Crystal violet

Pseudomonas putida (DSM 291), Pseudomonas moorei (DSM 12647), Sphingomonas mali (DSM 10565) and Bacillus subtilis (DSM 3657) were obtained from the German Collection of Microorganisms and Cell Cultures GmbH (Germany). Methylisothiazolinone, chloroxylenol, 3-indoleacetic acid, iron(III) chloride, phospholipid standards and ammonium format were purchased from Merck (Poland). SYTOX™ Green Nucleic Acid Stain, General Oxidative Stress Indicator (CM-H₂DCFDA, 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester) and AlamarBlue Cell Viability Reagent were obtained from Thermo Fisher Scientific (Poland). Difco™ nutrient broth and Mueller Hinton broth were purchased from Becton Dickinson (Poland). DMSO and phosphate-buffered saline (PBS) were obtained from BioShop (Canada). Methanol, ethyl acetate, hydrochloric acid, acetic acid, crystal violet and sodium sulfate anhydrous were obtained from Chempur (Poland). All of the other reagents with a high analytical purity grade were purchased from Avantor (Poland).

Five fluconazole-resistant C. albicans clinical isolates (Table 2) were selected to compare biofilm formation on voice prostheses (i) impregnated with a 10% solution of ceragenins in isopropyl alcohol (the CSA-impregnated group), (ii) incubated with isopropyl alcohol only (the alcohol-impregnated group), (iii) non-impregnated but treated with free ceragenins at a concentration of 2 × MBIC value (the CSA-treated group) (Table 2), and (iv) using non-impregnated and non-ceragenin-treated ones as the control group.
The biofilm cultures were carried out in 96-well microtiter plates in 200 µL of RPMI medium (Sigma-Aldrich, Saint Louis, MO, USA) under aerobic conditions for 48 h at 37 °C. After incubation, the planktonic cells were carefully removed and the biofilms were washed twice with PBS, dried at room temperature, and stained for 15 min using 150 µL of 0.1% crystal violet (Chempur, Poland). Next, the excess of stain was removed, and the biofilms were rinsed with deionized water and the plates left to dry. To solubilize the crystal violet, 100 µL of 98% ethanol was added to each well, and the optical density (OD) was determined at a wavelength of 570 nm using the Varioscan Lux microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) to estimate the number of bacteria in a biofilm.