Szx16 stereoscope

Regular light microscopy was performed using the Olympus CX41 microscope (Olympus Life Sciences, Tokyo, Japan). Colony images were photographed with a SZX16 stereoscope (Olympus Life Sciences, Tokyo, Japan). Images were acquired with an AxioCam MRm camera and processed with the software Zen pro.
Fluorescent microscopy was performed using a Zeiss Imager M2 microscope (Zeiss, Oberkochen, Germany). Images were acquired with an AxioCam MRm camera and processed with the software Zen pro. The fluorescence intensity of 50 individual cells from each strain imaged at ×63 magnification was quantified using Zen 2.6 Blue edition software (Zeiss, Oberkochen, Germany). Fluorescence intensity was quantified using the Zen Histo definition quantification software application. Each cell and its background were selected using the circular selection tool, and the average fluorescence intensity within that circle was recorded. The fluorescence intensity of the background around each cell was measured and served as a blank. The fluorescence intensity of each cell was normalized by subtracting the fluorescence intensity of the cell’s associated blank.

To determine the effectiveness of FGCs as a substrate for biofilm growth, 3 g of sterile, pebble sized (φ of − 3 to − 4, approximately 1 cm in diameter) FGCs were placed into 100 mL of sterile R2A media. This media was then inoculated with 1 mL of log phase cells, either BioTiger™, B. Thuringiensis, E. coli K-12, or the Chlorella spp. These initial samples were incubated at room temperature at 100 rpm for 1 week. After samples had become laden with cell mass a sterile solution of 80% glycerol was added bringing the overall concentration of glycerol to 20% and allowed to rest for 5 min. The FGCs were then removed from the solution and placed in a − 80 °C freezer (ThermoScientific). After freezing the samples were lyophilized (Labconco Benchtop 2.5 L Freeze dryer) and freeze-dried over a 48-h period. Samples were then stored in a refrigerator at 4 °C. Successful biofilm growth was determined by examining the samples using a SZX16 stereoscope (Olympus, Tokyo).
To determine the success of storage, 10 of the pebble sized treated FGCs were placed into 100 mL of R2A media 1, 3, 7, 14, 21, 28, 56, and 84 days after lyophilization. Growth was indicated by an increase in turbidity, OD₆₀₀, over the course of 72 h at room temperature and shaking at 100 RPM.

Zebrafish larvae (n = 60) were inoculated with each yeast and incubated as described above. Each day, zebrafish larvae were individually homogenized in sterile PBS and plated in YEPD agar supplemented with 0.05% chloramphenicol (Winkler) to obtain the yeast count (UFC/larvae). The interaction between yeast strains and the zebrafish larvae was performed through the inoculation of 5 × 10⁶ UFC/ml DTAF (Sigma) fluorescent yeast for 20 min. Observation of the larvae was performed at 0 and 5 dpi in an SZX16 stereoscope (Olympus) with a MicroPublisher 5.0 RVT camera (QImaging). Each experiment was independently performed three times.

Colony morphology was examined with a SZX16 stereoscope (Olympus). Colony images were captured with a GO-21camera and acquired using the QIMAGINE software. To determine the subcellular localization of mCherry-Crz1 or mCherry-Znf2, cells were observed with a Zeiss M2 epi-fluorescence microscope and images were acquired with the AxioCam MRm camera and processed with the software Zen 11 (Carl Zeiss Microscopy). The filter used for visualizing mCherry was the FL filter set 43 HE cy3 (Carl Zeiss Microscopy). GFP was visualized using the filter FL filter set 38 HE GFP (Carl Zeiss Microscopy). To visualize the nuclei, cells were fixed in a fixer solution (3.7% formaldehyde; 1X PBS; 1% Triton X) for 10 min and then stained with DAPI (0.4 μg/ml) for 15 min. The filter used to visualizing DAPI was FL Filter Set 49 DAPI (Carl Zeiss Microscopy).

Transmitted light images of the sugar films were collected using an Olympus BX-61 compound microscope, equipped with a 20x, 0.50 NA objective (UPlan FL N), and a tungsten-halogen light source. Image capture was via an Olympus DP72 color cooled CCD camera, at 2,048 × 1,536 pixels. Polarized light images were captured with this same configuration using the Olympus U-PO accessories. Macroscopic transmitted-light images of the sugar films were collected using an Olympus SZX-16 stereoscope, using a 1X, 0.15NA objective (SDFPLAPO1 x PF), and an Olympus DP71 cooled color CCD camera, at 2,048 × 1,536 pixels.