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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.

Studies in microbial ecology require accurate measures of cell number and biomass. Although epifluorescence microscopy is an accepted and dependable method for determining cell numbers, current methods of converting biovolume to biomass are error prone, tedious, and labor-intensive. This paper describes a technique with sedimentation field-flow fractionation to enumerate bacteria and determine their density, size, and mass. Using cultured cells of different shapes and sizes, we determined optimum values for separation run parameters and sample-handling procedures. The technique described can separate and detect 4',6-diamidino-2-phenylindole-stained cells and generate a fractogram from which cell numbers and their size or mass distribution can be calculated. A direct method for estimating bacterial biomass (dry organic matter content) which offers distinct advantages over present methods for calculating biomass has been developed.

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Abstract:The combination of flow and sedimentation field-flow fractionation Ž . FFF can be applied to the study of changes in the particulate nature of complex colloidal materials such as skim milk, where the aggregates were found to cover the diameter range 10᎐600 nm. The size distribution contained discrete peaks at about 2, 20, 200, and 400 nm. These have been interpreted as arising from simple aggregates of molecular caseins, aggregates of caseins stabilized by calcium phos-Ž phate, large hydrated micelles consisting primarily of caseins and calcium phos-. phate , and aggregates of the large hydrated micelles. Aging of the milk in the presence of a bacteriocide was accompanied by aggregation of the 200-nm particles. Complexing of calcium by the addition of small quantities of ethylenediamine Ž . tetracetic acid EDTA resulted in a reduction in the number of particles with a calculated diameter of ;400 nm; an increase in the number of particles ;200 nm; a reduction in the size of, and a sharpening of the peak corresponding to, the 10-nm particles; and a large increase in the number of 2-nm particles. Addition of excess EDTA resulted in the complete disappearance of all particles above 2 nm.