In vitro transformation frequencies (transformants per recipient) of B. subtilis 168. An overnight culture was diluted in MS media to OD450 = 0.25. The culture was grown under aerobic conditions at 37°C. Every hour, one ml was taken out and different amounts of plasmid (0.1 μg/ml [Δ], 1 μg/ml [◆], and 10 μg/ml [■] were added. The cultures were incubated for further 1 hr before plating on selective media for counting of recipients (LB) and transformants (LB + 5 μg/ml chloramphenicol).