ERP Micro December 2019

B astin et al . : J ournal of AOAC I nternational V ol . 101, N o . 5, 2018  1605

(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 trifluoracetic acid (conducted in a fume cabinet).—(a)  Transfer 50 μL of HPLC (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. (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 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. 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 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)  GuanidineHydrochloride (GdnHCl) Procedure. —Before each run, ensure that the target plate you are using has been cleaned properly. Prepare the solutions required for cleaning target plates 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. grade water into a 1.5 mL microcentrifuge tube. (b)  Carefully add 200 μL trifluoracetic acid. (c)  Close the tube tightly. (d)  Mix by inverting tube five times.

(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 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 target 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 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 dry completely for at least 15 min at room temperature (20–25°C). (m)  Store the clean target plate 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. F. 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 (Figure 2017.09A ). 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 and BTSmust be repeated.

Figure 2017.09A. 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).