AS 5013.24.1:2009 pdf free download – Food microbiology
9.4.4 After incubation for 24 h 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. After 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.
9.5.4 Motility test
Take an isolated colony obtained in 9.5.1.2 and sus- pend it in a tube containing TSYEB (5.6). Incubate in the incubator [6.3 a)] set at 25 °C for 8 h to 24 h until a cloudy medium is observed. Deposit a drop of the above culture using a loop (6.5) onto a clean glass microscope slide. Place a coverslip on top and examine it with the microscope (6.14). Listeria spp. appear as slim, short rods with tumbling motility. Cultures grown above 25 °C may fail to exhibit this motion. Always compare them to a known culture. Cocci, large rods, or rods with rapid swimming motility are nol Lisleria spp. Ac an alternative teet for motility, ueing an inoculating needle (6.5), stab the motility agar (5.9) with a culture taken from a typical colony on TSYEA (9.5.1 .2). Incu- bate it for 48 h in the incubator [6.3 a)] set 25 °C. Examine for growth around the stab. Listeria spp. are motile, giving a typical umbrella-like growth pattern. If growth is not sufficient, incubate for up to an additional 5 days and observe the stab again.
Simultaneously, streak control cultures of L. monocy- togenes, L. innocua and L. ivanovi. Itf blood agar (5.7) is used, incubate the plates at 35 °C or 37 °C for 18 h to 24 h. If double-layer plates (B.10.3) are used, incu- bate at35 °Cor37。C for12hto 18 h. An enhanced zone of i-haemolysis at the intersection of the test strain with each of the cultures of S. aureus and R. equi is considered to be a positive reaction. The positive reaction with R. equi is seen as a wide (5 mm to 10 mm)“arow-head” of haemolysis. The re- action is considered as negative if a small zone of weak haemolysis extends only about 1 mm at the in- tersection of the test strain with the diffusion zone of the R. equi culture. A positive reaction with S. aureus appears as a small zone of enhanced haemolysis extending only about 2 mm from the test strain and within the weakly haemolytic zone due to growth of the S. aureus cul- ture. Large zones of haemolysis do not occur in the area of S. aureus and L. monocytogenes.