An individual stock solution (2 mg/mL) of each of the 15 antifungal compounds (Fig. 1) was prepared by dissolving known amounts (4 mg) of each of the compounds in H2O or ACN (2 mL) as required. From these 15 individual stock solutions a standard mix solution of 100 µg/mL (100 ppm) concentration was prepared by transferring 100 µL of each solution into a 4 mL vial. H2O/ACN (90/10; 500 µL) was added to this vial bring the total volume to 2 mL.
Calibration curve standards (#1–5) at concentrations of 1 ppm, 5 ppm, 10 ppm, 30 ppm and 50 ppm were prepared weekly by accurately pipetting volumes of 10 µL, 50 µL, 100 µL, 300 µL and 500 µL respectively from the standard mix solution (100 ppm) into an amber vials (1.5 mL) and each vial was made up to a total volume of 1 mL with 90/10 (H2O/ACN). Three standard controls (#6–8) at concentrations of 7.5 ppm, 20 ppm, 40 ppm were also prepared weekly by accurately pipetting volumes of 75 µL, 200 µL and 400 µL respectively from the standard mix solution (100 ppm) into an amber vials (1.5 mL) and each vial was made up to a total volume of 1 mL with 90/10 (H2O/ACN).
Culture broth (MRS) uninoculated with LAB strain was used as control MRS broth. This was prepared as instructed by the manufacturer and pH adjusted to pH 4.3 (average LAB culture pH) with lactic acid and AA. The control MRS broth (10 mL) was then fortified with the 15 compound mix at the concentrations used for the preparation of both the calibration curve standards and three controls (see above). All weekly prepared standards and controls were stored between 2 and 4 °C in solution.
An individual stock solution (2 mg/mL) of each of the 15 antifungal compounds (Fig. 1) was prepared by dissolving known amounts (4 mg) of each of the compounds in H2O or ACN (2 mL) as required. From these 15 individual stock solutions a standard mix solution of 100 µg/mL (100 ppm) concentration was prepared by transferring 100 µL of each solution into a 4 mL vial. H2O/ACN (90/10; 500 µL) was added to this vial bring the total volume to 2 mL.Calibration curve standards (#1–5) at concentrations of 1 ppm, 5 ppm, 10 ppm, 30 ppm and 50 ppm were prepared weekly by accurately pipetting volumes of 10 µL, 50 µL, 100 µL, 300 µL and 500 µL respectively from the standard mix solution (100 ppm) into an amber vials (1.5 mL) and each vial was made up to a total volume of 1 mL with 90/10 (H2O/ACN). Three standard controls (#6–8) at concentrations of 7.5 ppm, 20 ppm, 40 ppm were also prepared weekly by accurately pipetting volumes of 75 µL, 200 µL and 400 µL respectively from the standard mix solution (100 ppm) into an amber vials (1.5 mL) and each vial was made up to a total volume of 1 mL with 90/10 (H2O/ACN).Culture broth (MRS) uninoculated with LAB strain was used as control MRS broth. This was prepared as instructed by the manufacturer and pH adjusted to pH 4.3 (average LAB culture pH) with lactic acid and AA. The control MRS broth (10 mL) was then fortified with the 15 compound mix at the concentrations used for the preparation of both the calibration curve standards and three controls (see above). All weekly prepared standards and controls were stored between 2 and 4 °C in solution.
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