ERP Micro December 2019

1616  B astin et al . : J ournal of AOAC I nternational V ol . 101, N o . 5, 2018

(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, 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. ( 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 homogeneousmixture by transferring themixture 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 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 4M aqueous GdnHCl (diluted 1:1 with a stock 8M 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 plate with plenty of tap water and wipe it carefully with a fiber-free cloth. (h)  Intensively wipe the target plate with 4M aqueous GdnHCl. (i)  Rinse the target with plenty 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 dry completely for at least 15 min at room temperature (20–25°C). (m)  Store the clean target plate in the container provided.

and two chromogenic agars (O&A, Bio-Rad or equivalent and RLM, Bio-Rad) were used. Organisms for testing must be subcultured as necessary to ensure purity. Testing should be conducted on a culture that has been grown for 24–48 h (follow specific temperature and incubation time as specified by manufacturer or reference method). Use only a single isolated colony (or colonies to get sufficient biological material) when performing identification on the MALDI Biotyper System. ( b )  Preparation of Bacterial Test Standard (BTS) .—Add 50μLofstandardsolventtoBTStube.Dissolvebygentlypipetting up and down 20 times; avoid generating bubbles. Let stand at room temperature (20–25ºC) for 5 min. Repeat pipetting 20 times. Centrifuge for 2 min at maximum speed (15 871– 21 130 g, equivalent to 13 000 to 15 000 rpm for Eppendorf tube centrifuged with a 5424R rotor). Aliquot 5 μL into 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 .—Prepare 70% formic acid by transferring 70 μL of formic acid to 30 μL of 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 (20–25ºC) 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. When using the 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 (conducted 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 (conducted in a fume cabinet). — (a)  Transfer 50 μL of HPLC E. Cleaning Target Plate: Reusable Polished Stainless Steel Targets Plates Only

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 plate cleaning procedure (conducted in a fume cabinet). — (a)  Transfer the target plate 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 it thoroughly under running tap water.