AOAC 2019 First Action Methods

( d ) If there are no typical colonies on the selective agar, proceed as follows: ( 1 ) Transfer 0.5 mL primary enrichment into 10 mL CSB broth. ( 2 ) After incubation for 18–24 h at 41.5±1°C, isolate 10 µL onto ESIA agar or CCI agar. ( 3 ) Identify the colonies using option ( a ) or ( b ) indicated above. J. QC External QC can be performed using one Cronobacter strain. ( a ) Add one isolated colony from a fresh and pure culture in 9 mL BPW. ( b ) Mix and incubate at 37±1°C for 18–24 h. ( c ) Dilute 1/100 of the culture in BPW in order to obtain a suspension containing approximately 10 6 cells/mL. ( d ) Follow the protocol from the Sample Lysis to Confirmation of Positive Results sections. ( e ) Check that the results obtained correspond to the characteristics of the tested strains. K. Limitations of the Method The GENE‑UP Cronobacter kit has been evaluated on a large number of matrices. However, given the wide variety of products and manufacturing procedures, it is recommended to check that the composition of the matrices tested does not affect the reliability of GENE‑UP results. References: (1)  ISO 6887-1:2017: Microbiology of the

Note : The lysis tubes can be removed from the GENE‑UP lysis tube holder using the GENE‑UP lysis tube remover tool. The GENE‑UP lysis tube holder is reusable, but the used lysis tubes should be disposed of according to appropriate biosafety procedures. ( i ) Refer to the appropriate GENE‑UP instrument user manual for instructions on how to start a run, view results, and use the Results are automatically interpreted once the PCR run is completed. The routine software interprets data for each sample and gives a positive, negative, or inhibited result as indicated in Table 2019.01B . H. PCR Inhibition Protocol ( a ) In the case of an inhibited result, dilute the lysate to 1:3 in the control buffer. ( b ) Transfer 10 μL control buffer in an adapted microtube. ( c ) Follow the same procedure as in the PCR Preparation section above, using 10 μL this dilution of lysate. Note : It is recommended to retest in parallel the lysate without dilution. In the case of inhibited results at 1:3, you can dilute the lysate to 1:10. I. Confirmation of Positive Results All positive results must be confirmed according to the ISO 22964 standard or following the bioMérieux GENE‑UP confirmation protocol. Confirmation should be performed using the enrichment broth stored at 2 – 8°C and should be initiated within 72 h following the end of the incubation period. Mix the enriched broth thoroughly by hand prior to starting the confirmation process. ( a ) Isolate 10 µL enrichment broth on ESIA or CCI agar. Incubate the agars following the instructions in the package insert. ( 1 ) Identify between one and five typical colonies using the conventional tests described in ISO 22964:2017 (including a purification step). ( 2 ) Test isolated colonies directly using a bioMérieux API strip ID32E (without a purification step). It is not necessary to perform the oxidase test. ( 3 ) Test isolated colonies directly using a bioMérieux Fast Crono confirmation test. ( b ) Perform the current ISO 22964 procedure. Transfer 100 µL enriched sample into 10 mL CSB broth and then follow the International Organization for Standardization (ISO) procedure to obtain isolated colonies (including the purification step). Refer to the latest edition of the standard EN ISO 7218 (4) for the confirmation procedures that can be used in standardized methods. ( c ) Use of any other AFNOR NF VALIDATION certified method that bases its technology on a different principle than the GENE-UP Cronobacter method and shares the same enrichment conditions as those used for the GENE-UP Cronobacter method, the full protocol for the candidate method must start from the same common step as the GENE-UP Cronobacter method. In the event of discordant results (positive with the candidate method, not confirmed by one of the options described above), the laboratory must take the necessary steps to ensure that the results obtained are accurate. For example, isolation may be performed again using a procedure that was not followed the first time. GENE‑UP Routine software. G. Results and Interpretation

Food Chain–Preparation of Test Samples, Initial Suspension and Decimal Dilutions for Microbiological Examination–Part 1: General Rules for Preparation of the Initial Suspension and Decimal Dilutions , International Organization for Standardization, Brussels, Belgium (2)  ISO 6887-4:2017: Microbiology of the Food Chain–Preparation of Test Samples, Initial Suspension and Decimal Dilutions for Microbiological Examination–Part 4: Specific Rules for the Preparation of Miscellaneous Products , International Organization for Standardization, Brussels, Belgium (3)  ISO 6887-5:2017: Microbiology of the Food Chain–Preparation of Test Samples, Initial Suspension and Decimal dilutions for Microbiological Examination–Part 5: Specific Rules for the Preparation of Milk and Milk Products , International Organization for Standardization, Brussels, Belgium (4)  ISO 7218:2007: Microbiology of Food and Animal Feeding Stuffs–General Requirements and Guidance for Microbiological Examinations , International Organization for Standards, Brussels, Belgium

J. AOAC Int . 103 , 184(2020) DOI: 10.5740/jaoacint.19-0122 Posted: July 2019 (pre-publication); January 2020 (First Action publication)

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