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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-mum 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 muCi of [(3)H]glucose per ml of sample for 4 h and the autoradiographs were exposed to NTB-2 emulsion at 7 degrees 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.

applied and environmental microbiology

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