ERP Micro December 2019

B astin et al . : J ournal of AOAC I nternational V ol . 102, N o . 5, 2019  1611

( e ) Rinse the target with tap water and wipe it with a fiber- free cloth. ( f   ) Transfer the MALDI target plate to a fume hood, cover the target plate with a layer of 80% aqueous trifluoracetic acid (prepared as described above) by adding 100 μL with a pipet, and thoroughly wipe all target plate positions with a fiber-free cloth or gloved hand. ( g ) Rinse the target with HPLC grade water and wipe it dry with a fiber-free cloth. ( h ) Let the target dry completely for at least 15 min at room temperature (20–25°C). ( i ) Store the clean target 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 target plates 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 plate. ( b )  GdnHCl procedure .—Before each run, ensure that the target plate you are using was cleaned properly. Please prepare the solutions required for cleaning targets as follows.—( 1 ) Preparation of 70% aqueous ethanol .—( a ) To prepare 100 mL solution, measure 30 mL deionized water with a graduated cylinder. ( b ) Transfer the water into a beaker. ( 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 it thoroughly under running tap water. ( d ) Using a fiber-free cloth, clean plate the target thoroughly with 70% aqueous ethanol. ( e ) Rinse the target plate with tap water and wipe it 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 them 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 it carefully with a fiber-free cloth. ( j ) Repeat steps ( h ) and ( i ) twice. ( k ) Rinse the target plates with deionized water and wipe it 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 )  Sample 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 (Figure 2017.09A 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 get 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, 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. 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 ) Make sure 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

Figure 2017.09A. Row A above shows appropriate amount of test organism for the DT procedure. Rows B and C show sub-optimal (“not enough” and “too much” for most organisms, respectively).