AOAC 2017 First Action Methods

( c ) Measure 70 mL absolute ethanol and mix with the water in a beaker. ( d ) Generate a homogeneous mixture by transferring the mixture from the beaker into the graduated cylinder and back again. ( e ) Repeat step ( d ) five times. ( 2 ) Target plate cleaning procedure.— ( a ) Transfer the target plate into a suitable container (e.g., 100 mm glass Petri dish or other suitable container) and pour in enough 70% aqueous ethanol (prepared as described above) to cover the target surface. ( b ) Incubate for 5 min at room temperature (20–25°C). ( c ) Remove the target plate and rinse thoroughly under running tap water. ( d ) Using a fiber-free cloth, clean the target plate thoroughly with 70% aqueous ethanol. ( e ) Rinse the target plate with tap water and wipe with a fiber- free cloth. ( f ) Cover the target plate with 4 M aqueous GdnHCl (diluted 1:1 with stock 8 M GdnHCl solution), incubate at room temperature (20–25°C) for 10 min, and then thoroughly wipe all target plate positions with a fiber-free cloth or gloved hand. ( g ) Rinse the target plates with plenty of tap water and wipe carefully with a fiber-free cloth. ( h ) Intensively wipe the target plates with 4 M aqueous GdnHCl. ( i ) Rinse the target plates with plenty of tap water and wipe carefully with a fiber-free cloth. ( j ) Repeat steps ( h ) and ( i ) twice. ( k ) Rinse the target plates with deionized water and wipe dry with a fiber-free cloth. ( l ) Let the target dry completely for at least 15 min at room temperature (20–25°C). ( m ) Store the clean target plates in the container provided. Cleaned target 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. E. Direct Transfer (DT) ( a )  Samples preparation procedure .—( 1 ) Using a sterile colony transfer device, smear an isolated colony of bacteria as a thin film directly onto a single sample position on a cleaned reusable steel target or disposable Biotarget 96 ( see Figure 2017.09 shows the appropriate amount of test organism to smear). Note : After incubation of bacteria on recommended isolation media for 24–48 h at the 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 obtain at least one valid BTS control per target and per run. Therefore, it is advised to select two BTS control positions. ( 3 ) 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, HCCAMatrix solution must be added within 30 min or the target must be cleaned and the inoculation of samples and BTS must be repeated. ( 4 ) Overlay each sample position and BTS control positions with 1 μL HCCAMatrix solution. Use a new pipet tip to add HCCA Matrix to each inoculated sample position. Note : Make sure that the

screw-cap tube containing HCCAMatrix 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. Note : The sample positioning on the target can be facilitated with the MBT Pilot™ workstation; the DT can be supported with the MBT Galaxy. (b)  Loading and ejecting target.— ( 1 ) Ensure that the target carrier is in the OUT position. ( 2 ) Open the load port lid and place the target 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. ( 3 ) Once the target 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 andACCESS LEDs go off. The green READYLED will be lit again when the loading procedure has been successfully completed. The MALDI target plate loading procedure typically takes 2 to 3 min. If the instrument is not ready after 5 min: ( a ) Press the MALDI target plate IN/OUT button once to eject the target. ( b ) When the green ACCESS LED is turned on, open and close the load port lid. ( c ) Press the MALDI target plate IN/OUT button once to reload the target. 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.— ( 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 plate, and close the load port lid. Note : The target carrier should only be moved to the OUT position when targets are being exchanged. Measured targets can remain in the instrument until the next target is ready to be processed. If no target is available, move the target carrier into the instrument (without a target in place), and move it out again only when the next target is ready to be loaded. Figure 2017.09. RowA 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).

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