Calcofluor

Candida albicans cells were also evaluated in terms of filamentous forms development. For that, at 6, 10 and 24 h, aliquots of each cellular suspension were diluted in PBS and the filaments were counted in an optical microscope using a Neubauer chamber. The results were presented as percentage of filamentous forms. In parallel, the morphology of cells was confirmed through fluorescence microscope (Olympus BX51 coupled with a DP71 digital camera; Olympus, Tokyo, Japan), after staining the cells with 1% (v/v) of calcofluor (Sigma-Aldrich, EUA) for 15 min in the dark. The excitation line 405 and the emission filters BA 430–470 (blue channel) were used, and images were acquired with the program FluoView FV100 (Olympus). The length of the filaments was also determined using the ImageJ Plug-in (Maryland, USA) software. All experiments were performed in triplicate and in a minimum of three independent assays.

3,3’-dihexyloxacarbocyanine iodide (DiOC₆) (Thermo Fisher Scientific) staining was carried out as previously described (Hughes and Gottschling, 2012 (link)). Briefly, 2 x 10⁶ log-phase cells were washed once in 10 mM HEPES, pH 7.6 + 5% glucose and then resuspended in 1 ml of the same buffer containing 175 nM DiOC₆. Cells were then incubated for 15 min at RT, followed by two washes with 10 mM HEPES, pH 7.6 + 5% glucose. Cells were resuspended in 10 mM HEPES, pH 7.6 + 5% glucose for imaging. Tetramethylrhodamine methyl ester (TMRM) (Thermo Fisher Scientific) staining was carried out exactly as DiOC₆ staining, except that cells were incubated with 50 nM TMRM.
For aging experiments, cell age was determined by calcofluor (Sigma-Aldrich) staining of bud scars. For this analysis, 5 μg/ml calcofluor was included in the first post-staining wash step prior to imaging. For each experiment, cells were grouped into 3 categories based on age range: Young (0–4 budscars); middle-aged (7–12); and old (>17).

Protoplasts were incubated in regeneration medium containing 0.001% Calcofluor (SIGMA) for 5 min and then washed once in regeneration medium. Calcofluor signals were observed under a confocal laser scanning microscope (FV-1000-D, Olympus) using a ×100 oil immersion objective lens (NA = 1.30). Fluorescent images were acquired with a 405 nm laser, a DM405/473/559/635 dichroic mirror and a 430–455 nm emission filter. Serial optical sectional images from the top to the center of the protoplast were acquired at 0.5 μm resolution. Fifty protoplast images were acquired per experiment and were repeated three times. Then, the following image analysis was performed using the image in which the regenerated cell wall was observed.

The ability of the E. coli strains from this study to express curli and cellulose phenotypic were evaluated according to [20 (link)], with some modifications. One colony was seeded as a spot culture on LB agar without NaCl, supplemented with 100 mg/L of Congo Red (Sigma-Aldrich, Saint Louis, MO, USA) and incubated aerobically at 37 °C for 48 h. After growth, the isolates were classified according to their morphology into four groups: rdar (red, dry and rough—curli expression and cellulose production); pdar (pink, dry and rough—cellulose production only); bdar (brown, dry and rough—curli expression only); and saw (smooth and white—no curli and cellulose expression) [21 (link)].
Cellulose production was characterized by streaking isolates onto LB plates containing 200 mg/L of calcofluor (fluorescent brightener 28, Sigma-Aldrich, Saint Louis, MO, USA) and incubation at 37 °C for 48 h. Cellulose production was judged by the presence of fluorescence under UV light (366 nm). Isolates that showed fluorescence were considered to be cellulose producers. The phenotypic expression of curli and cellulose was performed in biological and technical duplicate assays.

Free hand sections were taken with a sterile razor blade from surface sterilized ovaries or fruits. Slices were placed on detached leaves of cucumber or butternut gourd to allow infection. Other slices were boiled in ethanol for 10 minutes, placed for 24 h in basic aniline blue solution (0.05%, pH 8.9) at 4°C, stained with 0.01% calcofluor (Sigma), and examined with Olympus A70 epifluorescent microscope for the presence of sporangia and mycelia [10] . A similar procedure was employed to embryos taken from mature seeds.

The yeast cells were cultivated for 3 days at 37°C with shaking. The cells (10⁶ viable cells/mL) were fixed with 3.7% paraformaldehyde for 15 min, centrifuged at 3,000× g for 15 min, and resuspended again in 3.7% paraformaldehyde. After centrifugation, the cells were resuspended in PBS buffer with 0.05% Tween and 1% BSA (PBS-TB), containing the IgY polyclonal antibodies against anti-rPCN_full or anti-PCN_prep for 1 h at RT. Cells were then washed three times with PBS, and incubated for 1 h at RT with neutravidin AlexaFluor_488-conjugate (Life Technologies, Carlsbad, CA, USA) in PBS-TB for 1 h at RT in the dark. After 40 min, 10 µg/mL of calcofluor (Sigma-Aldrich) was added for the remaining 20 min of incubation. After washing with PBS, coverslips were placed onto the glass slides with Permount (Fisher Scientific, Pittsburgh, PA, USA) and fluorescence was analyzed using a fluorescence microscope. The slices were analyzed using a laser scanning confocal microscope (LSM-510 NLO; Carl Zeiss, Jena, Germany) using 40× NA 1.3 and 63× NA 1.4 plan apochromatic objectives. For phase-contrast microscopy studies, the samples were observed with an immunofluorescence microscope (DMI 4000B; Leica, Culver City, CA, USA). Z-series were processed with ImageJ (National Institutes of Health, Bethesda, MD, USA).

Cellulose production was detected by growing bacteria on LB agar supplemented with 200 μg/mL calcofluor (Sigma-Aldrich, Milan, Italy). Plates were incubated at 37 °C for 2–4 days. Colonies were visualized under a 366-nm light source [40 (link)]. Congo red binding was detected by growing bacteria on LB agar supplemented with Congo red (40 μg/mL; Sigma-Aldrich, Milan, Italy).