Simple method for plating Escherichia coli bacteriophages forming very small plaques or no plaques under standard conditions

Abstract

The use of low concentrations (optimally 2.5 to 3.5 microg/ml, depending on top agar thickness) of ampicillin in the bottom agar of the plate allows for formation of highly visible plaques of bacteriophages which otherwise form extremely small plaques or no plaques on Escherichia coli lawns. Using this method, we were able to obtain plaques of newly isolated bacteriophages, propagated after induction of prophages present in six E. coli O157:H(-) strains which did not form plaques when standard plating procedures were employed.

FIG. 1.

Formation of plaques by bacteriophages λcIb2 and T4D on lawns of E. coli MG1655 grown in LB medium or minimal FB medium supplemented with various carbon sources (0.4% glycerol, 0.2% glucose, or 0.6% succinate). The size scale is shown.

FIG. 2.

Formation of plaques by bacteriophages λpapa, P1Cm, and P1vir on lawns of E. coli C600 grown in LB medium or in the same medium supplemented with one of the following antibiotics: tetracycline (LB_tet), ampicillin (LB_amp), chloramphenicol (LB_cm), or kanamycin (LB_kan). These antibiotics were added to the bottom agar to final concentrations of 1.0, 3.5, 2.5, and 2.5 μg/ml, respectively. The size scale is shown. For plate preparation, 3 ml of the top agar and 1 ml of an overnight bacterial culture (1.6 × 10⁹ CFU) were used.

FIG. 3.

Formation of plaques of bacteriophages 933WΔtox (Δstx₂::cat gfp), 22Δtox (Δstx₂::cat gfp), 27Δtox (Δstx₂::cat gfp), 32Δtox (Δstx₂::cat gfp), ST2-8624 (Δstx₂::cat gfp), and φ24B (Δstx₂::cat) on lawns of E. coli C600 growing in the LB medium or in the same medium supplemented with one of the following antibiotics: tetracycline (LB_tet), ampicillin (LB_amp), or chloramphenicol (LB_cm). These antibiotics were added to the bottom agar to final concentrations of 1.0, 3.5, and 2.5 μg/ml, respectively. The size scale is shown. For plate preparation, 3 ml of the top agar and 1 ml of an overnight bacterial culture (1.6 × 10⁹ CFU) were used.

FIG. 4.

Changes in fractions of plaques of given diameter, generated by indicated bacteriophages on E. coli C600 grown on media containing the following antibiotics: none (open bars), 2.5 μg/ml ampicillin (bars with horizontal stripes), 1 μg/ml tetracycline (black bars), 2.5 μg/ml chloramphenicol (gray bars), and 2.5 μg/ml kanamycin (light gray bars). The absence of a bar indicates a lack of plaques. The results of each experiment are based on measurement of 100 to 300 plaques. Note also the information about EOP (Table 4).

FIG. 5.

Formation of plaques of bacteriophages induced by mitomycin C treatment (0.5 μg/ml) from E. coli O157:H⁻ strains 263, 272, 282, 298, 303, and 306 on lawns of E. coli C600 grown in LB medium or LB medium supplemented with ampicillin (LB_amp) to a final concentration of 3.5 μg/ml. The size scale is shown.