N hexadecane

The microbial adhesion to hydrocarbons (MATH) assay previously described by Rosenberg (28 (link)) was used to determine the cell surface hydrophobicity of C. albicans isolates. A loop full from overnight growth on SDA at 37°C was suspended and washed twice in phosphate buffer saline (PBS) and adjusted to display OD_600nm between 0.4 and 0.5 (A_o); then 3.0 ml adjusted suspension from each isolate was overlaid with 4.0 ml of the hydrophobic hydrocarbon, n-hexadecane (Sigma Aldrich, St. Louis, MO, USA) and vortexed vigorously for 30 s and left for 10 min at 30°C to allow phases separation. The lower aqueous phase was carefully separated using a glass Pasteur pipette and transferred to a clean polystyrene tube and its OD_600nm was measured (A₁). Results were reported as an average of three independent measurements, which were calculated according to the formula hydrophobicity (%) = [1–(A1/A0)] × 100. All assays are representative of at least three independent experiments, performed in triplicate, and the reference strain C. albicans ATCC 90028 was used as the study control.

CSH was assessed using the microbial hydrocarbon adhesion assay [22 (link)]. Yeast cells were grown overnight in Sabouraud dextrose broth (SDB) at 37 °C and then harvested and washed twice with phosphate-buffered saline (PBS). A yeast cell suspension with an optical density at 600 nm (OD600) between 0.4 and 0.5 was prepared in PBS (A0). The yeast suspension (3 mL) was overlaid by 0.4 mL of the hydrophobic hydrocarbon n-hexadecane (Sigma-Aldrich, Burlington, MA, USA). Cells were vortexed for 2 min and incubated for 10 min at 30°C to allow phase separation. The aqueous phase was measured (A1) at OD600. The decrease in absorbance was used to calculate the percentage of hydrophobicity of the cell surface (% hydrophobicity): hydrophobicity (%) = [1 − (A1/A0)] × 100. CSH tests were performed in at least three independent experiments, each carried out in duplicate to obtain an average value of % CSH value.

The hydrophobicity, i.e., the ability to adhere to hydrocarbons, was assessed according to [20 (link)] with some modifications. Fresh cultures of the strains (24 h of incubation at 37 °C in MRS broth with 0.05% L-cysteine) in anaerobic conditions (GasPak System; Oxoid Ltd., Basingstoke, UK) were harvested in the stationary phase by centrifugation at 6000 rpm for 10 min. The pellet was resuspended in NaCl 0.9% isotonic solution and was subsequently diluted to the absorbance value of 1 at 560 nm using a spectrophotometer (model 6705, Jenway, Stone, UK). Then, 3 mL of the bacterial suspension was vortexed with 0.6 mL of n-hexadecane (Sigma-Aldrich, Milan, Italy) for 4 min. The two phases were allowed to separate for 1 h at 37 °C. The aqueous phase was removed, and the absorbance (A) at 560 nm was measured. Finally, the hydrophobicity percentage was calculated with the following formula: (A0 − At)/A0 × 100, where A0 represents the absorbance at time 0 and At represents the absorbance at 560 nm after 1 h of incubation at 37 °C.
Hydrophobicity was also evaluated for L. rhamnosus GG ATCC^® 53103™, a commercial probiotic strain used as reference strain.

The hydrophobicity of the yeast cell surface [H(%)] was estimated using adhesion to n-hexadecane (Sigma, USA) according to the method by Rosenberg et al^{53 (link)} with a slight modification as follows: H(%) = [(1 − OD₄)/OD₀] × 100, where OD is the optical density at 0 and 4 h.

The 2,6-DCPIP (2,6-dichlorophenol indophenol) assay was performed on Bushnell-Hass (BH) mineral medium previously described [32 (link),33 (link)] with minor modifications, as follows. The isolates were pre-cultured in Luria Bertani (LB) medium at 30°C for 48 hours and then centrifuged at 4000 xg for 5 min and washed with 0.9% sterile saline. The final bacterial suspension was adjusted to 1.0 (OD_660nm), and 100 μL was transferred to a 1.5 mL tube containing 836.5 μL of BH medium, 53.5 μL of 2,6-DCPIP solution (375 μg/mL), and 10 μL n-hexadecane (Sigma) to final concentration of 1% (v/v). The assay was incubated at 30°C on a rotary shaker at 150 rpm for 21 days and observed every 24 hours. As negative controls for this assay, incubations were performed only with: 1) BH medium and DCPIP; 2) BH medium, DCPIP and n-hexadecane; 3) culture medium, DCPIP and bacterial suspension; and 4) BH medium, DCPIP and bacterial suspension inactivated by autoclaving. The assay was considered positive for n-hexadecane degradation if color changed from blue to colorless. No change in color was considered as negative result. Mycobacterium vanbaalenii PYR1 DSMZ7251 and Escherichia coli ATCC8739 strains were used as positive and negative controls, respectively.

Cell surface hydrophobicity (CSH) was assessed using the microbial adhesion assay to hydrocarbons (MATH) (Rosenberg, 1984 (link)). Briefly, yeast cells grown overnight at 37°C, were harvested and washed twice with PBS. A yeast cell suspension displaying an OD_{600 nm} between 0.4 and 0.5 was prepared in PBS (A₀); 3 ml of this yeast suspension was overlaid by 0.4 ml of the hydrophobic hydrocarbon, n-hexadecane (Sigma-Aldrich). After vigorous vortexing, phases were allowed to separate for 10 min at 30°C and the OD_{600 nm} of the aqueous phase was measured (A₁). The percentage of hydrophobicity was calculated as follows: hydrophobicity (%) = [1−(A₁/A₀)] × 100. All assays are a representative of at least three independent experiments, performed in triplicate.

Assimilation of hydrocarbons was determined according to Satow et al. [42 (link)] and modified by Zajc et al. [3 (link)]. Liquid YNB medium (pH 7.0) was prepared without any carbon source and, after autoclaving, supplemented with 20% (v/v) mineral oil and 20% (v/v) n-hexadecane (both Sigma Aldrich, USA) as the sole carbon source. Both mineral oil and n-hexadecane were filter-sterilized before use. The test tubes were inoculated with 100 µL of cell suspension or plugs of mycelium in three replicates and incubated without shaking for one month at room temperature.