Microscopic Cell and Stalk Characterization

Cells were spotted onto 1% agarose pads made in the corresponding growth medium. Phase microscopy was performed on a Nikon TiE inverted microscope equipped with a Prior Lumen 220PRO illumination system, Zyla sCMOS 5.5-megapixel camera, CFI Plan Apochromat 100× oil immersion objective (numerical aperture [NA] of 1.45 and working distance [WD] of 0.13 mm), and NIS Elements software for image acquisition. Cell and stalk dimensions were measured using Morphometrics (41 (link)) and ImageJ v. 1.48q (NIH), respectively. To measure membrane permeability, cells were grown in the presence of 1 µg/ml of propidium iodide. EPS production was assessed as previously described (29 (link)). Briefly, 500 microliters of cells grown in HIGG–1 µM phosphate were collected by centrifugation (14,000 × g, 5 min), and the pellet was resuspended in 30 µl of 0.5× phosphate-buffered saline (PBS). Ten microliters of the cell suspension was mixed with 5 µl of FITC-dextran (10 mg/ml; molecular weight [MW], 2 MDa; Sigma) and 1 µl of SlowFade Diamond mountant (Thermo Scientific). Two microliters of this mixture was spotted onto a glass slide, coverslipped, and sealed with vaseline-lanolin-paraffin (VALAP; 1:1:1) for imaging.