The methods for enumeration of faecal streptococci from natural waters have been compared by different authors (Volterra et al., 1986; Yoshpe-Purer, 1989). Dionisio and Borrego (1995) compared eight methods for the specific recovery of faecal streptococci from natural freshwater and marine waters on the basis of the following characteristics: accuracy, specificity, selectivity, precision and relative recovery efficiency. The results obtained indicated that none of the tested methods showed perfect selectivity. The methods that showed the best performance characteristics were the MPN technique (with Rothe and Litsky media) and the m-Enterococcus agar in conjunction with the MF technique. The latter is the only technique recommended in the “Standard Methods” for faecal streptococci in conjunction with membrane filtration (APHA/AWWA/WPCF, 1995). A rapid confirmation technique, based on the transplantation of the membrane from m- Enterococcus agar after incubation for 48 hours at 36 ± 1°C to Bilis-Esculin-Agar (BEA) for 4 hours additional incubation, improves the low specificity of the m-Enterococcus agar, enabling the confirmation of 100 per cent of the colonies (Figueras et al., 1996). A similar procedure has been proposed by ISO (Table 8.7).
Recently, Audicana et al. (1995) designed and tested a modification of the KEA agar, named Oxolinic acid-Aesculin-Azide (OAA) agar, to improve the selectivity in the enumeration of faecal streptococci from water samples by the MF technique. The OAA agar showed higher specificity, selectivity and relative recovery efficiencies than those obtained when using m-Enterococcus and KF agars. In addition, no confirmation of typical colonies was needed when OAA agar was used, which shortens the time taken significantly and increases the accuracy of the method. The excellent performance of this culture medium was recently reconfirmed in a routine monitoring programme for bathing waters (Figueras et al., 1998). A Europe-wide standardisation trial demonstrated that the m-Enterococcus agar with total confirmation of the colonies (Figueras et al., 1996), the OAA medium (Audicana et al., 1995), and the miniaturised MPN method (Hernandez et al., 1991) produced the best results (Hernandez et al., 1995).
8.5.2 Thermotolerant coliforms and E. coli
The presumptive detection of thermotolerant coliforms can be considered sufficient to give an estimation for the presence of E. coli. The EC and A-1 media are the most widely recommended for the presumptive detection of thermotolerant coliforms with the MPN technique (APHA/AWWA/WPCF, 1992). The differences between the two approaches are based on the incubation periods: 44.5 ± 0.2 °C for 24 hours for the EC medium, and 36 ± 1 °C for 3 hours and transfer to 44.5 ± 0.2 °C for 21 hours for the A-1 medium. The tubes containing gas and acid in EC medium are confirmed in the same medium by subsequent incubation at 44.5 ± 0.2 °C for 24 hours. The A-1 medium does not require a confirmation test. Table 8.3 details accepted media. Thermotolerant coliform density and the 95 per cent confidence limits can be estimated with the use of MPN tables (APHA/AWWA/WPCF 1995; Bartram and Ballance, 1996).
The mFC agar is the most frequent medium used to quantify thermotolerant coliforms in water samples when the MF technique is used. Petri dishes containing filters are incubated at 44.5 ± 0.2 °C for 24 hours. Typical thermotolerant coliform colonies appear various shades of blue, atypical E. coli may be pale yellow, and non-thermotolerant coliform colonies are grey to cream in colour. Table 8.5 details accepted standardised media.