AS 5013.10:2022 – Food microbiology
AS 5013.10:2022 – Food microbiology.
A recommended sampling method is given in ISO/TS 177281261 for food and animal Feed, In Iso 7071271 for milk and milk products, in ISO 133071281 for sampling at the primary production stage, in ISO 176041291 For sampling of carcasses. and In ISO 185931251 for sampling of surfaces.
It is important that the laboratory receives a sample which is representative and has not been damaged or changed during transport or storage.
8 Preparation of test sample
Prepare the test sample from the laboratory sample in accordance with the specific International Standard dealing with the product concerned. If there is no specific International Standard. it is recommended that the parties concerned come to an agreement on this subject.
9 Procedure (see diagrams in Annex A)
9.1 Test portion and Initial suspension
For preparation of the initial suspension, in the general case, use as diluent the preenrichment medium specified in 13.2 (buffered peptone water). Pre-warm the HPW to room temperature before use.
In general, an amount of test portion (mass or volume) is added to a quantity of BPW (mass or volume) to yield a tenfold dilution. For this, a 25 g test portion is mixed with 225 ml of HPW. However, for some type of samples (e.g. boot socks, dust), it may be necessary to use another ratio.
For specific products, follow the procedures specified in ISO 6887 (all parts).
This document has been validated for test portions of 25 g. A smaller test portion may be used without the need for additional validation/verification provided that the same ratio between (pre.)enrichmcnt broth and test portion Is maintained. A larger test portion than that Initially validated may be used if a validation/verification study has shown that there are no negative effects on the detection of Sa?rnor,eIIa spp.
NOTE I Validation can be conducted according to the appropriate parts of ISO 16140. Verification for pooling samples can be conducted according to the protocol described in ISO &8&ll•I:2017. Annex D138l.
For large quantities (e.g. I I or more), It Is recommended to pre•warm the BPW to 34 °C to 38 C before mixing It with the test portion,
NOTE 2 When more than one 25 g test portion from a specified lot of product is to be examined and when evidence Is available that combining test portions does not affect the result for that particular food, the test portions can be pooled More information on pooling of samples as well as a procedure to test the influence ci pooling on the sensitivity of the method can be found in ISO 6887.
92 Non-selectIve pre-enrichment
Incubate the initial suspension (9.1) between 34 C and 38 C (6.3) for 18 h ± 2 h.
It is permissible to store the pre-enriched sample after incubation at 5°C (6.8) for a maximum of 72 h (see References [301 to 1341).
9.3 Selective enrichment
Allow the selective enrichment media, RVS broth or MSRV agar (8.3 or 13.4), and MKTTn broth (13.5) to equilibrate at room temperature if they were stored at a lower temperature.
Allow the XLD agar (B.6) plates and the second selective plating medium to equilibrate at room temperature if they were stored at a lower temperature. If necessary, dry the surface of the plates before use (see Iso 11133).
9.4.2 Procedure for food, animal feed samples, and environmental samples tram the food production area
From the culture obtained in the RVS broth (9.3.2), inoculate by means ofa 10 p1 loop (6.10) the surface of an XLD plate (B.6) so that well-isolated colonies will be obtained, Proceed in the same way with the second selective plating-out medium.
From the positive growth obtained on the MSRV agar (9.3.2). determine the furthest point of opaque growth from the inoculation points and dip a 1 p1 loop (6.10) just inside the border of the opaque growth. Withdraw the loop ensuring that no large lumps of MSRV agar are extracted. Inoculate the surface of an XLD plate (B.6) so that well-isolated colonies will be obtained. Proceed in the same way with the second selective plating-out medium.
From the culture obtained in the MKTTn broth (9.3.2). inoculate by means of a 10 p1 loop (6.10) the surface of an X[.D plate (11.6) so that well-isolated colonies are obtained. Proceed in the same way with the second selective platingout medium.
NOTE I To obtain well.isolated colonies, large size Petri dishes with plating-out media (diameter approximately 140 mm) or two normal size plates (diameter approxImately 90 mm) can be used.
Incubate the X1.D plates Inverted at 37°C (63) for 24 h t 3 h.
Incubate the second selective plating-out medium in accordance with the manufacturer’s instructions.
If the selective enrichment media have been incubated for an additional 24 h. follow the same plating- out procedure as described above.
Typical colonies of Salmonella on XLD agar have a black centre and a lightly transparent zone of reddish colour due to the colour change of the Indicator.
NOTE 2 Salmonella H2S-negative variants grown on XLD agar are pink with a darker pink centre. Lactose- positive Salmonella grown on XLD agar are yellow with or without blackening. The occurrence of these phenotypes is summarized in Table 1.
Check the second selective plating medium after the appropriate incubation time for the presence of colonies which, from their characteristics, are considered to be presumptive Salmonella.