AS 5013.24.1:2009 Food microbiology
9.4.4 After incubation for 24h and for an additional 18 h to 24 h (if growth is slight or if no colonies are ob- served atter 24 h of incubation), examine the dishes (9.4.3) for the presence of colonies presumed to be Listeria spp.
9.4.4.1 Oxford agar; Typical colonies of Listeria spp. grown on Oxford agar for 24 h are small (1 mm) grey- ish colonies surrounded by black halos. After 48 h colonies become darker, with a possible greenish sheen, and are about 2 mm in diameter, with black halos and sunken centres.
9.4.4.2 PALCAM agar: For plates incubated micro- aerobically, after incubation expose the PALCAM agar plates to air for 1 h to allow the medium to regain its pink to purple colour. Atter 24 h Listeria spp. grow as small or very small greyish green or olive green colo- nies, 1,5 mm to 2 mm in diameter. sometimes with black centres, but always with black halos. After 48 h Listeria spp. appear in the form of green colonies about 1,5 mm to 2 mm in diameter, with a central de- pression and surrounded by a black halo.
Dry the agar surface well before use. Take a colony separated in 9.5.1.2 and plate and stab one space for each culture, using a wire (6.5). Simultaneously stab positive (L. monocytogenes) and negative control cul- tures (L. innocua).
After incubation at 35°C or 37 °C for 24h+2h, ex- amine the test strains and controls. L. monocytogenes show narrow, clear, light zones (B-haemolysis2); see figure 1); L. innocua show no clear zone around the stab. L. sellgeri show a weak zone of haemolysis. L. ivanovi usually show wide. clearly delineated zones of B-haemolysis. Examine the plates in a bright light to compare test cultures with controls.
NOTE 9 The haemolytic reaction may also be carried out using red blood corpuscles. Disperse the colony in 150 μl of TSYEB (5.6); incubate at 35 °C or 37。C for2 h. Add 150 μl of sheep red blood corpuscles in a 2%。solution of PBS (5.12). Incubate at 35C or 37 °C tor between 15 min and 00 min, then reflrigerate al 3 °C±2°C lur abul 2 h. Then examine for tho proconoo or aboencc of hacmolyaio. If the reaction is not definite, leave the culture at 3 °C±2 °C for upto24h.