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Aims: The effect of temperature (2-30°C), pH (4AE8-7AE4) and water activity (0AE946-0AE995) on the relationship between optical density (OD) at 600 nm and the plate count (CFU ml )1 ) was investigated for Listeria monocytogenes.Methods and Results: Calibration curves, relating OD with plate counts, were collected by measuring the OD of consecutive one-half dilution series, before determining the cell density by classic plate count methods. The calibration curves were observed to be shifting in a parallel way, with increasing stress levels. Especially pH influenced the curve in a great extent, while the other variables were showing more synergetic effects. The reason for the shift was investigated by a microscopic viability test, showing a viability decrease with increasing stress levels, causing the shift of the calibration curve. In a last step a model was made describing the effect of environmental factors on the calibration curve, with different data transformations being tested. A polynomial equation was fitted to the data, taking into account a set of constraints to incorporate microbiological knowledge in the black box model. Hence, illogical interpolation results and overfitting of the data could be avoided. Conclusions: Different stress factors are affecting the relationship between the OD and the cell count of L. monocytogenes by lowering the cell viability. These effects could be modelled using a constrained polynomial model. Significance and Impact of the Study: The observed phenomena are important when calculating growth parameters, like growth rate and lag phase, based on OD data.

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.

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