2.4 Antimicrobial study
L. innocua ATCC 33090 and E. coli ATCC 700728 were resuscitated by transferring the cultures into 10 ml TSB followed by incubation for 24 h at 37°C. The overnight liquid cultures were then streak plated onto TSA plate and incubated at 37°C for 24 h. A loopful of bacterial cells collected from a single colony on the TSA plate was inoculated into 10 ml fresh TSB and grew for 24 h at 37°C which served as the stock culture. Each time for minimum inhibition concentration (MIC)/minimum bactericidal concentration (MBC) analysis, 100 μl stock culture was mixed with 9.9 ml TSB and grew for 24 h at 37°C with shaking. The cultures were diluted to 106 and then used in the MIC/MBC analysis.
MIC and MBC were determined using 96 well microdilution plate method as described earlier (Ngo et al ., 2019). Briefly, bacteria were freshly grown from refrigerated stocks for 24 h at 37 °C with 100 rpm agitation. The cells were collected by centrifugation and resulted pellets were washed with 0.1% peptone water before being suspended in 10 ml 0.1% peptone water. Populations of bacteria were determined using direct plating on TSA after dilutions. Fatty acid arginate compounds were dissolved in 10% ethanol at 1 mg mL−1 and diluted with phosphate buffer saline (PBS, pH 10) to obtain concentrations of 1.8–512 μg mL−1. To the well of microdilution plates, 100 μL TSB, 100 μL test compounds at various concentrations and 20 μl of ~106 CFU mL−1bacterial suspension were added. Microplates were then covered and incubated for 24 h at 37 °C with 100 rpm orbital shaking. After the incubation, 20 μL of 0.1% thiazolyl blue tetrazolium bromide was added to each well, and plates were allowed to sit at ambient temperature (~22 °C) for 1 h. The MIC was the lowest concentration of the test compound for which no color change (no growth) occurred. MBC was determined by spreading 100 μL from each growth-negative well onto TSA and incubating at 37 °C for 24 h. MBC was the lowest concentration where no colony was observed. At least three independent replicates of quadruplicate tests were conducted for each compound.