Detection and analysis by dual-laser flow cytometry of bacteriophage T4 DNA inside Escherichia coli. |
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Authors: | C A Sanders D M Yajko P S Nassos W C Hyun M J Fulwyler W K Hadley |
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Institution: | Department of Laboratory Medicine, San Francisco General Hospital Medical Center, California 94110. |
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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. |
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