Optimization of conditions for culture of test bacteria used for direct bioautographic TLC detection. 2. Gram-negative test bacterium: Escherichia coli

Sándor Nagy, Tamás Koszegi, Lajos Botz, Béla Kocsis

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

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, OD600nm = 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 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 OD600nm = 0.1-0.4. - Dipping a developed TLC plate for 10 s gave acceptable results. - Incubation of the seede 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-diphenyltetrazolim bromide) reaction. - With our optimized conditions the total performance time of E. coli direct bioutography is 9.6 h instead of the originally reported 11.5 h. - Our procedure results in much sharper constrast of the inhibition zone than that without optimization.

Original languageEnglish
Pages (from-to)121-126
Number of pages6
JournalJournal of Planar Chromatography - Modern TLC
Volume16
Issue number2
DOIs
Publication statusPublished - Mar 1 2003

Keywords

  • ATP, adenosine-5′-triphosphate
  • ATP-DnaA, ATP-bound form of DnaA protein
  • Bioluminescent ATP assay
  • Dehydrogenase activity-detecting reagent
  • Direct bioautographic TLC
  • Escherichia coli
  • MTT, 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
  • TLC
  • Thin layer damage

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Clinical Biochemistry

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