ERP Micro December 2019

B astin et al . : J ournal of AOAC I nternational V ol . 102, N o . 5, 2019  1613

( j ) Dispense 1 μL sample onto the target, i.e., cleaned reusable steel target or disposable Biotarget 96. ( k ) As a valid BTS control is mandatory per target and run, it is advised to use two BTS control positions. Select the BTS control positions on a target to inoculate with BTS solution. ( l ) Pipet 1 μL reconstituted BTS solution onto the two selected positions and dry at room temperature (20–25°C). Note: After samples and BTS have dried, HCCA Matrix solution must be added within 30 min, or the target must be cleaned and the inoculation of samples BTS must be repeated. ( m ) Overlay each sample position and BTS control positions with 1 μL HCCA Matrix. Use a new pipet tip to add matrix to each inoculated sample position. ( n ) Dry the inoculated target at room temperature (20–25°C). ( o ) The inoculated target is now ready for use. Note: An inoculated target must be processed within 24 h of preparation, or the target must be cleaned and the inoculation of samples and BTS must be performed again. Note: The sample positioning on the target can be facilitated with the MBT Pilot workstation. J. Generating a Run Result Report ( a ) After the acquisition of the target has been completed, click View > Results to generate a PDF report. ( b ) The PDF report header contains the Run Identifier and Run Creation Date/Time from the Run Info section. ( c ) The PDF report footer contains the report creation date/ time, page count, and the appropriate area of application. The collaborative study involved a method comparison evaluation of the Bruker MALDI Biotyper method to the confirmation listed in the United States and ISO reference methods for Cronobacter and Salmonella species. For the Salmonella evaluation, a total of 15 laboratories throughout Europe participated in this study. The studywas organized andmanaged by ADRIADéveloppement. All 15 laboratories had one participating analyst. Each collaborator analyzed 24 blind-coded organisms, including 16 target organisms and 8 nontarget organisms. For the Cronobacter portion, a total of seven laboratories throughout the continental United States participated in this study. The study was organized and managed by Q Laboratories, Inc. Five of the laboratories had two separate analysts participate, one laboratory had three analysts participate, and one laboratory had one participant. Each participant analyzed 24 blind-coded organisms, including 16 target organisms ( Cronobacter or Salmonella ) and 8 non- Cronobacter or non- Salmonella organisms. The method extension to include Campylobacter species, involved a comparative evaluation of the Bruker MALDI Biotyper method to the confirmation listed in the United States and ISO reference methods for Campylobacter species. For the Campylobacter evaluation, there were a total of 17 participants from seven different laboratories throughout United States and one laboratory in Canada. Five of the laboratories had two analysts participate separately, two laboratories had three analysts participate, and one laboratory had one participant. Each collaborator analyzed 24 blind-coded organisms, including 16 Campylobacter organisms and 8 non- Campylobacter organisms. Results of Collaborative Study

H. eDT ( a ) If the identification score received is below 1.7, the isolate should be reanalyzed following the eDT. Note: After inoculation of bacteria on recommended isolation media, colonies are stable for up to 12 h when held at room temperature (20–25°C). If testing is not done within 12 h, subculture the test organism before testing on the MALDI Biotyper System. ( b ) Using a sterile colony transfer device, smear an isolated colony of bacteria as a thin film directly onto a sample position on a cleaned reusable steel target or a disposable Biotarget 96. ( c ) Overlay the sample spot with 1 μL 70% aqueous formic acid and allow to dry at room temperature (20–25°C). ( d ) Select a minimum of one BTS control position on a target to inoculate with BTS solution. It is mandatory to get at least one valid BTS control per target and per run. Therefore, it is advised to select two BTS control positions. Note: After samples and BTS have dried, HCCAMatrix must be added within 30 min, or the target must be cleaned and the inoculation of sample and BTS must be repeated. ( e ) Pipet 1 μL reconstituted BTS solution onto the selected positions and dry at room temperature (20–25°C). Note: Make sure that the screw-cap tubes containing HCCA Matrix are tightly closed after use to minimize solvent evaporation. ( f ) Overlay each of the sample positions and BTS control positions with 1 μL HCAA Matrix. Use a new pipet tip to add HCCAMatrix to each inoculated sample position. ( g ) Dry the inoculated target at room temperature (20–25°C). ( h ) The inoculated target is now ready for use. Note: An inoculated target must be processed within 24 h of preparation, or the target must be cleaned and the inoculation of samples and BTS must be performed again. Note: The sample positioning on the target can be facilitated with the MBT Pilot workstation, the EDT can be supported with the MBT Galaxy. I. Formic Acid/Ethanol (Tube) EXT ( a ) If the identification received is still below 1.7 after the eDT, the isolate should be reanalyzed following the EXT procedure. ( b ) Transfer 300 μL HPLC grade water to a microcentrifuge tube. Transfer colonies from the plate into the water to create a cell suspension of approximately 1.5–2 McFarlands. ( c ) Add 900 μL ethanol and mix suspension. ( d ) Centrifuge the suspension for 2 min at 15871–21130 × g (equivalent to 13 000–15000 rpm for Eppendorf tube centrifuged with a 5424R rotor). Decant the ethanol. ( e ) Repeat step ( d ) to remove all residual ethanol. Avoid contact with the pellet. ( f ) Air-dry the pellet for a minimum of 5 min at room temperature (20–25°C). ( g ) Add 25 μL 70% formic acid and pipet up and down to resuspend the pellet. Mix thoroughly on a vortex mixer and let stand for 5 min at room temperature (20–25°C). ( h ) Add 25 μL 100% acetonitrile and mix by pipetting up and down 2–3 times. ( i ) Centrifuge 2 min at 15 871–21130 × g (equivalent to 13000–15000 rpm for Eppendorf tube centrifuged with a 5424R rotor).