Enumeration and biomass estimation of bacteria in aquifer microcosm studies by flow cytometry

Flow cytometry was used to enumerate and characterize bacteria from a sand column microcosm simulating aquifer conditions. Pure cultures of a species of Bacillus isolated from subsurface sediments or Bacillus megaterium were first evaluated to identify these organisms' characteristic histograms. Counting was then carried out with samples from the aquifer microcosms. Enumeration by flow cytometry was compared with more-traditional acridine orange direct counting. These two techniques gave statistically similar results. However, counting by flow cytometry, in this case, surveyed a sample size 700 times greater than did acridine orange direct counting (25 (mu)l versus 0.034 (mu)l) and required 1/10 the time (2 h versus 20 h). Flow cytometry was able to distinguish the same species of bacteria grown under different nutrient conditions, and it could distinguish changes in cell growth patterns, specifically single cell growth versus chained cell growth in different regions of an aquifer microcosm. A biomass estimate was calculated by calibrating the total fluorescence of a sample from a pure culture with the dry weight of a freeze-dried volume from the original pure culture. Growth conditions significantly affected histograms and biomass estimates, so the calibration was carried out with cells grown under conditions similar to those in the aquifer microcosm. Costs associated with using flow cytometry were minimal compared with the amount of time saved in counting cells and estimating biomass.