Time-Lapse Imaging of Cell Division and Holdfast Formation

Cell division and holdfast synthesis timing were observed in live cells on agarose pads by time-lapse microscopy as described previously (Hoffman et al., 2015 (link)) with some modifications. A 1 µl aliquot of exponential-phase cells (OD₆₀₀ of 0.4 – 0.8) was placed on top of a pad containing 0.8% agarose in M2G or PYE and AF488-WGA (0.5 µg/ml final) sealed under a coverslip with valap. Time-lapse microscopy images were taken every 2 min for 8–12 h using a Nikon Eclipse 90i with a 100 x DIC oil immersion objective, a Nikon 83300 filter cube T-D-F and a Photometrics Cascade K1 camera, or an inverted Nikon Ti-E microscope and a Plan Apo 60X objective, a GFP/DsRed filter cube, and an Andor iXon3 DU885 EM CCD camera. Time-lapse movies were visualized in ImageJ (Schneider et al., 2012 (link)) to manually assess the time of cell division (t₀), as t = 0 when a cell newly divides, and the time of holdfast production (t_H), as the first frame where the fluorescent lectin signal is detected.