Effect of fixation on particle retention by microflagellates: underestimation of grazing rates

Abstract

The uptake of fluorescent particles by protists and filter-feeding metazoa is being used increasingly by microbial ecologists to study feeding behavior and measure grazing rates. Recent studies of microflagellate uptake of these inert particles have yielded inconsistent results. In particular. grazing rates determined from fluorescent particle uptake are often less than rates measured using other techniques. These low uptake rates have been attributed to osmotrophy, food quality or size selection, rapid egestion of inert particles, and the slower feeding by free-living, as opposed to attached, protists. We have found that a variety of flagellates egest food vacuole contents upon fixation with several commonly used agents including glutaraldehyde and formaldehyde. During time course experiments, the observed microsphere uptake rate for a small chrysomonad flagellate using 1 % glutaraldehyde was only 6 % of the rate obtained by using the fixation method of van der Veer (1982) (2 '10 acrolein, 2 % glutaraldehyde and 1 % tannic acid), modified for epifluorescence microscopy. Uptake rates of several mixed flagellate populations also were 2.4 to 3.1 times higher using the modified van der Veer method than with 1 % glutaraldehyde. The average number of ingested microspheres cell-' using this method was simllar to that observed in live cells immobilized with NiS04. Glutaraldehyde also caused the egestion of Synechococcus sp. cells and fluorescently labelled bacteria from the chrysomonad flagellate. We conclude that previous studies using common aldehyde fixation wlth particle uptake for measunng rates of microflagellate bacterivory have significantly underestimated actual rates of consumpt~on, and that these studes must be re-evaluated, and perhaps repeated, using effective fixation methods.

Distribution of heterotrophic nanoflagellates and their importance as the bacterial consumer in a eutrophic coastal seawater

“…Recently, some scientists indicated that protozoan zooplankton preferred living (Landry et al, 1991) or motile (Gonzalez et al, 1993) bacteria to non-living fixed ones. Moreover, fixation often caused flagellates to egest food vacuole contents (Sieracki et al, 1987). These informations suggest that it is possible that estimation of HNF grazing rates on bacteria obtained by heat-killed FLB method in the present study are underestimated.…”

Effects of flocculated humic matter on free and attached pelagic microorganisms

“…Although it has been et al. 1985), this typical pattern of batch culture has been achieved in several studies (Sieracki and Sieburth 1985;Tranvik and Hofle 1987) and indicates the absence of significant bacterivore populations.…”

“…For each grazing estimate, five subsamples of 5 ml were transferred from each of the cultures of the bacterivory experiment to test tubes containing FLB, adjusted to correspond to -8% of the total number of free bacteria in the test tube. Subsamples were fixed after 0, 5, 10, 15, and 20 min by adding an equal volume of a 4% glutaraldehyde and 4% acrolein solution (Sieracki et al 1987), prepared with 0.2-pm-filtered water from Barber Pond.…”

“…Subsamples were stained with the fluorochrome DAPI (Porter and Feig 1980) at a concentration of 5 mg liter-' (Sieracki et al 1985) for 10 min and filtered onto Irgalan black-stained, 0.2 Pm-Nuclepore filters for microscopic enumeration. At least 400 cells and 10 fields of view were counted for each sample at 1,200 x magnification.…”

Grazing rates and functional diversity of uncultured heterotrophic flagellates

“…In situ abundance of strains MED479 and MED134 was checked. At time 0 and after 40 min of incubation, aliquots for DAPI-stained microbial counts and FISH analysis were taken as before, with the exception that fixation was carried out with an equal volume of diluted fixative to reduce cell egestion (Sieracki et al, 1987) and that the final glutaraldehyde concentration was 2%. The incubation time (40 min) was chosen based on a previous time series that showed a plateau in the number of ingested bacteria at 45 min (Unrein et al, 2007).…”