AS 4276.3:2021 Water microbiology Method 3: Enumeration of heterotrophic microorganisms – Pour plate, spread plate, membrane filtration and most probable number techniques.
During the preparation of the pour plates the following precautions shall be taken:
(a) Keep the lids of the plates in place except when they are partially lifted for adding the inoculum and the medium.
(b) Do not exceed a time interval of 10 mm between pipetting into the plate and pouring the medium.
(c) Do not exceed a total time interval of 20 mm between the preparation of the first dilution and the pouring of the last plate.
7.1.3 Inoculation and pouring of plates
Plates shall be inoculated and poured as follows:
(a) Prepare plates by adding 1 mL of sample or appropriate dilution to each plate. When dilution is necessary, plate out appropriate dilutions.
(b) To each plate add molten agar, which has been cooled to 44 °C to 46 °C, to a depth of 3 mm to 5 mm.
NOTE Some organisms may suffer heat-shock above 47 °C.
(c) Immediately after pouring, keep the plate horizontal and, taking care not to wet the lid, thoroughly mix the medium and the inoculum by to-and-fro movements, followed by circular clockwise movements, followed by to-and-fro movements at right-angles to the first set, followed by circular anti-clockwise movements.
(d) Allow the plates to stand on a horizontal surface until the medium has set.
7.1.4 Incubation
Incubation shall be carried out as described in AS 4276.1. The plates should be incubated at 36 °C ± 2 °C for 44 ± 4 h and/or 22 °C ± 2 °C for 68 ± 4 h, as required.
For cooling tower waters, the incubation used shall be 36 °C ± 2 °C for 44 ± 4 h.
7.1.5 Counting of colonies
The total number of colonies shall be counted as follows:
(a) Register the count.
NOTE Use of a counter is recommended. If manually counting it is recommended to mark the counted colonies to avoid recounting. Use of an illuminated magnification source can also assist in counting.
(b) Count each spreading colony as a single colony.
Plates shall be rejected where 25 % or more of the medium is occupied by spreading organisms.
7.1.6 Calculation of results
The calculation of results shall be carried out in accordance with AS 4276.1.
7.2 Spread plate
7.2.1 General
Surface spread plating is an alternative technique for enumerating microorganisms in water samples.AS 4276.3 pdf download.