Variations in the fluorescence intensity of intact DAPI-stained bacteria and their implications for rapid bacterial quantification.

As current techniques for the quantification of bacteria are laborious and often imprecise, instrumental approaches such as sedimentation field-flow fractionation (SdFFF) are attractive. In this technique, fluorogenic dyes specific for nucleic acids are used to identify bacterial cells. Bacterial biomass can be quantified directly with SdFFF if the specific fluorescence of bacterial cells is constant. The effect of different growth conditions on the specific fluorescence of one strain each of Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis and Staphylococcus epidermidis stained with 4',6-diamidino-2-phenylindole was examined. Specific fluorescence varied over a 500-fold range, from 0.22 to 103 arbitrary fluorescence units per cell. Specific fluorescence was highest when cells were in log phase, and lowest when cells were in stationary phase. Specific fluorescence decreased when cells harvested in log phase were starved for 7 d in a carbon-free minimal medium, and increased rapidly (within 2 h) after cells were relieved from carbon limitation. Such variations in specific fluorescence must be considered when using gross fluorescence as a direct indicator of bacterial numbers in the SdFFF technique for quantifying bacterial biomass. Moreover, they have serious implications for the application of fluorescence techniques in other instrumental approaches for bacterial enumeration in environmental samples.