Optimization of the Culture Medium Used for Direct TLC-Bioautography. Application to the Detection of Antimicrobial Compounds from Cordia gilletii De Wild (Boraginaceae)

TLC-bioautography, a convenient and simple mean of testing plant extracts and pure substances for their effects on pathogenic microorganisms, enables easy detection of active fractions. Its application requires a medium fluid enough to cast suspensions of microorganisms but viscous enough to adhere to the TLC plate and maintain sufficient humidity for bacterial growth. Mueller-Hinton (MH) agar and broth are often used for this purpose. These media have major drawbacks, however - microorganisms can suffer thermal shock from the temperature required for agar casting, and agar can also dilute the antimicrobial compounds, and immersion broth does not conveniently adhere to the TLC plate and dries too rapidly. To overcome these problems, we investigated several combinations of MH broth and MH agar (proportions 95:5, 90:10, 85:15, 80:20, 75:25, 70:30, 60:60, and 50:50) to discover a medium sufficiently fluid to prepare bacterial suspensions at 37 degrees C (the temperature for optimum growth of many pathogenic bacteria) yet which solidifies at ambient temperature. The mixture MH broth-MH agar 90:10 fulfilled these requirements; media containing higher proportions of MH agar were solid at 37 degrees C and those containing lower proportions of MH agar flowed down the plate, partly carrying the active spots, leading to poor detection and resolution. This simple modification of the culture medium can be applied to most media amenable to TLC-bioautography. This 90:10 mixture was used to detect antimicrobial compounds in a Congolese medicinal plant, Cordia gilletii De Wild (Boraginaceae), affording superior chromatographic resolution compared with MH broth alone. The active extracts were further compared by a second TLC bioassay for compounds able to quench the luminescence of Vibrio fischerii, a bioindicator for toxicity and general biological activity.