Costar 96 well flat bottom

A commercial food grade lactic acid (88% v/v) was obtained from Xena Inc (Xena International Inc; Polo IL, USA) and lactic acid solutions were prepared in TSB at concentrations of 0.5, 1, 1.5, 2, 2.5, and 5% v/v. The pH of lactic acid was determined using a pH meter (Oakton pH 510 Benchtop Meter, Oakton Instruments, Vernon Hills, IL, USA) with a sensitivity of 0.01 and 2-point calibration. The concentration at which lactic acid could prevent survival and regrowth of the STEC strains was considered as the bactericidal concentration. The bactericidal concentration of lactic acid for all the bacterial strains was evaluated as follows: Each 900 μl of 0.5, 1, 1.5, 2, 2.5, and 5% v/v lactic acid was inoculated with 100 μl of bacterial culture (6.89 ± 0.72 log CFU/ml) for 300 s. Solution was centrifuged immediately after exposure for 1 min at 13000 × g using a Corning high speed microcentrifuge (Corning Inc., Corning, NY, USA). The supernatant was discarded, and pellets were resuspended in 1 ml sterile deionized water (SDW). From the resuspended solution, 100 μl was transferred to 100 μl of 2 × TSB in 96-well plates (Costar^® 96 Well Flat Bottom, Corning Life Sciences Inc. ME, USA) and incubated for 24 h at 37°C. The plates were observed for turbidity to determine survival and regrowth after incubation by determining the OD_{600 nm} using the BioTek Cytation 3 multi-mode plate reader.

A commercial food grade lactic acid (88% v/v) was obtained from Xena Inc (Xena International Inc; Polo IL, USA) and lactic acid solutions were prepared in TSB at concentrations of 0.5, 1, 1.5, 2, 2.5, and 5% v/v. The pH of lactic acid was determined using a pH meter (Oakton pH 510 Benchtop Meter, Oakton Instruments, Vernon Hills, IL, USA) with a sensitivity of 0.01 and two-point calibration. The concentration at which lactic acid could prevent survival and regrowth of the STEC strains was considered as the bactericidal concentration. The bactericidal concentration of lactic acid for all the bacterial strains was evaluated as follows: Each 900 μl of 0.5, 1, 1.5, 2, 2.5, and 5% v/v lactic acid was inoculated with 100 μl of the bacteria for 300 s. Solution was centrifuged immediately after exposure for 1 min at 13000 × g using a Corning high speed microcentrifuge (Corning Inc., Corning, NY, USA). The supernatant was discarded, and pellets were resuspended in 1 ml sterile deionized water (SDW). From the resuspended solution, 100 μl was transferred to 100 μl of 2 × TSB in 96-well plates (Costar^® 96 Well Flat Bottom, Corning Life Sciences Inc. ME, USA) and incubated for 24 h at 37 °C. The plates were observed for turbidity to determine survival and regrowth after incubation by determining the OD_{600 nm} using the BioTek Cytation multi-mode plate reader (Figure 1).

The minimum inhibitory concentration of lactic acid (LA, L-lactic acid, Xena International Inc., IL, USA) for the antibiotic-resistant and non-resistant bacterial strains was determined using a 96 well plate broth dilution method described by Dev Kumar et al. (2020) (link) with some modifications. Briefly, lactic acid stock solutions were serially diluted in 96 well plates −180 μl in each well—and inoculated with 20 μl of 6.83 ± 0.18 log CFU/ml of bacteria. Serial dilutions were performed from initial lactic acid concentrations of 5 and 3% to obtain lactic acid concentrations of 2.5, 1.5, 1.25, 0.75, 0.62, 0.37%, 0.31%, 0.18, 0.15, 0.09, 0.07, 0.04, 0.03, and 0.02% v/v. The 96-well plates (Costar^® 96 Well Flat Bottom, Corning LifeSciences Inc. ME, USA) were incubated for 24 h at 37 °C, and the growth kinetics were observed using the Bio-Tek Cytation 3 multi-mode plate reader (BioTek Instruments, Inc. USA). Conditions in the Bio-Tek Cytation 3 multi-mode plate reader were set as follows: the total runtime was set at 24 h with read intervals of 30 min, the shaker was set to an orbital shake every 10 s at a frequency of 283 cpm (3 mm), the read speed was set to Normal with a delay of 100 ms and the optical density was read at an absorbance of 600 nm. Un-inoculated blanks of TSB were used as a control for this experiment (Figure 1).