Several disadvantages of the MF technique have also been reported:
Inapplicability of the method to turbid samples which can clog the membrane or
prevent the growth of the target bacteria on the filter.
False negative results due to the inability of viable but non-culturable bacteria present
in environmental waters to grow with standard MF methods.
False positive results when non-target bacteria form colonies similar to the target
colonies (Figueras et al., 1994. 1996; Hernandez-Lopez and Vargas-Albores, 1994).
a) TTC agar with Tergitol-7 or Teepol b) Lactose agar with Tergitol-7 or Teepol c) Membrane enrichment Teepol broth d) m-FC medium with 1% rosolic acid in 0.2N NaOH added e) Laurylsulphate broth
m-FC medium with 1% rosolic acid in 0.2N NaOH added (if there is interference with background growth)
Incubation conditions 24 ± 2 h at 44.5 ± 0.2°C
18-24 h at 44 ± 0.25°C or 44.5 ± 0.25°C; a pre- incubation of 4 h at 30°C is recommended
Reaction Depends on the media selected Confirmatory media tests
Reaction Blue colonies = (+)
Verify a representative number of colonies. Two confirmatory methods can be used:
Verify by picking at least 10 typical colonies; by two tests:
A. With two steps 1. Lactose peptone water Incubation conditions 24 h at 44 ± 0.25°C or 44.5 ± 0.25°C Reaction Gas production = (+) for thermotolerant coliforms 2. Tryptone water Incubation conditions 24 h at 44 ± 0.25°C or 44.5 ± 0.25°C Reaction
1. Lauryl tryptose broth Incubation conditions 24-48 h at 35 ± 0.5°C Reaction Gas production = (+) 2. EC broth Incubation conditions 24 h at 44.5 ± 0.2°C Reaction
Indol production with indol reagent Kovacs = (+) for E. coli
Growth (+) and gas production = (+) for thermotolerant coliforms
Table 8.5 Standard methods for the determination of thermotolerant coliforms (E. coli presumptive) - MF methods
ISO 9308-1 (ISO, 1990b)1
B. With one step Lauryl tryptose mannitol broth with tryptophan Incubation conditions 24 h at 44 ± 0.25°C or 44.5 ± 0.25°C Reaction
Gas production = (+) and indol = (+/-) for thermotolerant coliforms; gas production = (+) and indol = (+) for E. coli