ERP Micro December 2019

B astin et al . : J ournal of AOAC I nternational V ol . 101, N o . 5, 2018  1607

J. Formic Acid/Ethanol (Tube) Extraction Sample Preparation ( 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 tube. Transfer colonies from the agar plate into the water to create a cell ( d ) Centrifuge the suspension for 2 min at 15871–21130  g (equivalent to 13000 to 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 ambient temperature (20–25°C). ( g ) Add 25 μL of 70% formic acid and pipette up and down to resuspend the pellet. Vortex thoroughly and let stand for 5 min at room temperature (20–25°C). ( h ) Add 25 μL of 100% acetonitrile and mix by pipetting up and down 2–3 times. ( i ) Centrifuge 2 min at 15 871–21130 g (equivalent to 13 000 to 15 000 rpm for Eppendorf tube centrifuged with a 5424R rotor). ( j ) Dispense 1 μL of sample onto the target, i.e., cleaned reusable steel target or disposable Biotarget 96. ( k ) Because 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 ) Pipette 1 μL of reconstituted BTS solution onto the two selected positions and dry at ambient temperature (20–25°C). Note: After samples andBTShavedried,HCCAMatrixportioned solution must be added within 30 min, or the target must be cleaned and the inoculation of samples and BTS must be repeated. ( m ) Overlay each sample position and BTS control positions with 1 μL HCCA Matrix. Use a new pipette tip to add HCCA Matrix to each inoculated sample position. ( n ) Dry the inoculated target plate at room temperature (20–25°C). ( o ) The inoculated target plate is now ready for use. Note: An inoculated target plate 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. K. Generating a Run Result Report ( a ) After the acquisition of the target plate 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. suspension of approximately 1.5–2 McFarland. ( c ) Add 900 μL ethanol and mix suspension.

Table 2017.09Q. Identification consistency category descriptions

Identification consistency category

Description

High

The best match is a high-confidence identification. The second-best match is: - a high-confidence identification in which the species is identical to the best match. - a low-confidence identification in which the genus is identical to the best match. - a nonidentification. The requirements for high consistency are not met. The best match is a high- or low-confidence identification. The second-best match is: - a high- or low-confidence identification in which the genus is identical to the best match. - a nonidentification. The requirements for high or low consistency are not met.

Low

None

( l ) If the identification score received is below 1.7, the isolate should be repeated. For 5–10% of the isolates, an eDT or EXT procedure might be required.

I. 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, HCCA Matrix portioned must be added within 30 min, or the target must be cleaned and the inoculation of sample and BTS must be repeated. ( e ) Pipette 1 μL of reconstituted BTS solution onto the selected positions and dry at room temperature (20–25°C). Note: Make sure that the screw-cap tube containing HCCA Matrix is tightly closed after use to minimize solvent evaporation. ( f ) Overlay each of the sample position and BTS control positions with 1 μL of HCCAMatrix. 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.

Results of Collaborative Study

The collaborative study involved a method comparison evaluation of the Bruker MALDI Biotyper method to the confirmation listed in the FDA/BAM, USDA/FSIS-MLG, and ISO reference methods for Cronobacter and Salmonella species. For the Salmonella evaluation, a total of 15 laboratories