Sorting and quantification of deactivated bacteria is an important way of quality control for whole-cell bacterial vaccines. In general, surface features of deactivated bacteria used for whole-cell bacterial vaccines affect the immunoresponse to bacteria-associated antigens. Enumeration of bacteria is also an important process development parameter for these vaccines. Field-flow fractionation (FFF) was previously applied to the separation of bacteria. For the first time, FFF is used for sorting bacteria strains of the same species on the basis of differences in bacterial membrane characteristics. Two FFF techniques, gravitational FFF (GrFFF) and asymmetrical flow FFF (AsFIFFF), are shown to be able to fractionate, distinguish, and quantify different deactivated Escherichia coli strains used for vaccines. E. coli can differ in the presence of fimbriae on the bacterial membrane. Fimbriae affect E. coli pathology and thus the use of E. coli for vaccines. GrFFF and AsFIFFF are able to fractionate fimbriated/ nonfimbriated cells in mixtures of different strains. While GrFFF is characterized by low cost and simplicity, As-FIFFF shows a higher performance in size fractionation with a high-speed separation. Coupled, on-line UV/visible turbidimetry yields the relative numbers of fractionated cells and sample recovery. Scanning electron microscopy and quasi-elastic light scattering are employed as uncorrelated techniques for size and morphology analysis of the E. coli strains.