A small amount of ampicillin solution should be streaked out on the surface of a nutrient agar plate and allowed to dry. Cross-streak the culture to be tested against the ampicillin. N.B. Ampicillin resistance is a convenient marker enabling testing for the presence of the plasmid but it has nothing to do with the increased sensitivity of the strains to reversion by mutagens. The rfa character of the tester strains is confirmed by determining crystal violet sensitivity. The rfa mutation permits large molecules, such as crystal violet, to enter and kill the bacteria.
Fresh culture is added to top agar, vortexed, and poured onto a nutrient agar plate.
Crystal violet is added to the centre of sterile filter paper and transferred to the seeded plates and the disc is pressed lightly to embed it slightly. The plate is incubated inverted. A clear zone of inhibition appears around the disc, indicating the presence of the rfa mutation. The uvrB deletion is quite stable and is not easily lost from bacteria but its presence can be easily confirmed by demonstrating UV sensitivity. Strains with the uvrB deletion will only grow on the un-irradiated side of the plate.