Flow cytometric measurements of cell volumes and DNA contents during culture of indigenous soil bacteria

Abstract

Indigenous soil bacteria were released from a clay loam soil by repeated washing and centrifugation followed by density gradient centrifugation to remove enough soil particles to allow a flow cytometric (FC) study of cell numbers, cell sizes, and DNA content in single cells. The bacteria were suspended in liquid soil extract medium and incubated at 15°C for 60 h, during which direct fluorescence microscopic counts (acridine orange direct counts, AODC) were done along with the FC measurements. Cells of Escherichia coli with a known number of whole genomes per cell (rifampicin treated) were used as a calibration standard both for the DNA measurements (mitramycin-ethidium bromide stain) and cell volumes (light scatter). In response to the nutrients in the soil extract medium, the indigenous soil bacteria increased in numbers and respiration rate after a lag period of about 17 h. The onset of growth was seen first as an increase in respiration rate, numbers of large cells, and the amounts of DNA per cell in the large cells. Respiration and direct microscopical determination of biovolume was used to calculate the average growth yield on the basis of cell carbon, which was found to be 20–30% during the period of active growth. For separate volume groups of the indigenous cells, the DNA content ranged from 1.5 to 15 fg DNA per cell, the majority being below 4 fg DNA. During growth in soil extract medium, the numbers of large cells (volume > 0.18 μm³) increased, and the frequency of cells with high DNA contents increased as well for this group. For the smallest sized cells (volumes < 0.065 μm³) it was not possible to detect any increase in numbers during the 60-h incubation, and the DNA contents of these cells remained virtually unchanged. Compared with cell volumes based on microscopy (AODC), the FC-light scatter data grossly overestimated the volume for indigenous cells but apparently not for the newly formed cells during growth in the suspension. This probably reflects differences in light scatter properties due to adsorbed materials on the indigenous cells. The FC-DNA measurements confirmed earlier findings in that the average DNA content per cell was low (around 2 fg DNA per cell), but demonstrated a positive relationship between cell size and DNA content for indigenous cells.