AS 5013.24.2:2020 pdf free.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).
8 Preparation of test sample
Prepare the test sample in accordance with the specific International Standard dealing with the product concerned [see ISO 6887 (all parts) and ISO 18593]. If there is no specific International Standard, it is recommended that the parties concerned come to an agreement on this subject.
9 Procedure
9.1 Test portion, initial suspension and dilutions
Buffered peptone water, as well as other appropriate diluents referred to in ISO 6887 (all parts) and any specific International Standard appropriate to the product concerned, may be used as diluent for the initial suspension.
Half-Fraser broth (as specified in ISO 11290-1), supplemented with selective agents or not, may he used as a diluent for the initial suspension when both the detection method (as specified in ISO 11290-1) and this enumeration method are carried out on the same test sample. The selective agents should be added (If required) to the suspension preferentially after enumeration, prior to the detection method.
If supplemented half-Fraser is used, inoculate the plates as soon as possible, up to 45 mm.
9.2 Inoculation and incubation
9.2.1 Distribute, by means of a sterile pipette (6.8), 0,1 ml of the initial suspension (or sample if liquid) and 0,1 ml of further decimal dilutions each inoculated onto the surface of a small dish (90 mm) of Agar Listeria according to Ottaviani and Agosti (see 8.2).
When, for certain products, it is desirable to estimate low numbers of L. monocytogenes and/or Listeria spp., the limits of detection may be raised by a factor of 10 by examining 1 ml of the test sample if the initial product is liquid, or I ml of the initial suspension for the other products. Distribute the 1 ml of inoculum either on the surface of a large Petri dish (140 mm) or over the surface of three small dishes (90 mm), dried beforehand if necessary In the incubator (6.2). If only the initial suspension is used, also prepare duplicate plates using an additional three small Petri dishes or one large dish of medium (see ISO 7218).
Repeat the procedure using 0,1 ml of the initial suspension (or sample if liquid) and 0,1 ml of further decimal dilutions if necessary each inoculated onto the surface of a small dish (90 mm) of agar medium.
9.2.2 Carefully spread the inoculum as quickly as possible over the surface of the agar plate without touching the sides of the dish with the spreader (6.6). Use a fresh sterile spreader for each dilution. Leave the plates closed and upright for about 15 mm at ambient temperature for the inoculum to be absorbed into the agar.
It is possible to use the same spreader for all the dishes of a given sample, by beginning with the higher dilution.
9.2.3 Incubate the Agar Listeria according to Ottaviani and Agosti plates prepared in 9.2.2 inverted at
37 °C (6.3) for 24 h ± 2 h and for an additional incubation at 37°C for 24 ± 2h.
9.3 Enumeration of characteristic colonies
9.3.1 After incubation for 24 h ± 2 h (before excessive development of colonies with large and overlapping opaque halos, which may make reading difficult), and for an additional 24 h ± 2 h (which may allow better development of colonies and of an opaque halo), examine the Petri dishes (9.2.3) for the presence of presumptive colonies of L. monocytogenes (see 9.3.2) and/or Listeria spp. (see 9.3.3).AS 5013.24.2 pdf download.