Autoradiography and Epifluorescence Microscopy Combined for the Determination of Number and Spectrum of Actively Metabolizing Bacteria in Natural Waters

Abstract

A technique is described for the determination of bacterial numbers and the spectrum of actively metabolizing cells on the same microscopic preparation by a combined autoradiography/epifluorescence microscopy technique. Natural bacterial populations incubated with [ 3 H]glucose were filtered onto 0.2-μm Nuclepore polycarbonate membranes. The filters were cut into quarters and fixed on the surface of glass slides, coated with NTB-2 nuclear track emulsion (Kodak), and exposed to the radiation. After processing, the autoradiographs were stained with acridine orange. A combination of overstaining on the slightly alkaline side and gradual destaining on the acid side of neutrality gave the best results. Epifluorescence microscopy revealed bright-orange fluorescent cells with dark-silver grains associated against a greenish-to-grayish background. Based on the standardization curves, detection of actually metabolizing cells was optimal when cells were incubated with 1 to 5 μCi of [ 3 H]glucose per ml of sample for 4 h and the autoradiographs were exposed to NTB-2 emulsion at 7°C for 3 days. In water samples taken immediately above sandy sediments at beaches of the Kiel Fjord and the Kiel Bight (Baltic Sea, FRG), between 2.3 and 56.2% (average, 31.3%) of the total number of bacteria were actually metabolizing cells. Spearman rank correlation analysis revealed significant interrelationships between the number of active bacteria and the actual uptake rate of glucose.