A 10-μl sample of bacterial culture was diluted 1 10 (approximate concentration: 10 cells/ml) and added to the 96 microtitre plate containing the commercially available standard substances solution (ferulic acid, coumaric acid, caffeic acid, SDG) and seedcake extract in MHB.
The initial concentration of the tested substance solutions was 300 mg crude drug/mL.
The MIC was defined as the lowest concentration of tested substance solutions, brazilin, protosappanin B and streptomycin at which no visible growth was observed.
Next, the desired amount of test substance was weighed and then filled up with the test substance vehicle PBS + BSA to obtain uniform test substance solutions of 10 wt% solutions.
In a 25 ml brown measuring flask, reference substance solutions (0.05 g gallic acid per ml) in aliquots of 1.0 ml, 2.0 ml, 3.0 ml, 4.0 ml, and 5.0 ml were separately placed.
Measurements of increase in absorbance at 234 nm for 30-90 sec after enzyme addition were done in 0.2 M borate buffer (pH 7.5) with linoleic acid (134 μM) as substrate and an enzyme concentration of 167 U/mL, using test substance solutions in DMSO or water or (for blanks) DMSO or water alone.
For this purpose, 5 g of yeast was suspended in 100 mL of toxic substance solution.
The titer of the antimicrobial substance solution, in AU/mL, was calculated as (1000/d) D, where D was the dilution factor, and d was the dose, the amount of antimicrobial substance solution added to each spot.
The mixed reference substance solution with the injection volume of 10 μL was determined by HPLC, respectively, according to the chromatographic conditions.
Normal donor plasma (PPP) was placed in the wells (90 µl/well) of a 96-well flat-bottom microtiter plate, after which 10 µl of the substance solution to be tested was added in different concentrations, along with 10 µl of the slow fluorogenic substrate (initial concentration 5 mM).
