Preparation of the Yeast Pichia pastoris for Transmission Electron Microscopy.

The methylotropic yeast Pichia pastoris is a model organism for the study of autophagy and peroxisome biogenesis. Being able to look at the organism via transmission electron microscopy (TEM) can yield valuable data on the morphology of the secretory pathway and many other organelles of interest. However, preparing the yeast for TEM work can be very arduous and costly. One of the reasons P. pastoris is so hard to prepare for visualization is because its cell wall is very thick and tough compared to the membrane of a mammalian cell. Thus, P. pastoris is notoriously difficult to infiltrate with fixatives, a step necessary to maintain its ultrastructure. This article outlines an efficient and cost effective way to prepare P. pastoris for TEM without the need for certain specialized equipment. With this protocol, excellent pictures can be obtained by using the buffers, KMnO4, sorbitol, and PIPES, along with glutaraldehyde. These components preserve the ultrastructure of the yeast without any apparent artifactual change in morphology