Colorimetric Method for Identifying Plant Essential Oil Components That Affect Biofilm Formation and Structure

Abstract

The specific biofilm formation (SBF) assay, a technique based on crystal violet staining, was developed to locate plant essential oils and their components that affect biofilm formation. SBF analysis determined that cinnamon, cassia, and citronella oils differentially affected growth-normalized biofilm formation by Escherichia coli. Examination of the corresponding essential oil principal components by the SBF assay revealed that cinnamaldehyde decreased biofilm formation compared to biofilms grown in Luria-Bertani broth, eugenol did not result in a change, and citronellol increased the SBF. To evaluate these results, two microscopy-based assays were employed. First, confocal laser scanning microscopy (CLSM) was used to examine E. coli biofilms cultivated in flow cells, which were quantitatively analyzed by COMSTAT, an image analysis program. The overall trend for five parameters that characterize biofilm development corroborated the findings of the SBF assay. Second, the results of an assay measuring growth-normalized adhesion by direct microscopy concurred with the results of the SBF assay and CLSM imaging. Viability staining indicated that there was reduced toxicity of the essential oil components to cells in biofilms compared to the toxicity to planktonic cells but revealed morphological damage to E. coli after cinnamaldehyde exposure. Cinnamaldehyde also inhibited the swimming motility of E. coli. SBF analysis of three Pseudomonas species exposed to cinnamaldehyde, eugenol, or citronellol revealed diverse responses. The SBF assay could be useful as an initial step for finding plant essential oils and their components that affect biofilm formation and structure.Selected natural products that originate in plants can influence microbial biofilm formation. For example, halogenated furanones, a class of compounds that inhibit biofilm formation by interfering with bacterial quorum sensing, were identified in a marine alga and are thought to have evolved to reduce biofouling (34). Other plant-derived compounds inhibit peptidoglycan synthesis (24), damage microbial membrane structures (10), modify bacterial membrane surface hydrophobicity (35), and modulate quorum sensing (14), all of which could influence biofilm formation. Terrestrial plants also support populations of surface-attached bacteria (3, 23) and could potentially produce phytochemicals that attenuate biofilm development through specific mechanisms. However, many plant essential oils, which are mixtures of numerous organic chemicals, contain compounds that inhibit microbial growth (2,8,32). Thus, a screening procedure to identify phytochemicals with specific antibiofilm activity must take into account the cytotoxicity of plant essential oils.Crystal violet (CV) staining, a colorimetric method, has been used widely to measure biofilm formation in part because of its amenability to large screening procedures (25, 26). For many applications, particularly screening assays for surface adhesion-deficient mutants (25,27), measuring the absolute amount of biofi...

Gold nanoparticles enhance methylene blue– induced photodynamic therapy: a novel therapeutic approach to inhibit Candida albicans biofilm

“…47 Crystal violet dye was used to measure percent inhibition on biofilm formation in the presence of different compounds because it affects diffusion as well as morphological and physiological differences in individual cells that influence dye binding. 48 formation after 24 hours' incubation, recorded as 63.2% and 82.2% by MB (20 µg/mL) and GNP-MB (20 µg/mL; MB) conjugate, respectively ( Figure 6). We used another tetrazolium salt (XTT) reduction assay for quantitative measurement of Candida biofilm formation.…”

Measurement of biofilm formation by clinical isolates ofEscherichia coliis method-dependent

“…The extent of biofilm formation was determined by applying three different formulas: (i) BF = AB ) CW, where BF is the biofilm formation, AB is the OD 540nm of stained attached bacteria and CW is the OD 540nm of stained control wells containing bacteria-free medium only (unspecific or abiotic factors) (Kadurugamuwa et al 2003); (ii) BF = AB ⁄ CW (Soto et al 2006); and (iii) SBF = (AB ) CW) ⁄ G in which SBF is the Specific Biofilm Formation index and G is the OD 620nm of cells growth in suspended culture (Niu and Gilbert 2004).…”

Potential Use of Cinnamomum burmanii Essential Oil-based Chewing Gum as Oral Antibiofilm Agent

“…Pohon kayu manis merupakan tumbuhan asli Asia Selatan, Asia Tenggara dan daratan Cina, Indonesia termasuk didalamnya. 3 Cinnamomum burmanii merupakan jenis kayu manis yang berasal dari Indonesia. Tanaman ini umumnya diusahakan oleh rakyat dan daerah penghasil utamanya adalah Sumatera Barat, Jambi, Sumatera Utara.…”