Fig 4. Attempts to develop more rapid screening methods for hypermutation in E. coli.

Panels A and B, using recA-lacZ reporter strain JLM281 on plates containing LB + 150 μg/mL X-gal. Panel A, compared to ciprofloxacin (positive control), the herbicide paraquat also induced recA expression; 10 μl of a 50 mg/mL paraquat solution was spotted onto a sterile blank test disk. Panels B- D, testing for hypermutation in EPEC strain B171-8 on LB + 5 μg/mL rifampin using antibiotic test disks. Plates were inoculated with a 1:5 dilution of an overnight culture of B171-8 using a sterile cotton swab and a criss-cross pattern over the entire plate. Panel B, 10 μl of 1 μg/mL ciprofloxacin was spotted on the disk. A ring of rifampin-resistant colonies grew up in the vicinity of the ciprofloxacin. Panel C, same as Panel B, except that 10 μL of 40 mM arsenic trioxide was spotted onto the blank disk. Panel D, same as Panels B and C, but using 10 mM 6-mercaptopurine. Panels E and F, attempt to create a semi-quantitative screening method for hypermutation. STEC Popeye-1 was grown in the absence or presence of 10 ng/mL ciprofloxacin ± 0.2 mM zinc acetate, then diluted into sterile saline to achieve an OD₆₀₀ of 0.2 for each culture. The diluted cultures were spread using a sterile cotton swab and a criss-cross pattern to cover the entire plate, then a trimethoprim E-test strip was applied to each plate. Each condition was plated in triplicate. Panel E, the ciprofloxacin-treated cultures (middle Petri dish) showed many more inlier colonies within trimethoprim’s zone of inhibition than in the control (left) or the ciprofloxacin + zinc condition (right). Panel F, the number of inlier colonies were counted in triplicate for each condition and are shown. *, significantly more than control; **, significantly fewer than ciprofloxacin alone, both by ANOVA.