ERP Micro December 2019

B astin et al . : J ournal of AOAC I nternational V ol . 102, N o . 5, 2019  1597

was incubated for 24–48 h at 41.5 ± 1ºC under microaerobic conditions. Isolated colonies were streaked to CBA plates and incubated for 24–48 h at 41.5 ± 1ºC under microaerobic conditions prior to being shipped to each site. Eight plates were then packaged into a BD GasPak ™ EZ Campy Gas Generating Pouch System (Part No. B260685; Thermo Fisher Scientific).

Table 1. Participation of each collaborating laboratory for Salmonella

Collaborator ID No.

Participating lab

Analyst

Target

Reusable a Reusable Reusable Reusable Disposable b Reusable Disposable Disposable Disposable Disposable Disposable Disposable Disposable Disposable

1 2 3 4 5 6 7 8 9

1 2 3 4 5 6 7 8 9

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

Isolate Distribution

All samples were labeled with a randomized, blind-coded 3-digit number affixed to the TSA slant tube. Isolates were shipped on a Wednesday via overnight delivery according to the Category B Dangerous Goods shipment regulations set forth by the International Air Transport Association (IATA). Upon receipt, samples were held by the collaborator at refrigerated temperature (2–8°C) until the following Monday, when analysis was initiated. For the Cronobacter evaluation, 14 collaborators from seven laboratories participated. For the Salmonella evaluation, 15 collaborators from 15 laboratories participated. For the method extension for Campylobacter species, all samples were labeled with a randomized, blind-coded 3-digit number affixed to the CBA plate. Isolates were shipped on a Saturday to arrive on Monday and according to the Category B Dangerous Goods shipment regulations set forth by the IATA. Collaborators were instructed to follow the appropriate preparation and analysis as outlined in the study protocol. One set of 24 isolates [16 target ( Cronobacter or Salmonella ) organisms and 8 non- Cronobacter or non- Salmonella organisms] were analyzed in each collaborative study by each participant. For the analysis of the Campylobacter method extension, upon receipt, each isolate was subcultured onto CBA for 24–48 h at 41.5 ± 1ºC under microaerobic conditions. The Salmonella organisms were streaked to XLD (Bio- Rad or equivalent), RSA, and TSA (Bio-Rad or equivalent). All plates were incubated at 34–38°C. The XLD and RSA were incubated for 20–26 h, and the TSA was incubated for 18–24 h. Following incubation, if growth was present on an agar plate, an isolated colony was analyzed by the Bruker MALDI Biotyper using the direct transfer (DT) technique. When needed, the extended DT (eDT) procedure was used. If necessary, tube extraction (EXT) was used to obtain a reliable result. Growth from each TSA plate was also analyzed by conducting a Gram stain, spot oxidase test (Thermo Fisher Scientific), Poly O and H antisera agglutinations (Bio-Rad or equivalent), and biochemical analysis using either API 20E (bioMérieux; AOAC OMA 978.24 ; 11) or GN VITEK 2 (bioMérieux; AOAC OMA 2011.17 ; 12). Salmonella Species Isolate Analysis

10 11 12 13 14 15

10 11 12 13 14 15

study was subcultured onto three agars: XLD (Bio-Rad or equivalent), RSA, and TSA (Bio-Rad or equivalent). All plates were incubated at 34–38°C. The XLD and RSA were incubated for 20–26 h, and the TSAwas incubated for 18–24 h. Following incubation, if growth was present on an agar plate, an isolated colony was analyzed by the Bruker MALDI Biotyper using the DT technique. When needed, the eDT procedure was used or, if necessary, the EXT to obtain a reliable result. To demonstrate the equivalency between the reusable target and the disposable target, both were used by different participants during the evaluation (Table 1). Growth from each TSA plate was also analyzed by conducting a Gram stain, spot oxidase test (Thermo Fisher Scientific), Poly O and H antisera agglutinations (Bio-Rad or equivalent), and biochemical analysis using either API 20E (AOAC OMA 978.24 ) or GN VITEK 2 (AOAC OMA 2011.17 ). The Cronobacter organisms were subcultured onto three agars [CCI (Thermo Fisher Scientific or equivalent), ESIA(Bio- Rad or equivalent), and TSA (Bio-Rad or equivalent)] from the blind-coded TSA slants. The CCI plates were incubated for 20–26 h at 44 ± 2°C, the ESIA plates were incubated for 20–26 h at 41.5 ± 2°C, and the TSA plates were incubated for 18–24 h at 35–37 ± 1°C. Following incubation, growth present on each plate was analyzed by the Bruker MALDI Biotyper using the DT technique. When needed, the eDT procedure was used. If necessary, EXT was used to obtain a reliable result. Growth from each TSA plate was also analyzed by conducting a spot oxidase test (Thermo Fisher Scientific) and by conducting biochemical confirmation analysis using either API 20E (AOAC OMA 978.24 ) or GN VITEK 2 (AOAC OMA 2011.17 ). Disposable a  Reusable = Stainless-steel target that can be cleaned and used again. b  Disposable = Target that is for single use, and the MBT adapter is required. Cronobacter Species

Exclusivity Organisms: Salmonella

The exclusivity organisms consisted of non- Salmonella Gram-negative organisms that were closely related to the target organisms. Each exclusivity organism in the Salmonella