Applications of flow cytometry on bacteria: cell cycle kinetics, drug effects, and quantitation of antibody binding.

Dual parameter flow cytometric analysis of bacteria has been found technically feasible. A microscope based flow cytometer with either a 100 W Hg-arc or a 5 W Ar laser as the excitation light source was used to record fluorescence and light scatter simultaneously to produce three dimensional histograms correlating the two parameters. Cells of Escherichia coli K-12 were fixed in ethanol and stained with mithramycin. Fluorescence (DNA)/light scatter histograms of cells in various phases of growth were recorded at a rate of up to 1 × 104 cells/sec with an instrumental resolution corresponding to coefficient of variation < 5%. Similar histograms of cells stained with FITC indicated that light scatter was approximately proportional to total cell protein. Histograms showing the relative cellular contents of DNA and protein in cultures of rapidly growing E. coli were in quantitative agreement with a current model of bacterial growth. Histograms of E. coli in slow growth indicated that a significant portion of the cells were in a nonreplicating phase. Four antibiotics, all ribosomal inhibitors of protein synthesis, were found to affect the cell cycle differently when present in concentrations sufficient to stop cell division. Mycobacterium leprae bacilli were coated with FITC-labeled M. leprae specific, human antiserum and with a BCG antiserum. The resulting FITC fluorescence/light scatter histograms yielded the amount of antibody per cell as related to cell size thus facilitating a determination of antigenicity per unit cell weight. The results exemplify bacteriological applications of flow cytometry and demonstrate the efficiency of the method in obtaining detailed and precise information on single bacteria in large numbers.