Optimization of conditions for culture of the test bacteria used for direct bioautographic detection.: 1.: The gram-positive test bacterium Bacillus subtilis

The main purpose of this study was to determine optimum conditions for culture of a test microbe Bacillus subtilis (ATCC 6633) which enabled us to establish its use for direct bioautography. The viability of the bacteria on TLC plates was measured on the basis of their adenosine-5'-triphosphate (ATP) content as determined by bioluminescent luciferin/luciferase assay, the data being referred to values for total bacterial protein. In the first experiments, we used a '20-h' culture of B. subtilis prepared by dilution of an optical density (OD) much greater than 0.4 culture to furnish a culture of OD = 0.4 (Method A). Later, on the basis of our optimization experiments we found that a '5-9-h' broth culture of A subtilis was suitable. Under these conditions the bacteria remained in the log phase (OD = 0.2-0.4) for 5-9 h (Method B) in immersion bacterial suspension. Because the test bacteria were in the log phase a much shorter incubation time (4-8 h) was sufficient for TLC plates instead of the original 18 h in a previous study. One advantage of this method, in addition to the shorter incubation time, is that we can use TLC plates coated with adsorbents other than silica.