Comparison of three methods for determining flagellate abundance, cell size, and biovolume in cultures and natural freshwater samples

Abstract

Flagellates of different nutrition modes (hetero-, mixo-, osmo-, autotrophic) from batch cultures and natural freshwater samples were examined for abundance, cell size and biovolume changes after fixation and staining. The methods used were formaldehyde fixation and staining with 4,6-diamidino-2-phenylindole (DAPI) or fixation with Bouin's solution combined with a quantitative protargol stain (QPS). The values estimated with these protocols were compared to a live drop counting method and inspection of living organisms. Cell dimensions were measured with a semi-automatic image analysis system. We observed cultured species of Bodo saltans, Chilomonas paramaecium-group, Cryptomonas sp., Entosiphonomonas cf. sulcatum, Goniomonas cf. truncata and Paraphysomonas cf. vestita. Chemical treatment caused cell loss of up to 56%. In contrast lower abundance of field samples was determined via live counting than via formaldehyde and DAPI treatment. The number of living flagellates <5 μm in length was severely underestimated due to lower magnifications recommended for live drop counting. Formaldehyde and DAPI gave the best results conceming the abundance whereas Bouin's solution followed by a QPS could not be used to distinguish between autotrophic and heterotrophic flagellates. Examinations of the effects of preservation on cell size differed markedly for the flagellate taxa examined, with shrinkage down to 19 % and inflation up to 137 % of live cell volumes. These effects, observed even within a group of Dinobryon species, lead us to conclude that no single conversion factor for computing biovolume on preserved flagellates from natural samples is applicable.