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.