Analytical Studies on Regeneration of Protoplasts of Geotrichum candidum by Quantitative Thin-Layer-Agar Plating

Abstract

A preparation of pure protoplasts of Geotrichum candidum became osmotically stable and colonies developed when the protoplasts were embedded in stabilizing thin-layer-agar and incubated with stabilizing basal medium. When growing protoplasts were exposed to distilled water and then reincubated with basal medium, the process of regeneration of protoplasts could be quantitatively demonstrated by counting colonies. The process was divided into three phases, lag, logarithmic, and stationary. Furthermore, the state of regeneration of protoplasts at each phase could be seen in detail by microscopic studies of protoplasts under similar growth conditions. In the lag phase, which lasted for 2 hr after inoculation, protoplasts were completely destroyed when placed in distilled water. During the logarithmic phase, from 2 to 5 hr after inoculation, protoplasts rapidly became osmotically stable and about 18% of them were growing. In the stationary phase, most protoplasts developed germ tubes within 2 hr. These results suggested that there are two main phases, although individual cells passed through three different conditions, osmotically labile, osmotically stable, and growing. No apparent structure of cell wall material could be detected by electron microscopy on the surface of the membrane of these osmotically stable cells. This suspension was contaminated with a large amount of ghosts and partially digested cells (Fig.

Magnesium‐Stimulated Resynthesis of Osmoresistant Layer by Candida albicans Spheroplasts

“…The regeneration curve ( Fig. 6) and stationary phase, as described by Fukui et al [16]. Regeneration rate upto the stationary phase varied between 70 and 90% of the inoculated spheroplasts with different spheroplast preparations.…”

“…A previous paper from this laboratory reported that protoplasts of Geotrichum candidum could be regenerated quantitatively by thin-layer-agar plating and that the regeneration process had 3 phases [16] . During this regeneration process the protoplasts became resistant to osmotic shock, but there was no apparent structure of a cell wall on the surface of the membrane in the logarithmic phase, even when the protoplasts had become insensitive to osmotic shock.…”

“…Since then there had been many studies on bacterial and fungal protoplasts [1-3, 5, 8, 19, 20, 23, 25, 45, 46, 52, 53, 57]. Recent studies on those osmolabile spherical bodies had thrown much light on the mechanism of their regeneration [10,12,13,16,18,24,26,38,41,42,47,60] and on cellular physiology. On the other hand, studies on the lysis of cell wall structures by these lytic enzymes had provided information on cell wall structures of bacteria and fungi, the mode of action of lytic enzymes and the mechanism of action of antibacterial and antifungal antibiotics [4,11,29,30,31,34,36,39,48,51].…”

Regeneration of Escherichia coli Giant Protoplasts to Their Original Form

“…Among these GPs, we observed many irregular shapes during division, as well as the formation of three or more poles. The regeneration process showed a great deal of similarity to that of B acillus megaterium , a Gram-positive bacterium [22], and Geotrichum candidum , a type of fungus [20]. Identical pole formation has been observed in the protoplasts of E. coli [25], suggesting that GPs and protoplasts have similar elongation processes.…”

“…Spheroplasts and protoplasts exhibit metabolic activity, but cannot undergo cell division due to the absence of a cell wall [17,18]. When placed in media, the bacteria regenerate to their original form prior to forming colonies [19,20,21]. In addition, the process of regeneration to the original bacteria by microscopic observation has also been obtained [22].…”