Direct bioautography is a potent means of obtaining information about the antimicrobial activity of a compound separated from a complex mixture. In this process the developed TLC plate is dipped into a broth culture of a test bacterium and the bacterium will grow directly on the plate. Optimum experimental conditions must, however, be used for each test bacterium.The main purpose of this study was to find optimum culture conditions for a Gram-negative test bacterium,
(ATCC 25922) enabling us to establish a direct bioautographic method with the shortest possible performance time. Because the intracellular adenosine-5′-triphosphate (ATP) level is a direct and sensitive measure of bacterial well-being, ATP assay was used for this purpose. As far as we know this is the first report of the use of an ATP method for optimization of direct bioautography with
. Our optimizing experiments on
culture showed that the bacteria had to be in the log phase (optical density,
= 0.1–0.4) in the bacterial suspension used for dipping. TLC plates immersed in the log-phase culture needed a shorter incubation time for bacterial growth on the TLC plate (3 h) than for the original ‘overnight’ culturing suggested in studies by others.In this paper we will show that:
ATP assay is a valid method for optimizing
Bacterial ATP level oscillates during the growth phase in culture media.
TLC plates should be immersed in
dipping suspension with
Dipping a developed TLC plate for 10 s gave acceptable results.
Incubation of the seeded TLC plate at 37°C for 3 h was found to be optimum.
An ATP/protein ratio of 10–15 nmol mg
in dipping culture and ∼5 nmol mg
on seeded TLC plates were the minimum threshold values for visualization of living bacteria by means of the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) reaction.
With our optimized coditions the total performance time of
direct bioautography is 9.6 h instead of the originally reported 11.5 h.
Our procedure results in much sharper contrast of the inhibition zone than that without optimization.
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