Figure 3. Fluctuation test.

A. Shows the protocol used. The test was designed to determine if Hly^- clones arose prior to exposure to km or specifically in response to exposure. A primary culture of strain MG1655HY (pANN202-312R) was grown in LB medium containing cm but lacking km. Eleven secondary cultures were performed by transferring small amounts of the primary culture (final cell concentration in the secondary culture, 10³ cells/ml) into the same culture medium. These cultures underwent many rounds of cell division. From one of the cultures, three replicate subsamples were plated on blood agar plates containing cm and km. From the rest of the cultures, a single sample was plated onto identical plates. B. The percentage of Hly^- clones in the different viable counts on blood agar medium containing cm and km.