Determination of the Total Microbial Abundance in Black Sea Bottom Sediments Using Flow Cytometry

Abstract

The known approaches to sample preparation have been improved to achieve a more complete detection of microorganisms of the Black Sea bottom sediments using flow cytometry of SYBR Green I-stained cells. Total microbial abundance in the samples from the shelf and deep-sea sediments varied from 0.03 to 1.54 × 10⁸ cells g^–1 and from 0.002 to 1.24 × 10⁸ cells g^–1 dry weight, respectively. This is comparable to the data reported previously for various areas of the oceans, including the Black Sea. Application of sodium pyrophosphate was shown to be the most universal method for treating sediments of various types; along with this, using hydrofluoric acid is possible for the deep-sea reduced sediments, whereas treatment with methanol was preferable for the sediments of coastal waters with a normal degree of aeration of the bottom layer. For samples of various types, optimal sample preparation procedures were proposed (choice of chemical reagent, mode of ultrasonic processing and centrifugation, and additional washing procedures). These procedures resulted in significantly more efficient enumeration of bacterial cells, while application of flow cytometry ensured rapid determination of the total number of microorganisms in the bottom sediments.