Aims:The minimum inhibitory concentration (MIC) of oregano essential oil (OEO) and two of its principle components, i.e. thymol and carvacrol, against Pseudomonas aeruginosa and Staphylococcus aureus was assessed by using an innovative technique. The mechanism of action of the above substances was also investigated. Methods and Results: The applied technique uses 100-well microtitre plate and collects turbidimetric growth data. To produce the inhibition pro®les, a wide range of concentrations were tested for each of the three compounds, as well as for carvacrol±thymol mixtures. Following a speci®c mathematical analysis of the observed inhibition pro®les from all compounds, it was suggested that mixtures of carvacrol and thymol gave an additive effect and that the overall inhibition by OEO can be attributed mainly to the additive antimicrobial action of these two compounds. Addition of low amounts of each additive: (a) increased permeability of cells to the nuclear stain EB, (b) dissipated pH gradients as indicated by the CFDA-SĒ uorescent probe irrespective of glucose availability and (c) caused leakage of inorganic ions. Conclusions: Mixing carvacrol and thymol at proper amounts may exert the total inhibition that is evident by oregano essential oil. Such inhibition is due to damage in membrane integrity, which further affects pH homeostasis and equilibrium of inorganic ions. Signi®cance and Impact of the Study: The knowledge of extent and mode of inhibition of speci®c compounds, which are present in plant extracts, may contribute to the successful application of such natural preservatives in foods, since certain combinations of carvacrol± thymol provide as high inhibition as oregano essential oil with a smaller¯avour impact.

Biofilms are widespread in nature and constitute an important strategy implemented by microorganisms to survive in sometimes harsh environmental conditions. They can be beneficial or have a negative impact particularly when formed in industrial settings or on medical devices. As such, research into the formation and elimination of biofilms is important for many disciplines. Several new methodologies have been recently developed for, or adapted to, biofilm studies that have contributed to deeper knowledge on biofilm physiology, structure and composition. In this review, traditional and cutting-edge methods to study biofilm biomass, viability, structure, composition and physiology are addressed. Moreover, as there is a lack of consensus among the diversity of techniques used to grow and study biofilms. This review intends to remedy this, by giving a critical perspective, highlighting the advantages and limitations of several methods. Accordingly, this review aims at helping scientists in finding the most appropriate and up-to-date methods to study their biofilms. ARTICLE HISTORY

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