Microbiology Methods for ERP Review 3-2020

196

5.0 1 2 Each collaborator will receive a complete set of test materials (uncontaminated, low, and high) for each 3 method (candidate and reference) and will also receive one known uncontaminated sample to 4 determine the aerobic plate count (4). All participating collaborators will initiate sample analysis and 5 aerobic plate count for each matrix on the same day. One additional control (negative) test portion will 6 be provided to each collaborator for each matrix to determine the total plate count on the day of 7 analysis. Analysis and Confirmation 5.1 Each collaborator will receive one set of 375 g test portions (12 uncontaminated, 12 low, 12 high) to run on the Solus One Salmonella method, and one set of 25 g test portions to run on the FDA BAM method. Each set will be blind coded so that the 8 9 5.2 The 375 g set of test portions will be enriched in Solus One Salmonella 1125 mL Solus One enrichment broth (BPW + supplement) at 41.5 ± 1°C for 21–22 h. Collaborators who have the Dynex DS2 instrument in their laboratories will conduct the Solus One Salmonella method using the Dynex instrument (automated procedure). Collaborators who do not have the Dynex DS2 instrument in their laboratories will conduct the Solus One Salmonella method following the manual procedure. It is anticipated that there will For each 25 g test portion, aseptically add into sterile beaker (250 ml) or other appropriate container. Using sterile glass or paper funnel (made with tape to withstand autoclaving), pour 25 g analytical unit gently and slowly over surface of 225 mL brilliant green water contained in sterile 500 mL Erlenmeyer flask or other appropriate container. Prepare brilliant green water by adding 2 mL 1% brilliant green dye solution per 1000 mL sterile distilled water. Let container stand undisturbed for 60 ± 5 min. Incubate loosely capped container, without mixing or pH adjustment, for 24 ± 2 h at continue to selective enrichment. One (1) mL of each enriched portion will be transferred to 10 mL TT broth and 0.1 mL will be transferred to 10 mL Rappaport Vassiliadis (RV) broth. Note: BAM Method RV medium must be made from individual ingredients according to BAM formulation. Do not use commercial formulations. RV tubes will be incubated at 42 ± 0.2°C for 22–26 h using a circulating, thermostatically controlled water bath. TT tubes will be incubated at 35 ± 2°C for 22–26 h. Secondary enrichments will be streaked to bismuth sulfite (BS), xylose lysine desoxycholate (XLD), and Hektoen enteric (HE) agar plates and incubated at 35°C for 22 ‐ 26 h. Isolated colonies will be transferred to TSI and LIA slants and incubated 35 ± 2°C for 22 ‐ 26 h. Salmonella colonies will be confirmed using serological (Somatic O and poly H agglutination) and biochemical procedures according to FDA BAM Ch. 5. The recovered isolates will be identified by the API20E (AOAC Official Method 978.24 ), VITEK2 GN be an even mix of collaborators (automated and manual). 5.3 35°C. . 5.4 After primary enrichment, ALL test portions (Solus One Salmonella and FDA BAM) will 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 contamination level is unknown to the collaborator.

6