Monitoring Amphora sp. growth by flow cytometry

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.