AS 4276.5:2019

This Standard sets out a method, using membrane filtration, for enumerating coliforms, E. coli and thermotolerant coliforms in water other than packaged water.
For the enumeration of total coliforms and E. coli 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. coli 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. coli and thermotolerant coliforms in water samples in 24 h or less, on the basis of their
specific enzyme activities.
MI agar contains two enzyme substrates:
(a) fluorogenic 4-Methylumbelliferyl-ß-D-galactopyranoside (MUGal); and
(b) a chromogen Indoxyl-ß-D-glucuronide (IBDG),
to detect the enzymes ß-galactosidase and ß-glucuronidase, produced by most coliforms and E. coli,
respectively.
Chromogenic E. coli/coliform selective agar contains two chromogenic agents:
(i) 6-chloro-3-indolyl-ß-D-galactopyranoside (Rose-Gal) that detects ß-galactosidase activity of the
coliform group; and
(ii) 5-bromo-4-chloro-3-indolyl-ß-D-glucuronide cyclohexylammonium salt (X-Glu) that detects ß-glucuronidase activity in E. coli 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. coli is a better indicator of faecal contamination.
NOTE 3 A flow diagram of the procedure is shown in Appendix A.

Originated as AS 1095.4.1.5-1981.
Previous edition AS 4276.5-1995.
Jointly revised and designated as AS/NZS 4276.5:2007.
Amalgamated with AS/NZS 4276.7:2007 and revised and redesignated as AS 4276.5:2019. Originated as AS 1095.4.1.5-1981. Previous edition AS 4276.5-1995. Jointly revised and designated as AS/NZS 4276.5:2007. Amalgamated with AS/NZS 4276.7:2007 and revised and redesignated as AS 4276.5:2019.