This Standard sets out a method, using membrane filtration, for enumerating coliforms, E. coil and
thermotolerant coliforms in water other than packaged water.
For the enumeration of total coliforms and E. coil where waters are expected to contain low numbers of bacteria (e.g. potable waters, treated water), this method provides the option to use either MI agar or chromogenic E. coli/coliform selective agar, with incubation at 36 °C ± 2.0 °C.
For the enumeration of thermotolerant coliforms or E. coil where waters are expected to contain high
numbers of bacteria (e.g. untreated waters, surface waters and effluents), the use of Chromogenic E.
coli/coliform selective agar incubated at 44 °C ± 0.5 °C is required. MI agar is not suitable for samples
with high bacterial load due to the likelihood of diffusion of fluorescence of colonies on membranes
with high counts.
Both media used in this method have the ability to simultaneously detect and enumerate total
coliforms, E. coil and thermotolerant coliforms in water samples in 24 h or less, on the basis of their
specific enzyme activities.
M1 agar contains two enzyme substrates:
(a)  fluorogenic 4-Methylumbelliferyl-(3-D-galactopyranoside (MUGal); and
(b) a chromogen lndoxyl-(3-D-glucuronide (IBDG),
to detect the enzymes P-galactosidase and 1i-glucuronidase, produced by most coliforms and E. coil,
respectively.
Chromogenic E. coli/coliform selective agar contains two chromogenic agents:
(i)  6-chloro-3-indolyl- 3-D-galactopyranoside (Rose-Gal) that detects (3-galactosidase activity of the
coliform group; and
(ii) 5-bromo-4-chloro-3-indolyl-(3-D-glucuronide cyclohexylammonium salt (X-Glu) that detects
(3-glucuronidase activity in E. coil bacteria.
NOTE 1  Membrane filtration is suitable for enumerating microorganisms only when the turbidity of the water is low.
NOTE 2  Historically, thermotolerant coliforms, previously referred to as faecal coliforms, were considered to be faecally derived. More recent evidence indicates E. coil is a better indicator of faecal contamination.
NOTE 3  A flow diagram of the procedure is shown in Appendix A. AS 4276.5:2019 A 0 t N N O t 0 Australian STANDARD Water microbiology Method 5: Coliforms, Escherichia coli and thermotolerant coliforms- Membrane filtration method STANARD Australio AS 4276.5:2019 This Australian Standard@ was prepared by FT 020, Water Microbiology. It was approved on behalf of the Council of Standards Australia on 12 February 2019. This Standard was published on 4 March 2019