Aims: To develop a protocol to isolate single cells in wells of a microtitre plate, having a high certainty of individual cells, combined with a sufficient yield. Methods and Results: Single cells were obtained using 1/2 dilution series in microtitre plates. Seventy-two Lactococcus lactis dilution series were checked by plate counting. When the last five columns of the plates were observed, the chance of having one single cell was 80%, while the yield was 75 wells containing cells. A simulation model confirmed these results. This method was compared with the commonly applied method. Conclusions: This method makes it possible to combine a higher chance of having one cell in a microtitre well with a slightly higher yield. Significance and Impact of the Study: A tool is developed to isolate single cells to provide a suitable base for investigating and modelling the individual cell lag phase.