Application of Flow Cytometry to Assess the Abundance and Physio Logical State Bacteria of High Bog Peat
Year: 2024
Type: article
Abstract: Background. Peat from upland bogs, as a substrate, is characterized by a number of specific conditions that make it difficult to separate microbial cells for cytometric analysis. At the same time, the absence of up-to-date protocols for sample preparation suitable for capillary cytometers necessitates the selection of optimal conditions for cell isolation and staining. Purpose. To investigate the influence of pre-treatment conditions on cytometric assessment of the total count of prokaryotic cells (TCP) and the viable count of prokaryotes (TVP). Materials and methods. To separate bacterial cells from their substrate, we used a combination of physical and chemical treatments, including ultrasound, surfactants, and ethanol in various forms, for both fixed and fresh samples. West and the cells with fluorochromes such as Acridine orange, PI (propidium iodide), DAPI (4,6-diamidino-2-phenylindole), and SYBR Green. To maximize cell isolation, we performed stepwise flushing, ultrasound and centrifugation, followed by filtration through a 50 μm filter. We then determined the number of cells using a Luminex Guava® EasyCyte 12 HT capillary flow cytometer, and luminescent microscopy with black membrane filters served as a control. Results. The optimal concentration and duration of staining of samples using various fluorochromes were determined. For acridine orange – 20 mcg/ml, 30 minutes; PI and DAPI – 10 mcg/ml, 60 minutes; SYBR Green I – 1 x, 60 minutes. The optimal duration of ultrasound treatment has been established, which allows the maximum number of cells to be isolated from the substrate while maintaining their integrity: for TCP (15 min) and TVP (30 min). At the same time, additional treatment of the sample with surfactants can significantly increase the number of cells isolated from the substrate. It is shown that a 3-stage washing of the substrate is necessary to increase the number of isolated cells from 50-60 % to 89-93 %. Conclusion. The adaptation of the cytometric method for determining the total number and abundance of viable prokaryotes for poorly decomposed peat of upland bogs has been carried out. It was found that the preferred method of sample preparation in determining the total number of bacteria is physico-chemical treatment with surfactants (30 min ultrasound, Triton X-100 (0.1 %), PF (0.1 %)). The need to increase the ultrasound treatment time to 30 minutes, without significant loss of the number of cell fractions, has been confirmed. EDN: DYTKGL
Authors: Dmitriy A. Shpanov, Ivan N. Zubov
Institution: Russian Academy of Sciences
Language: English
Subfield: Ecology
Field: Environmental Science
Domain: Physical Sciences
Open Access status: gold
Estimated APC paid: $350
OpenAlex ID: W4409851747