This chapter describes techniques used in the study of the vacuole and focuses on the procedures used in the laboratories. The yeast vacuole is a large, dynamic organelle that can be detected by fight microscopy. It is most prominent when cells are viewed using differential interference-contrast optics. Reliable detection of vacuoles using vital staining with fluorescent dyes in conjunction with fluorescence microscopy is described in the chapter. Several methods are available for purification of the yeast vacuole, all of which take advantage of the low buoyant density of the vacuole. The method of choice is a modified version of the procedure used by involves enzymatic removal of the cell walls, osmotic lysis of the spheroplasts, followed by flotation of the vacuoles on a discontinuous Ficoll gradient. Other methods use polybase induced lysis of spheroplasts under isotonic conditions followed by density gradient centrifugation. The procedure that follows has consistently given 10–20% yield of vacuoles and low contamination of other organelles.
ASJC Scopus subject areas
- Molecular Biology