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Chapter III Material and methods
USA) according to the manufacturer’s instructions. Cells were energized with glucose (20
mM final concentration) for 10 min at 37°C prior to treatment with test compounds. The pore
former nisin (25 mg/l final concentration) was used as a positive control and untreated cells
served as negative controls. Cell suspensions (0.5 ml) were treated with bacteriocin
preparation ~ 0.5 mg/l final concentration), and positive and negative controls at 37°C and
300 µl aliquots of each sample were removed at various intervals over 60 min. Cells were
kept on ice during sampling. Cells were stained by addition of 100 µl live/dead BacLight TM
staining reagent to 100 µl samples in triplicate in 96-well flat-bottomed microtitre plates.
Samples were mixed thoroughly by pipetting and the Microtiter plate was incubated in the
dark at an ambient temperature for 15 min. The fluorescence emission of green (excitation at
485 nm, emission at 530 nm) to red (excitation at 485 nm, emission at 630 nm) ratio was
measured with a Fluorescence Spectrophotometer. The effect of bacteriocin on membrane
integrity was calculated by taking the green to red fluorescence ratios of the untreated
samples as 0% permeable and the nisin treated samples as 100% permeable. Membrane
permeability was expressed as a green to red fluorescence ratio (530/630 nm) (Cleveland et
al., 2001).
3.12.3 Measurement of intracellular K+ content
The intracellular K + concentration of bacteriocins treated S. Typhimurium ATCC
19585 cells was determined as described previously (Olivia et al., 1998) with the following
modification. Cells were energized with glucose (20 mM final concentration) for 10 min at
37°C prior to treatment with test compounds. Nisin (25 mg/l final concentration) was used as
a positive control while untreated cells served as negative controls. Cells were treated with
either bacteriocin (20 mg/l final concentration) or the positive and negative controls at 37°C,
0.5 ml samples were taken in duplicate after 10 min incubation. These samples were
centrifuged (13000 x g, 5 min, 4°C) through 0.3 ml of silicon oil (1:1 ratio, BDH, UK). The