Stringent Analysis of Whole Microcolonies: Analyzing All Cells in a Microcolony and Reducing Contamination by Unrelated Neighboring Bacteria

<p>To analyze all cells in a microcolony, very young microcolonies were harvested (10³–10⁴ cfu/microcolony; see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020399#pbio-0020399-g001" target="_blank">Figure 1</a>D; <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020399#pbio-0020399-t001" target="_blank">Table 1</a>). To reduce contamination with neighboring Lac⁻ bacteria and unrelated Lac⁺ microcolonies from the minimal-lactose selection plate, first, a minority of the Lac⁻ cells plated carried an antibiotic-resistance marker, and only resistant microcolonies were analyzed, and, second, sectored colonies observed were retested for instability (to eliminate sectored colonies that are not unstable, shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020399#pbio-0020399-g001" target="_blank">Figure 1</a>C, which may result from accidental overlap of blue and white cfu). Procedures described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020399#s3" target="_blank">Materials and Methods</a>, “whole microcolony analysis.” Cam^R, chloramphenicol resistant; Kan^R, kanamycin resistant; Tet^R, tetracycline resistant. Streptomycin resistance (not shown) was also used.