Feasibility of using GFP-expressing Escherichia coli, coupled with fluorimetry, to determine protozoan ingestion rates

The feasibility of using a live Escherichia coli population, which had been engineered to express the green fluorescent protein (GFP), coupled with fl uorimetry, was tested as a means for determining protozoan ingestion rates. Its potential use was based on evidence that once cells are acidified. e.g . in a food vacuole, the fluorescence is lost. Of the 29 protozoa tested, o ver 85% ingested the GFP-expressing E. coli and a detailed experiment with the ciliate Tetrahymena pyriformis was carried out, principally to assess t he performance of the live bacterium against two commonly used surrogate pr ey, i.e. fluorescently Labelled bacteria (FLB) and fluorescently labelled m icrospheres (FLMs). A decrease in GFP-expressing E. coli fluorescence and, hence, concentration, was recorded by fluorimetry and epifluorescence micro scopy, with calculated ingestion rates being equivalent. A higher ingestion rate was determined by counting the number of fluorescent E. coli within t he ciliate over 120 s, but this was equivalent to that obtained for the sta ined E. coli using the same direct method of analysis. However, the ciliate was shown to process the stained and unstained E. coli cells differently, with only the latter resulting in an increase in ciliate abundance. (C) 200 1 Federation of European Microbiological Societies. Published by Elsevier S cience B.V. All rights reserved.