Biofilm formation is a major pathogenicity strategy ofBacteria employ a variety of strategies to escape killing by antibiotics, including mutation, phenotypic variation, and change to a biofilm growth mode 1-3 . One form of phenotypic variation, known as persistence, is characterised by the presence of a subset of antibiotic-tolerant cells within a bacterial population. Persister cells pre-exist in most bacterial populations, including cultures at mid-log phase, stationary phase, and in biofilms [4][5][6] . The prevalence of persister cells in a population depends on its growth mode, the age of inocula, strain background, growth medium, and time course chosen for selection [6][7][8] . Persister cells display heterogeneity in growth rates and tolerance to various antibiotics 6,9,10 , though multidrug tolerance is not a consistent trait 11 . Transcriptome analysis suggests that persister cells have reduced expression of genes involved in metabolic pathways, biosynthesis pathways, and energy production 12,13 , which often leads to a dormancy status of cells. However, being entirely dormant is not necessarily a prerequisite for the formation of persister subpopulations [14][15][16] .In clinical settings, bacteria grow predominantly as biofilms, following attachment and accumulation on biotic or abiotic surfaces; and they present group dynamics [17][18][19] . Bacteria within biofilms are highly tolerant to antibiotics, but the exact mechanisms behind this tolerance are complex and no single factor can fully account for this specific trait [20][21][22] . Persister cells are more prevalent in biofilms than in log-planktonic cultures and are thought to be responsible for the recalcitrance of many chronic infections, such as cystic fibrosis and chronic wound infections,
Background: The ability of the human fungal pathogen Candida albicans to form biofilms, for example on indwelling medical devices, is a major pathogenic mechanism and has been the focus of intense studies in the fungal pathogenesis field. A key research tool used is the quantitative methods for measuring biofilm formation of C. albicans. Objective: We sought to optimize the conventional crystal violet (CV) staining assay for quantification of biofilm formation by C. albicans and evaluate its performance. Methods: Individual modifications included (i) submerge-washing of microplates to remove non-adherent cells, (ii) heat-fixation, (iii) short-term staining for 3 min, (iv) submerge-washing to remove unbound CV dye, and (v) short-term destaining for 15 min were compared with the standard procedure, and those were superior were incorporated. Performance analysis was carried out for the modified CV assay, in comparison to the conventional CV assay and the XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide] reduction assay. Results: The modified CV assay demonstrated several advantages in quantitative assessment of biofilm formation of C. albicans over the conventional CV assay, including greater accuracy and reproducibility, shorter experimental time and reduced labor intensity, and was at least comparable to the XTT reduction assay.Conclusion: The modified CV method can be used as an alternative to the XTT reduction assay in quantification of biofilm growth by C. albicans.
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