Exciting opportunities exist for the application of simple fluorescence-activated cell sorting (FACS) to microbiology. The technology is widely available, but critical reports on the efficiency of cell sorting using benchtop instruments are lacking. It is vital that single cell sorting be of the highest purity possible. If purity is compromised detrital material or unwanted cells will be captured along with target cells of interest. Here, the isolation of fluorescent bacteria using a benchtop FACSCalibur-sort flow cytometer is described. The efficiency and purity of isolated cells was determined using fluorescence microscopy, culturing, and molecular analysis. To achieve high purity it was essential that the total event rate did not exceed 300 cells per second. This instrument was capable of recovering >55% sorted Escherichia coli cells, coupled with a purity exceeding 99%. However, the purity of recovered cells was substantially reduced (<25%) when the event rate increased. Cell sorting onto polycarbonate membranes did not reduce the ability of E. coli to form colonies, and sorting of ∼1000 E. coli cells was sufficient for 16S rDNA amplification. Additionally, as few as 100 isolated Erwinia sp. carrying the gfp gene were amplified using seminested PCR targeting the single copy gfp gene. With such low numbers of bacteria being required for molecular identification, FACS can be achieved without the requirement for high-speed droplet cell sorters.