AS 5013.24.2:2020 Food microbiology Method 24.2: Microbiology of the food chain – – Horizontal method for the detection and enumeration of Listeria monocytogenes and of Listeria spp. – Enumeration method (ISO 11290-2:2017, MOD).
9.3.2 Consider as L. monocytogenes the blue-green colonies surrounded by an opaque halo (typical colonies). Colonies of L. ivanovii are also blue-green and surrounded by an opaque halo.
NOTE 1 Some strains of L. monocytogenes exposed to stress conditions, particularly acid stress, may show a very weak halo (or even no halo).
NOTE 2 Some rare L. monocytogenes are characterized by a slow P1 PLC (phosphatidyl inositol phospholipase C) activity. Such bacteria are detected when the total duration of incubation is more than, for example, 4 days. Some of these strains could be pathogenici’°J No L. monocytogenes strains have been described as PIPLC negative.
9.3.3 Consider as presumptive Listeria spp. the blue-green colonies with or without opaque halo. NOTE Some organisms other than Listeria spp. may produce blue colonies on this medium. See Annex C.
9.3.4 Count all the colonies presumed to be L. monocytogenes (9.3.2) on each Petri dish containing less than 150 L. monocytogenes characteristic colonies, or less than 360 L. monocytogenes characteristic colonies if 140 mm Petri dishes are used.
9.3.5 Count all the colonies presumed to be Listeria spp. (9.3.3.) on each Petri dish containing less than
150 Listeria spp. characteristic colonies, or less than 360 Listeria spp. characteristic colonies if 140 mm
Petri dishes are used.
In case of mixed cultures of blue-green colonies with or without opaque halo, or in case of blue- green colonies with large and overlapping opaque halos, it is preferable to count the colonies on each Petri dish containing less than 100 Listeria spp. characteristic colonies, or less than 240 Listeria spp. characteristic colonies if 140 mm Petri dishes are used.
9.4 Confirmation of L. monocytogenes or Listeria spp.
9.4.1 Selection of colonies for confirmation
18.104.22.168 Consider each group of three 90 mm Petri dishes used for the initial suspension as one dish. If on one dish there are fewer than five presumptive colonies, take all of them for confirmation.
22.214.171.124 For confirmation of presumptive L. monocytogenes, take from each Petri dish, representing each dilution, five colonies in total, representative for each colony type (e.g. with large halo and small halo).
Streak the selected colonies onto the surface of pre-dried plates of a non-selective agar, for example blood agar, nutrient agar, tryptone soya yeast extract agar (TSYEA) (B.1) in a manner which will allow isolated colonies to develop.
Use of blood agar for pure culture enables interpretation of haemolysis, when positive, already at that stage (see 126.96.36.199 and Annex D). If streaking on blood agar does not show haemolysis, then the haemolysis test shall be done by stabbing (188.8.131.52.2) or in liquid medium (184.108.40.206.3).
Place the Petri dishes in the incubator set at 37°C for 18 h to 24 h or until growth is satisfactory.
If the colonies are not isolated, pick a typical L. monocytogenes colony onto another non-selective dish.
Carry out the following tests (9.4.2) from colonies of a pure culture on the non-selective agar.
220.127.116.11 For confirmation of presumptive Listeria spp. take, from each Petri dish, representing each dilution, five colonies in total, representative for each colony type (e.g. large and small colonies, with or without halo).
For confirmation o Listeria spp. use plates o TSYEA.AS 5013.24.2 pdf download.