The Effect of the Quantitative Protargol Stain and Lugol's and Bouin's Fixatives On Cell Size: A More Accurate Estimate of Ciliate Species Biomass

Abstract

ABSTRACT. The quantitative protargol stain (QPS) is used to estimate ciliate biomass and species composition from mixed field samples. Length, width, breadth and volume of live Euplotes sp., Eutintinnus sp., Strobilidium spiralis, Strombidium acutum, and Gymnodinium sanguineum were compared with 0.6% acid Lugol's fixed, 5% Bouin's fixed, and QPS cells. Cells shrank due to treatments (ANOVA and Tukey's test, α= 0.05). Protistan post‐fixation cell volume (as a percentage of live volume) was 55%‐80% for acid Lugol's fixed, 40%‐70% for Bouin's fixed, and 30%‐65% for QPS. Each species shrank to a different extent; cytostructural elements apparently alter the effect of fixation. Egestion is likely not the main cause of shrinkage since the autotroph, G. sanguineum, shrank to the same extent as the heterotrophs when stained by QPS. If field studies do not consider fixation effects on cell size, biomass may be underestimated. We recommend, for studies on planktonic ciliates, either acid Lugol's and QPS be used concurrently or QPS be used alone and biovolume values divided by 0.4 to correct for shrinkage. We stress that this is a rough estimate as this value ranges from 0.3 to 0.45 for planktonic protists.

Indirect effect of environmental factors on interactions between microbial and classical food webs in freshwater ecosystems

“…Obvious shrinkage of stained ciliates was noticeable after silver preparation. Therefore, the calculated cell volumes were multiplied with a correcting factor of 0.4 (Jerome et al, 1993). In the case of Crustacea (Cladocera and Copepoda), double samples of the volume 0.5 dm 3 were collected and pooled to reduce heterogeneity in crustacean distribution and sampling variability, thus the final volume of a sample measured 1 dm 3 .…”

Diversity and vertical distribution of planktonic ciliates in a stratified mesotrophic lake: relationship to environmental conditions

“…Ciliates are highly perishable, and their motility is a species-specific feature, so species determination and measurements were carried out on live material immediately on return to the laboratory, following silver impregnation (Augustin et al 1984). The stained ciliates had suffered obvious shrinkage following the impregnation, so calculated cell volumes were multiplied by a correcting factor of 0.4 (Jerome et al 1993). Taxonomic identification was primarily based on the key in Foissner & Berger (1996).…”

Effect of acid Lugol solution as preservative on two representative chitineous and gelatinous zooplankton groups

“…This has also been demonstrated in previous studies for ciliates (Putt and Stoecker 1989, Ohman and Snyder 1991, Jerome et al 1993, Stoecker et al 1994, flagellates (Borsheim and Bratbak 1987, Choi and Stoecker 1989, Menden-Deuer et al 2001, several phytoplankton groups Fig. 1.…”

“…(Verity et al 1992, Montagnes et al 1994, and cyanobacteria (Hawkins et al 2005), with an average protozoan volume reduction of 33% to 40%. Maximum shrinkage rates are published, with a 40% volume reduction for cyanobacteria (Hawkins et al 2005), 57% for naked heterotrophic flagellates (Borsheim and Bratbak 1987), and 45% for ciliates (Jerome et al 1993). Studies of seven different phytoplankton groups showed a mean length reduction by 10.7% (ranging from 3.1% to 22.2%), including three Bacillariophyceae species (mean size reduction 7.1%) (Montagnes et al 1994).…”

“…When applying an empirical length to carbon regression for larvaceans (Jaspers et al 2009), carbon biomass is underestimated by 46% when shrinkage effects due to Lugol solution are ignored. We therefore suggest that shrinkage should be carefully addressed and different rates in body size reduction be applied as suggested in the common methodology for ciliates (Putt and Stoecker 1989, Ohman and Snyder 1991, Jerome et al 1993, Stoecker et al 1994) and flagellates (Borsheim and Bratbak 1987, Choi and Stoecker 1989, Menden-Deuer et al 2001.…”