Cultivation-independent, semiautomatic determination of absolute bacterial cell numbers in environmental samples by fluorescence in situ hybridization

Abstract

Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes has found widespread application for analyzing the composition of microbial communities in complex environmental samples. Although bacteria can quickly be detected by FISH, a reliable method to determine absolute numbers of FISH-stained cells in aggregates or biofilms has, to our knowledge, never been published. In this study we developed a semiautomated protocol to measure the concentration of bacteria (in cells per volume) in environmental samples by a combination of FISH, confocal laser scanning microscopy, and digital image analysis. The quantification is based on an internal standard, which is introduced by spiking the samples with known amounts of Escherichia coli cells. This method was initially tested with artificial mixtures of bacterial cultures and subsequently used to determine the concentration of ammonia-oxidizing bacteria in a municipal nitrifying activated sludge. The total number of ammonia oxidizers was found to be 9.8 x 10(7) +/- 1.9 x 10(7) cells ml(-1). Based on this value, the average in situ activity was calculated to be 2.3 fmol of ammonia converted to nitrite per ammonia oxidizer cell per h. This activity is within the previously determined range of activities measured with ammonia oxidizer pure cultures, demonstrating the utility of this quantification method for enumerating bacteria in samples in which cells are not homogeneously distributed.

FIG. 1

Principle of the cell quantification method developed in this study. See text for an explanation of steps 1 to 7. (A) Microscopic picture of activated sludge from the Munich II wastewater treatment plant. (B and C) Aliquots of the same activated sludge after spiking with 10⁸ (B) and 10⁹ (C) E. coli cells per ml. FISH was performed with probe GAM42a (red) and the EUB338 probe mix (green). The images containing the fluorescence conferred by the probes were superimposed, and E. coli cells appear yellow due to color blending. (D) Calibration curve generated from corrected cell area fractions of E. coli in spiked sludge aliquots. (E) The same microscopic field as in A, showing simultaneous FISH using probes NEU and Nso1225 (red) and the EUB338 probe mix (green). Cells of ammonia oxidizers appear yellow due to color blending. (F) Graph showing the use of the calibration curve (D) for converting the measured area fraction of an autochthonous population to the equivalent E. coli concentration. (G) Highly magnified optical section through a cell aggregate of ammonia-oxidizing bacteria in activated sludge stained by FISH using probes NEU and Nso1225 (red). (H) Highly magnified optical section through E. coli cells from a pure culture stained by FISH using probe GAM42a (red).

FIG. 2

Calibration curves used to convert cell area fractions to equivalent E. coli concentrations for the determination of cell concentrations in pure culture mixtures of C. testosteroni and G. asaii (A), in activated sludge from the Munich II wastewater treatment plant (B), and in the same activated sludge after addition of 1.7 × 10⁸ N. europaea cells per ml (C). The general linear equation of these curves is log C_eq = m × log A + b, where C_eq is the equivalent E. coli concentration, A is the cell area fraction, m is the slope, and b is the ordinate intercept of the line. The values of m are 1.253317 (A), 1.105818 (B), and 1.004006 (C). The values of b are 5.27404 (A), 6.429176 (B), and 6.83411 (C). Error bars which are smaller than the marker symbols are not shown.

FIG. 3

Concentrations of ammonia nitrogen in the influent (⋄) and the effluent (▵) of the nitrification basin of the Munich II wastewater treatment plant measured during the last 2 weeks before sampling.