The Use of Autoradiography to Determine the Proportion of Bacteria Metabolizing in an Aquatic Habitat

Abstract

Autoradiographic methods have been developed to determine the proportion of metabolizing bacteria on leaves of Elodea canadensis. Detection with a light microscope of tritium labelling of pure cultures of bacteria was optimal when samples were incubated with 20 μCi of [3H]glucose or [3H]thymidine/ml for 1.75 to 2 h and the bacteria exposed to K2 emulsion for 14 days. Two fluorescent pseudomonads did not form autoradiograms when labelled with [3H]thymidine. For autoradiograms of bacteria from E. canadensis, L4 emulsion was more suitable than K2 emulsion. High-resolution autoradiograms were prepared of a pure culture of a pseudo-monad and of bacteria from moribund leaves of Elodea canadensis. Counts of labelled and unlabelled cells in light-microscopic and high-resolution autoradiograms suggested that valid counts of labelled bacteria could be obtained from light-microscopic autoradiograms. Direct counts of bacteria from E. canadensis leaves of different ages revealed greater increases as leaves matured and died than did indirect methods. Autoradiographic data from young leaves indicated that the percentage of bacteria labelled with [3H]glucose was similar to the percentage of the total population which was estimated by the plate method. On the mature and moribund leaves larger proportions of the populations were labelled with [3H]glucose than were included in the plate counts.