AME 25:55-63 (2001)  -  doi:10.3354/ame025055

Abstract: We studied the influence of methodology on the variability of bacterial direct counts and biomass estimates. Two frequently used fluorochromes, 4,6-diamidino-2-phenylindole (DAPI) and 3,6-bis(dimethylamino)acridine (acridine orange [AO]), were applied to determine bacterial abundances and sizes along a vertical profile in a oligo-mesotrophic freshwater lake (Piburger See, Austria). Only 90 ± 11% of AO stained bacteria were detected with the fluorochrome DAPI. On average, DAPI stained cells were half as large (48 ± 11% of mean cell volume) as cells stained with AO. The observed discrepancies in abundance and cell volumes were significantly related to community DNA synthesis rates, as suggested by lower differences at higher uptake rates of [³H]thymidine. In addition, a decrease in the relative DNA content cell^-1 with increasing cell size was found in the bacterioplankton assemblage. Considering the staining properties of the 2 dyes, this may partially account for the observed differences in mean cell sizes. We summarized and evaluated most linear and allometric volume-to-carbon conversion factors published during the past 2 decades. Total bacterial biomass was estimated by applying several of these conversion factors to data sets determined from DAPI and AO stained preparations. Depending on the dye and conversion factor, bacterial biomass, averaged over the total water column, ranged between 5 μg C l^-1 and 165 μg C l^-1. As a result of this comparison we recommend the use of allometric conversion formulae specifically elaborated for a particular dye, i.e., CC = 218 x V^0.86 (Loferer-Krößbacher et al. 1998) for DAPI stained bacteria and CC = 120 × V^0.72 (Simon & Azam 1989, recalculated by Norland 1993) for AO stained cells (where CC is cellular carbon content [fg C], and V is bacterial volume [μm³]). In addition, these 2 formulae produced biomass estimates closest to the median values of estimates by all the investigated conversion factors.

KEY WORDS: Bacterial size · Epifluorescence direct counting · Volume-to-carbon conversion