirmatory testing.
Categorical performance characteristics were determined based on the analysis of 466 suspected
Confirmatory tests for
Feature | |
---|---|
Reduction of sulphites to sulphides | black or grey colonies |
Motility | none |
Reduction of nitrate to nitrites | orange/red-colored medium after the addition of the Nessler reagent |
Lactose fermentation | orange or yellow-colored medium with lactose (acidification of the medium and gas formation) |
Protease formation | gelatine liquefaction |
For colonies that grew on the mCP, those that caused its yellow discoloration (sucrose fermentation) were considered characteristic. The plate with the colonies grown was placed in ammonium hydroxide vapors to confirm the ability of the bacteria to produce acid phosphatase. Positively confirmed colonies changed color from yellow to pink (Table II). A summary of the results of confirmatory tests for bacterial strains determined towards
Confirmatory tests for
Feature | |
---|---|
Sucrose fermentation | sucrose-positive – yellow and matte colonies |
Possess acid phosphatase | change of color of the colony from yellow to pink in ammonium hydroxide vapors |
Comparison of the results of confirmation tests for
Characteristics of |
TSC ISO/CD 6461-2:2002 | mCP Directive 98/83/EC |
---|---|---|
Number of all confirmed colonies | 466 | 381 |
Typical colonies, confirmed positive (true positive) | 150 | 107 |
Atypical colonies, confirmed positive (false negative) | 33 | 11 |
Typical colonies, confirmed negative (false positive) | 196 | 79 |
Atypical colonies, confirmed negative (true negative) | 87 | 184 |
Typical colonies, non-detection error | 0.18 | 0.09 |
The following categorical performance characteristics were determined: sensitivity, specificity, selectivity, false-positive rate, false-negative rate, and efficiency. For the mCP and TSC media, the determined method sensitivity reached a high value of 90.7% and 82.0%, respectively. At the same time, it turned out that, for the surface water samples tested, the method according to Council Directive 98/83/EC (1998) with the mCP medium is more specific (70.0%) than the method according to ISO/CD 6461-2:2002 (2002) based on the TSC medium (30.7%). Method specificity is determined, among other things, through error introduced by false-positive colonies growing on the media used (mCP – 42.5%, TSC – 56.6%) and error resulting from non-detection of typical colonies (mCP – 0.09, TSC – 0.18). The results presented in Table IV show that the false-positive rate values, i.e., the fraction of observed positive (typical) colonies selected for confirmation that were not confirmed to be
Comparison of performance parameters of procedures used for detection of
Performance parameters | TSC ISO/CD 6461-2:2002 | mCP Directive 98/83/EC |
---|---|---|
Sensitivity | 82.0% | 90.7% |
Specificity | 30.7% | 70.0% |
Selectivity | 32.2% | 28.1% |
False negative rate | 27.5% | 5.6% |
False positive rate | 56.6% | 42.5% |
Efficiency | 50.9% | 76.4% |
According to literature data, the false-positive rate is usually lower for the TSC medium because it is considered more selective than the mCP medium, leading to fewer false-positive results (Lamy et al. 2020). During the present study, non-typically-looking colonies, which, in part, were confirmed as
Regarding selectivity, for both media compared, a similar value was obtained for this parameter, which was 28.1% (Council Directive 98/83/EC 1998) and 32.2% (ISO/CD 6461-2:2002 2002), respectively. Available literature data indicate that, when testing real samples, the mCP usually performs worse than the TSC medium. However, the authors point out possible differences in results that are caused by different types of water samples tested (groundwater, surface water) and the testing direction, i.e. determination of the number of spores or vegetative forms (Bisson and Cabelli 1979; Sartory 1986; Sartory et al. 1998; Araujo et al. 2004; Sartory et al. 2006).
As part of the study, the efficiency of the methods was also determined, and was expressed as the number of colonies correctly selected for confirmatory testing. In this case, a higher efficiency was achieved by the method based on the mCP (76.4%) compared with the method based on TSC (50.9%). This parameter is strongly influenced by the experience of the analyst and the degree of contamination of the sample tested.
Methods for detecting and enumerating of
The results were analyzed using the approach in PN-EN ISO 17994:2014 (2014). A relative difference (RD) was determined for each pair of confirmed counts, i.e., the difference between the two results, a and b, measured on a relative (natural logarithmic) scale. The equation used was:
The tests performed to detect
Furthermore, it is worth paying attention to the technical aspects and the speed of obtaining results in the procedures compared. A significant advantage of the method for detecting
In contrast to the procedure from the Directive, it is not possible to obtain the final results of tests carried out on the TSC medium according to ISO/CD 646-12:2002 (2002) until after 72 hours, and sometimes even after 96 hours. This is mainly due to the need to carry out confirmatory tests requiring two successive steps: transferring isolated colonies onto blood medium or nutrient agar and performing culture on the appropriate media that make up the biochemical pathway after another day of incubation. However, this procedure is supported by the higher recovery achieved, which is essential in health safety, especially for drinking water samples. In this case, testing as part of ongoing surveillance for
The results showed that some performance characteristics for the procedure based on the mCP medium were significantly better than those using the TSC medium. At the same time, a statistical analysis of the results showed that the recovery of target bacteria from the samples tested using the TSC medium was significantly higher. Based on literature data and the presented results, it can be concluded that the procedure using the TSC medium may be more suitable for testing surface water samples for the enumeration of
In summary, when it comes to legal regulations, at the level of the laws of Europe and Poland, the requirement for the enumeration of
It can therefore be considered that the current standardized method for the determination of