Estimation of the point in time for development of maximal natural competence of B. subtilis 1012. A) Growth curve of B. subtilis 1012 which was grown under vigorous shaking at 37°C in HS medium at 37°C. After 5 h 15 min at an optical density of 5, the culture reached the early stationary phase. From a cultivation time of 4 h 15 min to 6 h 15 min, aliquots were taken at 15-min time intervals. Transformation of aliquots from each point in time with the reference plasmid pHT43 showed that B. subtilis developed natural compentence in the period from 5 h 15 min to 6 h of cultivation (grey column). B) Estimation of transformation competence of B. subtilis 1012 at different growth phases. After transformation with plasmid pHT43, the number of colonies on selective plates containing chloramphenicol revealed that maximal transformation rates could be obtained by using aliquots taken after 6 h of cultivation at an optical density of 5.2.