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Chapter 8*: SANITARY INSPECTION AND MICROBIOLOGICAL WATER QUALITY - page 20 / 52

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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).

Isolation media

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

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

APHA

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

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