Populations of symbiotic Rhizobium meliloti extracted from alfalfa nodules were shown by flow microfluorometry to contain a significant number of bacteroids with higher nucleic acid content than the free-living rhizobia. Bacteroids with lower nucleic acid content than the free-living bacteria were not detected in significant quantities in these populations. These results indicate that the incapability of bacteroids to reestablish growth in nutrient media may not be caused by a decrease in nucleic acid content of the symbiotic rhizobia.It has been shown that the growth of symbiotic rhizobia (bacteroids) cannot be reestablished in nutrient media (1, 3). One reason for the nonviability of bacteroids has been attributed to a decrease in nucleic acid content of the symbiotic rhizobia (7). However, previous attempts to determine the nucleic acid content of symbiotic rhizobia has provided conflicting results (3, 7, 10, 12). Bergersen (2) observed no difference in deoxyribonucleic acid (DNA) content per microorganism between cultured Rhizobiumjaponicum and soybean bacteroids. Dilworth and Williams (7) reported a twoto threefold decrease in DNA content per bacteroid in Lupinus species during the first few weeks of nodulation. Sutton (12), working with rhizobia associated with Lotus species, found no difference in DNA content among bacteroids and cultured rhizobia for a slow-growing Rhizobium strain and a lower DNA content per bacteroid for a fast-growing strain. Reijnder et al.(10), on the other hand, found the DNA content of symbiotic R. leguminosarum increased about three-fold as compared to cultured rhizobia. These chemical studies have been conducted on the total bacteroid populations isolated from nodules. Bacteroids in these populations are known from microscopic examinations to be extremely heterogeneous in size and in nucleoid morphology (6,8). Chemical analyses on the total population, therefore, gave only a mean value for all individuals in the population and failed to provide information on the nucleic acid content of individual microorganisms. In this communication, we report the use of flow microfluorometry (FMF) to compare the nucleic acid content of individual rhizobia in populations of free-living (broth-cultured) and sym-biotic (bacteroid) Rhizobium meliloti. This technique has the advantage of being able to analyze the nucleic acid of individual rhizobia in a population and categorize them according to their relative nucleic acid content. FMF has recently been used to investigate the nucleic acid content of large mammalian cells (5,11). We are not aware of previous applications of this technique to the study of microorganisms.Nodules of alfalfa (Medicago sativa var. Buffalo) were harvested 10 weeks after inoculation with free-livingR. meliloti F28. The conditions forR. meliloti and alfalfa plant growth, and the preparations of free-living and symbiotic rhizobial samples were as previously described (4, 9).The samples were fixed overnight at 40C in 63% ethanol. The organisms were then rinsed with distille...