60x n a 1.4 oil

An in vitro hatching assay was prepared as described above using S. aureus-GFP. After incubating the plate at 37°C, eggs with bacteria were fixed using 4% PFA, 0.5% glutaraldehyde in PBS at room temperature for 1 hr. Plate contents were then transferred to 1.5 ml Eppendorf tubes. Eggs in this mixture were pelleted by centrifugation at 1000 rpm for 5 mins with a slow brake speed (4). Samples were then washed twice with PBS and then resuspended in PBS for imaging.
Fixed samples were imaged on 35 mm Petri dishes with No. 1.5 coverglass at the bottom using a Nikon 60x N.A. 1.4 oil immersion objective lens on a Nikon Eclipse Ti microscope. Number of puncta on the sides and on the poles were enumerated by eye.

2 μl of purified A. algerae spores obtained from H. zea (6x10⁷ spores/ml) or purified E. hellem and E. intestinalis spores from tissue culture (~10⁸ spores/ml) were mixed with 10 μl of germination buffer. The reaction was placed on ice to prevent PT firing prior to imaging. 2 μl was placed on a poly-L-lysine-coated glass slide (Fisher Scientific, catalog #12-545-78) and sealed with a #1.5 18 x 18 mm coverslip (Fisher Scientific, catalog #12-519-21A). Polar tube firing typically occurred ~2–5 minutes after mixing the spores with the germination buffer. PT firing was imaged using a Nikon Eclipse Ti microscope with a Nikon 60x N.A. 1.4 oil immersion Plan Apochromat Ph3 phase-contrast objective lens. An Andor Zyla 5.5 megapixel sCMOS camera was used, which provided a wide field of view at 14–50 frames per second with 3–35 ms exposure time, no binning was applied. The microscope was equipped with an environmental chamber which was set at 30°C for A. algerae[24 (link)] and 37°C for Encephalitozoon species[54 (link)].