AS 3896:2017 Waters- -Examination for Legionella spp. including Legionella pneumophila.
8.2.2 Untreated sample
Inoculate 0.1 mL of the sample directly onto plates of BMPA or BCYE+AB and MWY or GVPC.
8.2.3 Heat-treated sample
The procedure shall be as follows:
(a) Heat 5 mL of the (untreated) sample at 50±1°C for 30 ±2 mm.
(b) Inoculate 0.1 ml. of the heat-treated sample onto plates of BMPA or BCYE+AB and MWY or GVPC.
8.2.4 Acid—treated sample
The procedure shall be as follows:
(a) Transfer 1 mL of the sample into 9 mL of acid buffer (pH 2.2).
(b) Mix well and leave for 5 ±0.5 mm at ambient temperature.
(c) Inoculate 0.1 mL of this sample onto plates of BMPA or BCYE+AB and MWY or GVPC.
NOTE: If greater sensitivity is desired from the acid treatment step, either or both of the following may be used:
(a) Transfer I mL of the sample into I mL of acid buffer, mix well and leave for 15±0.5 mm at ambient temperature (see Bibliography).
(b) Use larger plating volumes with the following label:
8.3 Incubation
All six plates shall be incubated (see Clauses 8.2.2, 8.2.3 and 8.2.4) in a humid atmosphere
at 36 ±2°C for 7 to 10 days.
8.4 Examination
The procedure shall be as follows:
(a) Examine the incubated plates, visually under lOx to 20x magnification, on at least two occasions; the examinations preferably occurring on day 4 and at the end of the incubation period. On day 4, many Legionella colonies will display an opalescent or ground glass appearance, with many variant colours. By day 7, colony morphology changes: the surface appearance becoming more granular and irregular colony edges develop.
1 Some legionellac may be slow growing and require longer incubation.
2 Colonies ofkn have a speckled blue appearance but green, brown, pink and red coloured colonies have also been observed. See Appendix C for examples of typical Leg ionella colonies.
3 The colony morphologies described above may be affected by the presence of large numbers of Leg ione!!a colonies.
4 The same Legionella strain may look different on different media. MWY contains dyes which may be taken up by some legionellac.
5 Different Legionella serogroups/strains cannot be reliably differentiated on the basis of colony morphology.
6 Excessive growth of background microorganisms, even following heat and acid treatment. may interfere with the detection of presumptive lcgionellae. If the colonies are not well defined (crowded or spreading colonies), a purity check on BCYE is recommended.AS 3896 pdf download.