Fig. 2

Detection of temperate phage contamination in a phage lysate or transduced strain. a The spot agar assay was performed using a serially diluted P1 phage lysate starting from the un-diluted lysate (labelled as 1×) to the dilution factor of 10¹¹ (labelled as 10⁻¹¹). The results reveal that plaques are observed at every concentration down to the 10^7th dilution. The P1 lysate stock was thereby determined to have an infection titer value of 7 × 10⁹ pfu mL⁻¹. b A schematic diagram of a typical LB agar plate used for the cross-streak agar assay: The vertical dark region at the center represents the zone of P1 lysate. The dot represents the location where the tested cells are inoculated at a safe distance from lysate zone, and the horizontal solid lines represent either the temperate phage-infected cells that are growing across the lysate zone or the phage-free cells that are not growing beyond the lysate zone. c The two representative plates of the cross-streak agar assay performed with the colonies obtained from phage transduction experiment demonstrate that 14 of the 18 colonies tested are infected with temperate phages. The plate on the right side indicates that the donor (D) and recipient (R) strains used are devoid of phages. d A scheme of the steps involved in Evans Blue-Uranine (EBU) plate assay, explaining the principle of this technique to screen for temperate phage contamination. e Temperate phage-containing cells verified using cross-streak agar assay grew as dark green colonies on EBU plate. f Cells verified to be free of phages grew as pale green colonies on EBU plate. g An EBU plate assay was performed with the diluted cultures of colonies obtained from a P1 phage transduction experiment. A representative result plate and an enlarged view of the colonies obtained from various EBU plates are shown. A mix of uninfected colonies (pale yellowish green color) containing no temperate phages and colonies containing temperate phages (dark green) were observed on the plates