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2.8.2 Bioscreen assay for measuring Salmonella Typhimurium and P.
aeruginosa growth inhibition
The Bioscreen device (Oy Growth Curves Ab Ltd) was used for measuring the
influence of the chemical compounds on the planktonic growth of Salmonella
Typhimurium and P. aeruginosa. The Bioscreen is a computer controlled
incubator/reader/shaker that uses 10x10 well microtiter plates and measures light
absorbance of each well at a specified wave length in function of time. An overnight
culture of S. Typhimurium ATCC14028 (grown up in LB medium) or P. aeruginosa
(grown up in TSB) was diluted 1:200 in liquid broth (TSB 1/20). 300 µl of the diluted
overnight culture was added to each well of the 10x10 well microtiter plate.
Subsequently, serial dilutions of the chemical compounds were prepared in DMSO or
EtOH. 3 µl of each diluted stock solution was added to the wells (containing the 300
µl bacterial culture) in 3-fold. As a control 3 µl of the appropriate solvent was also
added to the plate in 3- or 4-fold. The microtiter plate was incubated in the Bioscreen
device at 25°C for at least 24 h, with continuous medium shaking. The absorbance of
each well was measured at 600 nm each 15 min. Excel was used to generate the
growth curves for the treated wells and the untreated control wells.
The effect of each compound concentration on the planktonic growth was classified
into one of the following categories:
1) The planktonic growth is not or only slightly affected, indicated by the symbol
‘-‘.
2) The planktonic growth is retarded, indicated by the symbol ‘+’.
3) The planktonic growth is completely or almost completely inhibited, indicated
by the symbol ‘o’.
The following criterium was used to decide between the first and the second category:
If the absorbance (measured at 600 nm) of the bacterial culture treated with the
compound is at least 0.5 (for Salmonella) or 0.8 (for Pseudomonas) units lower
than the absorbance of the untreated culture during 4 consecutive hours, then the
effect on the planktonic growth is classified in category 2.