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The MIC values of CBD against C. albicans were determined using the twofold serial microdilution method based on the CLSI protocol . The fungi were incubated with various concentrations of CBD (3.25–400 µg/mL) in RPMI in 96-well plates for 24 h at 37 °C. The MIC was determined as the lowest concentration of CBD showing no turbidity after 24 h, where turbidity was interpreted as visible fungal growth. In addition, the metabolic activity of the planktonic fungi was determined quantitatively using a standard 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reduction assay (Calbiochem, Burlington, MA, USA) in an Infinite M200 PRO plate reader (Tecan Group Ltd., Männedorf, Switzerland). The assay was performed in triplicates.
The fungus Candida albicans is a commensal microorganism found in various sites of the human body such as the oral cavity, vagina, and the lungs. It is the most common fungal pathogen in humans, often associated with serious invasive mucosal and systemic infections. One of the most important virulence properties of C. albicans is attributed to biofilm formation on either biotic or abiotic surfaces [1,2,3,4]. Formation of biofilm is a complex process that involves attachment to the surface, colonization, and maturation. Biofilms consist of highly structured multilayer network of various cell types: yeast-form cells, pseudohyphal cells, and hyphal cells enwrapped in a protective extracellular matrix [5,6,7,8]. The matrix components include glycoproteins, carbohydrates, lipids, nucleic acids and exopolysaccharides (EPS) .
2.2. Fungal Strains and Growth Conditions
CBD powder ( Figure 1 ) (purity 99.4%, CAS Number: 13956-29-1) was purchased from NC labs (Czech Republic) and prepared as 10 mg/mL stock solution in ethanol.
A total of 10 µL of overnight grown C. albicans inoculum exposed to CBD in 96-well plate as described above was plated on PDA and incubated at 37 °C for 3–4 days. Untreated fungi served as positive control. No colonies appearance on agar plates indicates fungicidal effect of CBD. The assay was performed in triplicates.