A dual-laser flow cytometer was used to analyse different species of bacteria for the molar percentage of guanineplus-cytosine (% G + C) without the need for DNA extraction or purification. Ethanol-fixed bacterial cells were stained with a combination of DNA-specific fluorochromes, Hoechst 33258 and chromomycin A3, which bind to AT-and GC-rich regions of DNA, respectively. A linear relationship (r = 0.99) was demonstrated between the log of the ratio of chromomycin A3 to Hoechst 33258 fluorescence and the log of the % G + C as determined by thermal denaturation (Tm) or buoyant density centrifugation (Bd) methods. Linearity was maintained for all bacterial species tested over the range of 2847% G + C. A standard curve was constructed using five strains whose % G + C had been determined by other methods. From the equation describing this line, the % G + C values of nine other strains with known DNA base composition, together with the five strains used to construct the curve, were calculated using the chromomycin A3 to Hoechst 33258 ratio and were in agreement with values obtained by Tm, IBd or HPLC. The reproducibility of flow cytometric analysis (mean error 0.7% G + C) compared well with the reproducibility of other methods. Mixtures containing two species were also analysed. Two cell populations could be discerned in mixtures containing two species which differed in base composition by as little as 4 % G + C.Dual-laser flow cytometric analysis of stained bacteria is a rapid, simple and accurate method for determining the % G + C of bacterial DNA and can be used to distinguish populations of bacteria with differing % G + C content.

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:l 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, ethanolfixed, 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 T4DNA inside E. coli. A value of 35.6 k 0.2% was obtained, which agrees with % G + C values determined by traditional methods. These results demonstrate that duallaser flow cytometry can be used to study viral DNA inside the bacterial host.Key terms: Hoechst 33258, chromomycin A3, DNA base composition, viral DNA, determination of % G + C The combination of two DNA-specific f luorochromes, Hoechst 33258 (H33258) and chromomycin A3 (CA3), has been used to study mammalian chromosomes and bacterial DNA (5, 6, 9, 10,17,22). H33258 binds preferentially to regions of DNA rich in A-T base pairs (3, 14) while CA3 binds preferentially to G-C-rich regions of DNA (2). The ratio of CA3:H33258 relative fluorescence measured by dual-laser flow cytometry has been shown to be related to the base composition of bacterial DNA (22) and has been used to determine the % G + C of DNA in bacteria (17).Using H33258 and CA3 in combination, it may be possible to detect bacteriophage DNA inside of its bacterial host and to determine the % G + C of the bacteriophage DNA using the CA3:H33258 fluorescence ratio. It has been shown that the combination of H33258 and CA3 can be used to differentiate bacterial populations in mixtures containing two species of bacteria which differ in DNA base composition by as little as 4% G + C (17). The DNA of bacteriophage T4 has a % G + C of 34-36.1 (12, 15, 18, 21) which differs in base composition from the DNA of its host, E . coli 148-52% G + C (1611, by greater than 4% G + C. Because of the wide difference in the % G + C, it should be possible, in theory, to distinguish T4 DNA from E. coli DNA using CA3 versus H33258 fluorescence.Flow cytometric analysis of T4 DNA inside E. coli cells is somewhat more complex than analysis of bacterial DNA by flow cytometry. The presence of both T4 DNA and host cell DNA within E. coli cells contribute to the fluorescence associated with CA3-and H33258-stained E . coli cells infected with T4. However, relatively early in the course of infection of E . coli by T4, the chromosome of the host is degraded by viral nucleases (13). As degradation of host cell ...