Recent procedures for quanhfying the ingestion of non-nutritive bacterial sized particles by bacterivores lack the necessary resolution for the accurate determination of ingestion rates. Here, we descnbe how the filter-transfer-freeze procedure of Hewes & Holm-Hansen (1983) permits concomitant epifluorescence and phase contrast microscopy which lets us unambiguously discriminate between lngested and non-ingested fluorescent microspheres (FM) in 4 specles of apochlorotic nanoflagellates. This rapid procedure reduces the subjectivity of grazing rate determlnations while improving precision. We have used this procedure to observe non-continuous feeding, egestion processes and the effect of growth inhibition on phagotrophy.

Nuclepore filters of 0.6-1.0μm pore size have been used to prepare "protist-free" water for a number of studies in microbial ecology. This procedure has been called into question by a recent study claiming that a significant portion of bacterial loss in filtrates could be due to uncharacterized predators passing through 0.6μm filters. We were unable to directly observe protists in 0.6μm filtrates using phase contrast, epifluorescence, or transmission electron microscopy. Using the culture techniques of rice grain enrichment and most probable number, however, we were able to observe and quantify several species of bacterivorous nanoflagellates that developed not only in 0.6μm, but also in 0.4μm seawater filtrates. The ability of predacious nanoflagellates to squeeze through bacteria-sized pores questions studies of bacterial production and chemical cycling that have assumed protist-free filtrates.

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