AS 4276.3:2021 pdf Water microbiology
1 Scope and general
This Standard specifies methods for enumeration of heterotrophic microorganisms in water using the
(a) Pour plate.
(b) Spread plate.
(c) Spiral plate.
(d) Membrane filtration.
(e) Most probable number (MPN).
These techniques are intended to measure the operational effectiveness of treatment processes
and be used for the general examination of many types of water to evaluate and/or note changes in
This document does not cover the comparison of results obtained from different techniques.
1.2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document:
NOTE Documents for informative purposes are listed in the Bibliography.
AS 2031, Water quality — Sampling for microbiological analysis (ISO 19458:2006, MOD)
AS 5140, Microbiology of food, animal feed and water — Preparation, production, storage and performance testing of culture media (ISO 11133:2014, MOD)
AS 4276.1, Water microbiology, Method 1: Water quality — General requirements and guidance for
microbiological examinations by culture (ISO 8199:2018, MOD)
1.3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
observed number of objects, such as colonies, determined by direct counting, or most probable number
(MPN) estimation based on statistical calculation using the number of positive units in a dilution series
of a test sample
indicates the existence of an option
3.1 Laboratory samples
Laboratory samples shall conform to AS 2031.
Dilutions of the laboratory sample shall be prepared, as required, in accordance with the procedure
in AS 4276.1.
The applicable apparatus is required depending on the technique used:
(a) Apparatus for preparation of dilutions — Refer to AS 4276.1.
(b) Petri dishes — 50 mm to 90 mm or up to 150 mm in diameter.
(c) Pipettes, volume measuring devices.
(d) Colony counter, microscope — Provide with a lens for magnification and a means for the
illumination of the prepared plates.
(e) Tally counter, automated spiral plate counters, automated counters.
(f) Incubators, water baths.
(g) Membrane filtration apparatus — Refer to AS 4276.1.
(h) Membrane filters — 0.45 µm pore size, refer to AS 4276.1.
(i) Sterile filter funnels.
(k) Spiral plater and associated equipment.
(l) Sterile spreaders.
(m) Circulating water bath — Able to maintain temperature of 45 °C ± 1 °C.
(n) MPN — Sealers and trays in accordance with manufacturer’s instructions or requirements.
(o) Apparatus for media preparation — Include an autoclave, see Appendix B.
(p) Refrigerator — Able to maintain temperatures of 2 °C to 8 °C for media storage.
5 Culture media and diluents
The culture medium shall be chosen from the following:
(a) Plate count agar (PCA).
(b) Reasoner’s 2A agar (R2A).
(c) m-Heterotrophic plate count agar (m-HPC).
(d) Tryptone soya agar (TSA).
(e) Yeast extract agar (YEA).
(f) Nutrient agar (NA).
NOTE For formulations and procedures for preparing the culture media, see Appendix B.
Dilutions of the laboratory sample shall be prepared as required, using the procedure given in AS 4276.1.
6 Quality assurance
The laboratory shall have a clearly defined quality control system to ensure that the apparatus, culture
media, reagents and technique are suitable for the test.
NOTE The use of controls and blanks, as appropriate, is part of the quality control system.
6.2 Performance testing of culture media
The performance of the culture medium shall be tested in accordance with the method and criteria in
AS 5140. Table 6.1 provides a summary of expected performance results.
NOTE Refer to AS 5140 for the following:
(a) Definitions of productivity.
(b) Preparation of the working cultures and test suspensions.
AS 4276.3:2021 pdf Water microbiology