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  • Author or Editor: Béla Nagy x
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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 ) ≫ 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 B. 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.

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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, Escherichia coli (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 E. coli . Our optimizing experiments on E. coli culture showed that the bacteria had to be in the log phase (optical density, OD 600nm = 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:

  1. ATP assay is a valid method for optimizing E. coli direct bioautography. Bacterial ATP level oscillates during the growth phase in culture media. TLC plates should be immersed in E. coli dipping suspension with OD 600nm = 0.1–0.4. 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 −1 in dipping culture and ∼5 nmol mg −1 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 E. coli 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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Optimum conditions have been established for culture of the fungus Candida albicans (ATCC 90028) for microbial detection of zones in direct bioautographic TLC. Bioluminescent ATP assay is a highly sensitive method for optimizing the viability of Candida albicans test fungus in bioautographic TLC detection. A suspension of microbes (OD 600nm = 0.5–0.7, in Mueller-Hinton broth with 5% glucose) in the log phase of growth can be used for dipping TLC plates. On the basis of our results with Candida albicans , we can differentiate between microbiostatic (bacteriostatic or fungistatic) and microbiocidal (bactericidal or fungicidal) effects on TLC plates. Our micrographs clearly show the borders of inhibition zones in bioautograms. This technique leads to new possibilities in studies of the interactions between microbes and antimicrobial compounds on bioautographic silica gel TLC plates by using scanning electron microscopy. In this paper we describe an optimization procedure for bioautographic TLC detection.

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