Detection and analysis by dual-laser flow cytometry of bacteriophage T4 DNA inside Escherichia coli.
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.
Sanders
C A
CA
Department of Laboratory Medicine, San Francisco General Hospital Medical Center, California 94110.
Yajko
D M
DM
Nassos
P S
PS
Hyun
W C
WC
Fulwyler
M J
MJ
Hadley
W K
WK
D001482:Base Composition; D006690:Bisbenzimidazole; D014128:Chromomycin A3; D004261:DNA Replication; D004279:DNA, Viral / Q000032:analysis*; D004926:Escherichia coli / Q000032:analysis*; D005434:Flow Cytometry / Q000295:instrumentation; D005456:Fluorescent Dyes; D007834:Lasers; D013604:T-Phages / Q000032:analysis* / Q000502:physiology; D014779:Virus Replication
D004279:DNA, Viral; D005456:Fluorescent Dyes; D014128:Chromomycin A3; D006690:Bisbenzimidazole