ERP Micro December 2019

B astin et al . : J ournal of AOAC I nternational V ol . 101, N o . 5, 2018  1617 (3)  Once the target plate has been loaded, close the lid. (4)  Press the MALDI target plate IN/OUT button once. The MALDI target plate loading procedure starts and the green READY and ACCESS LEDs go off. The green READY LED will be lit again when the loading procedure has been successfully completed. The MALDI target plate loading procedure typically takes 2–3 min. If the instrument is not ready after 5 min: (5)  Press the MALDI target plate IN/OUT button once to eject the target plate. (6)  When the green ACCESS LED is turned on, open and close the load port lid. (7)  Press the MALDI target plate IN/OUT button once to reload the target plate.

Cleaned targets plates can be stored before use in a dry place at room temperature (20–25°C) in the container provided. Avoid exposing cleaned targets to potential contamination (e.g., dust) or corrosive atmospheres. Note: Do not place any adhesive labels on the target. Do not drop or scratch the target.

F. Direct Transfer (DT)

( a )  Sample preparation procedure .— (1)  Using a sterile colony-transfer device, smear an isolated colony of bacteria or yeast as a thin film directly onto a single sample position on a cleaned reusable target or disposable Biotarget (Figure 2017.10A ). Note : After incubation of bacteria on recommended isolation media for 24–48 h at the reference method–specified temperatures following the appropriate reference methods, 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. (2)  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. (3)  Pipette 1 μL of 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. (4)  Overlay each sample position and BTS control positions with 1 μL HCCA Matrix solution. Use a new pipet tip to add HCCAMatrix to each inoculated sample position. Note: Make sure that the screw-cap tube containing HCCA Matrix is tightly closed after use to minimize solvent evaporation. (5)  Dry the inoculated target at room temperature (20–25°C). (6)  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. ( b )  Loading and ejecting target plate .— (1) Make sure that the target plate carrier is in the OUT position. (2)  Open the load port lid and place the target plate onto the target platform. Note: If the lid does not open easily, the target carrier may be in the IN position. If this is the case, move the target carrier to the OUT position by pressing the MALDI target plate IN/OUT button once.

Note: The green READY LED will be off and the yellow WARM-UP LED will be lit if the laser is in standby mode. The laser will enter this mode after 10 min with no acquisition. The green READY LED will be lit again when a new acquisition sequence is started. ( c )  To eject a target plate. — (1) Press the MALDI target plate IN/OUT button once. The MALDI target plate ejection procedure starts and the green READY and ACCESS LEDs are off. (2)  When the green ACCESS LED is lit again, open the load port lid, remove the target, and close the load port lid. Note: The target carrier should only be moved to the OUT position when target plates are being exchanged. Measured target plates can remain in the instrument until the next target plate is ready to be processed. If no target is available, move the target carrier into the instrument (without a target plate in place) and move it out again only when the next target plate is ready to be loaded. Note: When exchanging target plates, do not leave the load port lid open for any longer than necessary. Leaving the load port lid open for extended periods does not damage the instrument but will prolong target loading by as much as 30 min. ( a ) Log on to the MALDI Biotyper System computer. ( b ) Start the MALDI Biotyper System software by first clicking on the Flex Control icon. Then click onto the RTC Software icon on the desktop. ( c ) After successful start-up, the main application window is displayed. ( d ) Prepare a test run according to the MALDI Biotyper System Instructions for Use. G. Operating the MALDI Biotyper System ( a ) MALDI Biotyper System identification consists of two steps, which are performed on each test and BTS control position. ( b ) Amass spectrum is acquired from the test or BTS control position (measurement step). ( c ) The resulting mass spectrum is processed, and the resulting peak pattern is matched against reference patterns in the MALDI Biotyper reference library (identification step). ( d ) The identification step is started immediately after the associated measurement step has been completed and a mass spectrum is available. H. Results

Figure 2017.10A. Row A above shows appropriate amount of test organism for the DT procedure. Rows B and C show suboptimal (“not enough” and “too much” for most organisms, respectively).