Monitoring Amphora sp. growth by flow cytometry

Abstract

A protocol on flow cytometry (FC) was developed for monitoring the growth of Amphora sp. in non-axenic and near-axenic (<15% of bacteria) cultures, enabling the discrimination of aggregates and suspended bacterial cells. Autofluorescence emitted by diatoms was gated on the FL3 red channel and bacteria stained with SYTO-BC were detected on the FL1 green channel. In the FL3 positive region, two subpopulations were gated, one corresponding to a homogeneous high-density region, consisting mostly of single diatoms (R1), and a subpopulation of events with up to one log decade fold of FL3 fluorescence intensity and increased relative cell size, suggesting the presence of aggregates of two or more diatoms in the culture (R2). Percentages of single diatoms, diatoms with attached bacteria and free-suspended bacteria were determined in each quadrant (A, B, C) at different growth phases. The aggregates of diatoms with attached-bacterial cells increased with culture age, and the percentage of free-suspended bacteria accounted for less than 10% in this controlled experiment. It is expected that growth monitoring by FC will allow the detection of bacterial contamination or overgrowth at early stages of Amphora sp. cultures.

Keywords:

• flow cytometry
• benthic microorganisms
• diatoms
• growth monitoring
• autofluorescence

Additional information

Funding

This study was supported by the Portuguese Science and Technology Foundation (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit, COMPETE 2020 (POCI-01-0145-FEDER-006684) and the BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 – Programa Operacional Regional do Norte.