Plan apochromat objective dm 100x 1.40 oil ph3

To visualize cells during exponential growth starter cultures were grown overnight and then diluted 1∶100 into fresh medium and allowed to achieve at least three doublings before observation. Cells were mounted on ∼1.2% agar pads (0.25× minimal medium base) and a 0.13–0.17 mm glass coverslip (VWR) was placed on top. To visualize individual cells the cell membrane was stained with either 2 µg/ml Nile Red (Sigma) or 0.4 µg/ml FM5-95 (Molecular Probes). To visualize nucleoids DNA was stained with 2 µg/ml 4′-6-diamidino-2-phenylindole (DAPI) (Sigma). Microscopy was performed on an inverted epifluorescence microscope (Nikon Ti) fitted with a Plan-Apochromat objective (Nikon DM 100x/1.40 Oil Ph3). Light was transmitted from a 300 Watt xenon arc-lamp through a liquid light guide (Sutter Instruments) and images were collected using a CoolSnap HQ² cooled CCD camera (Photometrics). All filters were Modified Magnetron ET Sets from Chroma and details are available upon request. Digital images were acquired and analysed using METAMORPH software (version V.6.2r6). Analysis was performed using ImageJ software: foci counting utilized the particle analysis plugin; cell lengths and widths were measured using the ObjectJ plugin.

To visualize cells during the exponential growth phase, starter cultures were grown overnight in transformation medium supplemented with xylose and then diluted 1:100 into transformation medium supplemented with xylose, 0.1% glutamate and 0.2% casein hydrolysate. Cultures were allowed to achieve at least three doublings before observation. To visualize spores, strains were grown in Schaeffer's medium supplemented with xylose and grown at 37°C for 48 h.
Cells were mounted on ~1.4% agar pads (in sterile ultrapure water) and a 0.13‐ to 0.17‐mm glass coverslip (VWR) was placed on top. Microscopy was performed on an inverted epifluorescence microscope (Nikon Ti) fitted with a Plan Apochromat Objective (Nikon DM 100x/1.40 Oil Ph3). Light was transmitted from a 300 Watt xenon arc lamp through a liquid light guide (Sutter Instruments), and images were collected using a Prime CMOS camera (Photometrics). The GFP filter set was from Chroma: ET470/40x (EM), T495Ipxr (BS) and ET525/50m (EM). Digital images were acquired using METAMORPH software (version 7.7) and analysed using Fiji software (Schindelin et al, 2012). All experiments were independently performed at least twice, and representative data are shown.