Examination of Direct Counting Methods for Bacteria in Lake Sediment by Epifluorescence Microscopy.

Abstract

Direct counting of bacteria in lake sediments by epifluorescence microscopy was examined. To evaluate the counting efficiency we prepared synthetic sediment samples of known bacterial density. In the case of acridine orange (AO) staining, it was impossible to estimate bacterial numbers accurately owing to the difficulty of discriminating bacterial cells from abiotic particles in the samples. However, staining with 4' 6-diamidio-2-phenylindole (DAPI) gave satisfactory results, i.e., recovery of the bacterial counts was more than 93% ; the best efficiency was obtained at the final concentration of the dye of 1.0 μg⋅m1-1. Treatment by ultra sonication with pyrophosphate addition (10 mM) was effective to deflocculate and disperse the bacteria attached to sediment particles. The level of dilution of sediment samples used to prepare microscopic slides was shown to be important to obtain accurate counts. Optimal dilution level should be decided to reduce masking by sediment particles and, in turn, to assure a statistically satisfactory number of bacteria in an optical field. However, the optimum level was variable among samples which had different sediment textures (e.g. water content, particle size, detritus forms and volume) and bacterial density.Vertical profiles of bacterial density in Lake Suwa sediment were compared by the direct counting and plate viable counting. Total bacterial counts were 2.94 × 1010 cells⋅g dw-1 and 3.74 × 109 cells⋅g dw-1 at 0-3 cm and 45-50 cm respectively, and these counts were 600 and 1, 500 times higher than those obtained by the plate method. The size distributions of bacteria at 0-3 cm and 45-50 cm, which were measured using an image analysis system, were concentrated in the range of 0.2 to 0.8 μm, whereas the mode of cell length was 0.46 and 0.39 μm at 0-3 cm and 45-50 cm respectively. At 0-3 cm larger cells existed and the distribution at both depths was significantly different. The relationships of bacterial numbers, size distribution and organic carbon contents in sediment are discussed.