Improved Assays to Identify the Antibiotic Effects on Planktonic and Sessile Bacteria Using the Example of 1.8-Cineol

Screening of antibiotic substances is a mandatory working step during drug development. A variety of methods are available to test their efficiency, they can be divided into diffusion and dilution methods. Diffusion methods in agar based media are rather qualitative approaches, whereas dilution methods, commonly executed in polystyrene microtiter plates, are frequently used to determine the minimal inhibitory concentration (MIC) and the minimal biofilm inhibitory concentration (MBIC50) in a quantitative way. During these standardized assays the physical properties of the agent, e.g. its hydrophobic properties and thermal instability, are often neglected. This study compares different diffusion assays for their sensitivity and improved dilution assays in respect to the thermal sensitivity and the hydrophobic character of antibiotics. We applied 1.8-cineol, a hydrophobic antibacterial component of essential oils, on the pathogen Staphylococcus aureus and investigated the influence of incubation time, cell culture vessels and commonly employed surfactants on the assay. The presented study describes an optimized diffusion assay and a protocol for the exact determination of the MIC and MBIC50 of thermally instable hydrophobic antibiotic substances. Our assays can be easily executed since they are based on optical density measurements and simple crystal violet staining. We conclude that preliminary screenings of hydrophobic substances can be executed by the well diffusion method. However, for the determination of the MIC and MBIC50 we highly recommend the application of cleaned and etched glass tubes instead of polystyrene cell culture plates. The usage of the surfactants Tween 80 or Tween 20 was found unnecessary and furthermore falsifying the results. Taken together our improved standard techniques may help to better quantify the antimicrobial potential of hydrophobic antibiotics, e.g. essential oils. This may give new insights into the mode of action and furthermore enable the development of new antimicrobial substances urgently needed to fight resistances against common antibiotics.