Detection and analysis by dual‐laser flow cytometry of bacteriophage T4 DNA inside Escherichia coli

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Abstract
Bacteriophage T4 DNA was detected and analyzed inside E. coli by dual‐laser flow cytometry using a dye combination of Hoechst 33258 (H33258) and chromomycin A3 (CA3) which bind to A‐T‐ and G‐C‐rich regions of DNA, respectively. An exponentially‐growing culture of E. coli ATCC 11303 was infected with T4 bacteriophage at a 1:1 multiplicity of infection. Samples were taken immediately and at 5 min intervals and placed on ice to interrupt viral replication. The samples were then centrifuged, ethanol‐fixed, stained with H33258 and CA3, and analyzed by flow cytometry. Twenty‐five minutes post‐infection, a population of cells which contained T4 DNA began to appear on both a bivariate contour plot and a frequency histogram plot of the data. By 35 min, T4 DNA‐containing cells could be distinguished from E. coli cells containing little or no T4 DNA. The ratio of CA3:H33258 fluorescence was then used to calculate the % G + C value for T4 DNA inside E. coli. A value of 35.6 ± 0.2% was obtained, which agrees with % G + C values determined by traditional methods. These results demonstrate that dual‐laser flow cytometry can be used to study viral DNA inside the bacterial host.