AS 5013.13:2018 pdf download.Food microbiology Method 13: Microbiology of the food chain-Horizontal method for the detection of Cronobacter spp. (ISO 22964:2017, MOD).
8 Sampling
Sampling is not part of the method specified in this document. See the specific International Standard dealing with the product concerned. If there is no specific International Standard dealing with sampling of the product concerned, it is recommended that the interested parties come to an agreement on this subject. A recommended sampling method is given in ISO/TS 17728111 for food and animal feed, and ISO 18593L4i for sampling of surfaces. It is important that the laboratory receives a sample which is truly representative and which has not been damaged or changed during transport or storage (see ISO 7218).
9 Preparation of test sample
Prepare the test sample from the laboratory sample in accordance with the specific International Standard dealing with the product concerned: see ISO 6887 (all parts). If there is no specific International Standard, it is recommended that the parties concerned come to an agreement on this subject.
10 Procedure (as shown in Annex A)
10.1 Test portion
In general, to prepare the primary dilution, add 10 g or 10 ml of the test sample (Clause 9) to 90 ml of preenrichment medium (lfl) (BPW), to yield a tenfold dilution. Pre-warm the BPW to room temperature before use. For specific products, follow the procedures specified in ISO 6887 (all parts). For dry milk, follow ISO 6887-5.
This document has been validated for test portions of 10 g. A smaller size of the test portion may be used without the need of additional validation/verification providing that the same ratio between preenrichment 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 Cronobacterspp. NOTE 1 Validation can be conducted in accordance with the appropriate documents of ISO 16140 (all parts). Verification for pooling samples can be conducted in accordance with the protocol described in ISO 6887-1:2017, Annex D (verification protocol for pooling samples for qualitative tests). NOTE 2 Large sample sizes can compromise the recovery of stressed Cronobacter spp. when interfering microflora are present, such as probiotics. For preparing quantities larger than 10 g, BPW should be pre-warmed between 34 °C and 38 °C (7.2) before inoculated with the test portion.
10.2 Pre-enrichment
Incubate the inoculated pre-enrichment medium prepared in accordance with 10.1 between 34 °C and 38 °C (7.2) for 18 h ±2 h.
10.3 Enrichment
After incubation of the inoculated pre-enrichment medium, mix well and transfer 0,1 ml of the obtained culture 10.2 into 10 ml of CSB (B.2) and mix well. Incubate at 41,5 °C (7.2) for 24 h ± 2 h.
10.4 Isolation of presumptive Cronobacterspp.
Allow the CCI (B.3) plates to equilibrate at room temperature if they are stored at a lower temperature. If necessary, dry the surface of the plates (7.13) following the procedure given in ISO 11133. From enrichment culture, mix well and inoculate, by means of a 10 lil loop (7.3), the surface of the CCI agar (B.3) to obtain well-separated colonies. Incubate the plate at 41,5 °C (7.2) for 24 h ± 2 h.
After incubation, examine the chromogenic plate for the presence of typical colonies of presumptive Cronobac tee: Typical Cronobacter colonies on CCI are small to medium-sized (1 mm to 3 mm) and blue to blue-green in colour. Non-Cronobacter colonies are often white or white with a green centre, grey or black. Some naturally pigmented colonies of non-Cronobactercan appear yellow or red.
10.5 Confirmation
10.5.1 General
For confirmation, sub-culture from the selective medium CCI (see 10.4) five marked typical or suspect colonies. In the case that colonies are not well separated, it might be necessary to streak a typical colony first on the selective agar (B.3) again. If on the dish there are fewer than five typical or suspect colonies, take all the marked colonies for confirmation. Use pure cultures for biochemical confirmation.AS 5013.13 pdf download.