Bacterial growth on surfaces: automated image analysis for quantification of growth rate-related parameters.

A fast routine method for estimating bacterial cell growth rates by using the metachromatic dye acridine orange is described. The method allows simultaneous estimates of cellular RNA and DNA contents of single cells. Acridine orange staining can be used as a nonspecific supplement to quantitative species-specific hybridizations with fluorescence-labelled ribosomal probes to estimate the single-cell concentration of RNA. By automated analysis of digitized images of stained cells, we determined four independent growth rate-related parameters: cellular RNA and DNA contents, cell volume, and the frequency of dividing cells in a cell population. These parameters were used to compare physiological states of liquid-suspended and surface-growing Pseudomonas putida KT2442 in chemostat cultures. The major finding is that the correlation between substrate availability and cellular growth rate found for the free-living cells was not observed for the surface-bound cells; in contrast, the data indicate an almost constant growth rate for attached cells which was independent of the dilution rate in the chemostat.