ERP Micro December 2019

( b )  GdnHCl procedure.— Before each run, ensure that the target plate being used has been cleaned properly. 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 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 it with a fiber- free cloth. ( f ) Cover the target plate with 4 M aqueous GdnHCl (diluted 1:1 with a stock 8 M GdnHCl solution) and incubate at room temperature (20–25°C) for 10 min. Then thoroughly wipe all target plate positions with a fiber-free cloth or gloved hand. ( g ) Rinse the targets plates with plenty of tap water and wipe it carefully with a fiber-free cloth. ( h ) Intensively wipe the target plate with 4 M aqueous GdnHCl. ( i ) Rinse the target plate with plenty of tap water and wipe it carefully with a fiber-free cloth. ( j ) Repeat steps ( h ) and ( i ). ( k ) Rinse the target plate with deionized water and wipe it dry with a fiber-free cloth. ( l ) Let the target plate dry completely for at least 15 min at room temperature (20–25°C). ( m ) Store the clean target plate in the container provided. 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.

1.5 mL microcentrifuge tubes (screw caps recommended) and store at –20°C or below for up 5 months; do not refreeze once thawed. ( c ) Preparation of 70% formic acid.— Transfer 70 µL formic acid to 30 µL HPLC grade water. ( d ) Preparation of HCCA Matrix.— Add 250 µL of standard solvent. Vortex for 1 min or until dissolved. Once reconstituted, store in the dark at room temperature (20–25°C) up to 1 week. Check for crystal formation on bottom of tube each day before use, and vortex for a minimum of 1 min or until dissolved. E. Cleaning Target Plate (Reusable Polished Stainless Steel Targets Plates Only) When using reusable polished stainless steel targets plates, two procedures are available to guarantee an appropriate and efficient cleaning of the reusable polished stainless steel targets plates, one using trifluoroacetic acid and one using GdnHCl. ( a ) Trifluoroacetic acid procedure (conduct in a fume cabinet).— Before each run, ensure that the target plate has been cleaned properly. Prepare the solutions required for cleaning targets as follows: ( 1 )  Preparation of 70% aqueous ethanol.— ( a ) To prepare 100 mL solution, measure 30 mL HPLC grade 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 )  Preparation of 80% aqueous trifluoroacetic acid (conduct in a fume cabinet).— ( a ) Transfer 50 μL HPLC grade water into a 1.5 mL microcentrifuge tube. ( b ) Carefully add 200 μL trifluoroacetic acid. ( c ) Close the tube tightly. ( d ) Mix by inverting tube five times. ( 3 )  Target cleaning procedure (conduct in a fume cabinet).— ( a ) Transfer the target into a suitable container and pour in enough 70% aqueous ethanol (prepared as described above) to cover the target plate 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 with tap water and wipe it with a fiber-free cloth. ( f ) Transfer the MALDI target plate to a fume hood and cover the target with a layer of 80% aqueous trifluoroacetic 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. Note : Conduct in a fume cabinet. ( g ) Rinse the target plate with HPLC grade water and wipe it dry with a fiber-free cloth. ( h ) Let the target plate dry completely for at least 15 min at room temperature (20–25°C). ( i ) Store the clean target plate in the container provided. 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 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.

Figure 2017.10. 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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