Transmission Electron Microscopy of Pichia pastoris

EM of P. pastoris cells was performed as previously described (Levi et al., 2010 (link)). Briefly, a 50-ml culture of yeast cells was grown in rich glucose medium to an OD₆₀₀ of ∼0.5. The culture was concentrated to a volume of <5 ml with a bottle-top vacuum filter and was fixed by adding 40 ml of ice-cold 50 mM KPi, pH 6.8, 1 mM MgCl₂, and 2% glutaraldehyde and leaving on ice for 1 h. The cells were washed repeatedly and then resuspended in 0.75 ml of 4% KMnO₄ and mixed for 1 h at room temperature. The cells were washed, resuspended in 0.75 ml of 2% uranyl acetate, and mixed for 1 h at room temperature. Finally, the cells were washed and dehydrated and embedded in Spurr’s resin. The resin was polymerized for 2 d at 68°C. Thin sections were stained with uranyl acetate and lead citrate and were viewed with a JEOL 100CX II electron microscope.