Calcofluor fluorescent brightener 28

Yeast strain YHUM2959 carrying a wsc1∆ mutation was obtained by deletion of the WSC1 gene in yeast strain ESM356-1 (S288c, MATα, ura3-52, leu2∆1, his3∆200, trp1∆63), [33 (link)] following a previously described protocol, and plasmid pFA6a-natNT2 [34 (link)].
For growth tests under cell wall stress conditions, yeast strains ESM356-1 or YHUM2959 were freshly transformed with appropriate plasmids (Table 1). Transformants were grown to logarithmic phase in liquid SC-Trp medium, and fivefold serial dilutions of the cultures were spotted onto solid YPD medium supplemented with either 0.4 µg/mL caspofungin (Sigma-Aldrich, Taufkirchen, Germany), 30 µg/mL Congo red (Carl-Roth, Karlsruhe, Germany), 100 µg/mL calcofluor (fluorescent brightener 28, Sigma-Aldrich, Taufkirchen, Germany), or 2 mg/mL caffeine (Sigma-Aldrich, Taufkirchen, Germany). Growth on plates was documented by photography after incubation at 30 °C for 3–5 days. Standard methods for yeast culture medium and transformation were used as described previously [35 ].

Expression of curli fimbriae was assessed by examining colony morphology of K. pneumoniae when grown on Congo red agar. Congo red agar was prepared as described by Römling and Rohde (1999 (link)). In brief, MH agar was supplemented with 40 μg/mL Congo red (Sigma-Aldrich, Ireland) and 20 μg/mL Coomassie brilliant blue R-250 (Thermo Fisher Scientific, Waltham, MA). Plates were then inoculated with 3 μL of each overnight bacterial culture grown in MH broth and then incubated for 72 h at 25 and 37°C after which colony morphology was inspected and recorded. Salmonella Typhimurium ATCC™14028 was included as a control for a RDAR phenotype (Römling and Rohde, 1999 (link); Finn et al., 2013 (link)).
Calcofluor agar was used to assess these isolates for their ability to produce cellulose (Zogaj et al., 2001 (link)). In this case, the plates were prepared by supplementing MH agar with 40 μg/mL calcofluor fluorescent brightener 28 (Sigma-Aldrich). The calcofluor agar plates were inoculated with 3 μL of overnight culture and incubated for 72 h at 25 and 37°C. Salmonella Typhimurium ATCC™14028 was included as a control. The colony morphology was observed and recorded under a 366-nm UV light source to detect the binding of calcofluor to the cellulose produced. Both assays were carried out in triplicate for each isolate.

Expression of the rdar morphotype was visualized on LB without salt plates supplemented with 40 μg/ml Congo Red (Sigma) and 20 μg/ml Coomassie Brilliant Blue G‐250 (Sigma). Upon expression of cellulose and curli fibers, dye binding is staining the bacterial colony. Development of the colony morphology was investigated at 28°C and 37°C and documented by photographing at distinct time points. Strains of interest were streaked for single colonies and 5 μl of a suspension of OD₆₀₀ 5 was spotted onto the agar plates.
For visualization of cellulose expression, strains were spotted onto LB without salt plates supplemented with 50 μg/ml Calcofluor (Fluorescent Brightener 28, Sigma). Fluorescence was observed under UV light of wave length 365 nm.