J. Gen. Appl. Microbiol., 57, 177‒182 (2011)

In the past three decades, several methods have been reported for the growth of yeast spheroplasts in liquid culture. When spheroplasts of Saccharomyces cerevisiae were inoculated in rich medium containing 0.4 M KCl as an osmotic stabilizer and cultured for 7‒8 h with occasional manual shaking, they entered the fi rst round of mitosis at low cell density. Spheroplasts inoculated at higher cell density showed signifi cant retardation of mitosis (Doi and Doi, 1979, 1982). Doi and Doi (1982) further showed that spheroplasts underwent the fi rst round of DNA synthesis at roughly the same time as normal cells, but underwent the fi rst round of nuclear division signifi cantly later than normal cells. Spheroplasts cultured in YPG (1% yeast extract, 2% peptone, 2% glucose) medium supplemented with 1 M sorbitol and zymolyase for 18 h increased their diameter threefold, resulting in giant spheroplasts with an average diameter of 13.4 μm (Tamai et al., 1983). Our previous study showed that mitotic nuclear division could be visualized more clearly in giant spheroplasts than in whole cells by DAPI staining and immunofl uorescence microscopy using anti-tubulin antibody (Sando et al., 1987; Miyakawa, 2000). The formation of giant spheroplasts of microbial cells overcomes the technical problems that are otherwise encountered owing to their small size. Giant spheroplasts of E. coli were successfully generated by spheroplast incubation methods (SI methods) to measure membrane potentials of plasma membrane by a patch-clamp method (Kuroda et al., 1998). Yabe et al. J. Gen. Appl. Microbiol., 57, 177‒182 (2011)