| Scanning Electron Microscopy (SEM) with Morphology of yeasts | ||||||||
|---|---|---|---|---|---|---|---|---|
| C. albicans | C. tropicalis | Non-parametrical statistical analysis Inter-species | ||||||
| Assays | n a | Mean size of the yeast cell area, µm2 (SD) d |
Wilcoxon test p-value |
Mean size of the yeast cell area, µm2 (SD) d |
Wilcoxon test p-value |
Wilcoxon test p-value |
||
| 48h | 3 | 1.80E-02 (3.00E-03) |
2.22E-01 | 1.00E-02 (2.00E-03) |
4.44E-01 | 3.05E-05 | ||
| 72h | 3 | 1.60E-02 (2.00E-03) |
1.10E-02 (2.00E-03) |
3.00E-05 | ||||
48 and 72h time samples were selected to compare the total cell counts, number of live/ dead cells, as well as biofilm structure and cell area for each Candida species using epifluorescence microscopy (EM) and scanning electron microscopy (SEM) analysis, respectively. In EM, the percentages of dead and alive cells within images were measured through ImageJ by Fiji version 1.57 (Schindelin et al., 2012) using the macros Biofilms Viability checker (see methods); while, the total cells counting in DAPI images were processed by a sequence of modules forming a pipeline in Cell Profiler software (Mcquin et al., 2018), which the applied pipeline can be revised in the Supplemental Material. DAPI images were used to obtain total cells per image and the average was then calculated as the mean of yeasts per cm2. Morphology of the yeast was also obtained from the best images by Fiji ImageJ version 1.57 (Schindelin et al., 2012). Wilcoxon test was applied to evaluate statistical differences between 48 and 72h samples in each Candida species (Intraspecies). In non-parametrical statistical analysis (Interspecies), the Wilcoxon test was calculated using the results between C. albicans and C. tropicalis at 48 and 7h samples. aNumber of assays realized on different days; in each assay, we collected at least 12 photographs for cell counting. bAverage of Candida cells obtained by pictures from triplicate assays and their standard deviation (SD). cThe estimation of yeast/cm2 calculated by the following formula: average of Candida cells (SD)* (1E+08/12880). dThe mean of yeast area (µm2) measured through the average area of Candida cells obtained by each picture in SEM analysis and their standard deviation (SD).