Oxford ct1500 cryostation

Wild-type, N402, and ΔtppA were grown for 1 week on AMM. Margins of colonies containing conidiophores were excised with a surgical blade and carefully transferred into a copper cup (diameter 10 mm, height 8 mm). Dislodging during snap freezing was prevented by gluing agar blocks in the copper cup with frozen tissue medium (KP-Cryoblock, Klinipath, Duiven, the Netherlands). The sample was snap-frozen in nitrogen slurry and immediately transferred in a vacuum transfer device to an Oxford CT1500 Cryostation attached to a JEOL 5600LV scanning electron microscope (JEOL, Tokyo, Japan). Electron micrographs were acquired from uncoated frozen samples, or after sputter-coating with gold three times during 30 s. Micrographs of uncoated samples were taken at an acceleration voltage of 2.5 kV, and consisted of 30 averaged fast scans (SCAN 2 mode). Coated samples were observed at 5 kV using F4 scans.

Small rectangular 5- by 5-mm blocks of agar medium topped with a filter were transferred to copper cups for snap-freezing in nitrogen slush. They were glued to the copper surface with frozen tissue medium (KP-Cryoblock; Klinipath, Duiven, The Netherlands) and sputter coated 3 times for 1 min each by using a gold target. Cryo-scanning electron microscopy (cryo-SEM) was done with a JEOL 5600LV scanning electron microscope (JEOL, Tokyo, Japan) equipped with an Oxford CT1500 Cryostation. Electron micrographs were taken at an acceleration voltage of 5 kV.