Synchronous Growth and Sporulation of Bacillus megaterium

Abstract

Filtration of late log-phase cultures of Bacillus megaterium ATCC 19213 grown on defined sucrose salts medium (SS) or SS plus glutamate medium (SSG) through nine layers of Whatman no. 40 filter paper in a fritted-glass disc Buchner funnel resulted in filtrates containing cells which showed synchronous growth and proceeded to sporulation. SS cells completed one synchronous division after filtration; sporulation ensued after the cessation of growth. SSG cells completed two synchronous divisions and sporulation occurred during the second division. A high degree of synchrony of vegetative growth of SSG cells was evident by the stepwise pattern of growth, by the doubling of cell numbers at each division, the high division index, and by the rapid formation of sporulation cell types and homogeneity of cell types in the filtered cultures when compared with asynchronous cultures. Because the described system gives both good growth and sporulation synchrony, the method should be useful in delineating early events in sporulation and their regulation.

Fine Structure of Bacillus megaterium During Synchronous Growth

“…The new cross wall begins to appear at the base of the mesosome, projecting into the cytoplasm at right angles to the old cell wall (Fig. 3, 4,5,18,19). At the same time, the space at the base of the mesosome narrows considerably, so that the only connection between the contents of the mesosome and the extracytoplasmic region is through the 30 to 60 A space which separates the plasma membrane from the cell wall ( Fig.…”

“…At this time, sporulation began and was complete by the 10th hr after inoculation. Details of growth kinetics are published elsewhere (19). Cells sectioned during the period of synchronous division were observed to be in various stages of cross wall formation and to possess a varying number of mesosomes.…”

“…Culture technique. Details of the culture procedures and the modified filtration method of Maruyama and Yanagita (25) which were used to obtain division synchrony have been previously reported (19). In the present experiments, when the culture reached late log phase (166 Klett units, measured with a no.…”

“…Although a statistical analysis is lacking, it appears from the micrographs (Fig. 3, 4, [15][16][17][18][19] that, when the cell is actively dividing, the chromatin is attached at one end to a mesosome located at or near the cell pole and at the other end to the mesosome involved in transverse septum formation. The nature of this attachment is not apparent, but, in some instances (Fig.…”

Interference Contrast and Phase Contrast Microscopy of Sporulation and Germination of Bacillus megaterium

“…Culture techniiques. For studies on the growth and sporulation of B. megaterium, a portion of a standard spore suspension (6) was heated at 70 C for 0.5 hr, inoculated into a defined sucrose salts medium (SS) (14) containing the germinants L-alanine and inosine (each 100 pg/ml; NBC), incubated at 30 C with shaking to a turbidity of 70 Klett units (about 2 X 108 cells/ml), and measured in a Klett-Summerson photoelectric colorimeter with a no. 54 filter.…”