Figure 5.

Fluorescence micrographs of porcine kidney cells (PK-15) exposed as resting cells in the monolayer to crude extracts (13 µg mL⁻¹ and 200 µg mL⁻¹) from A. calidoustus MH34. The cells were stained with the triple-stain Hoechst 33342 + calcein-AM + propidium iodide. The DNA stain Hoechst 33342 stains nuclei in both living and dead cells and emits blue fluorescence. Calcein-AM is cleaved by intracellular esterases to calcein, emitting green fluorescence in live cells with intact plasma membranes. The DNA stain propidium iodide permeates only dead cells, and nuclei in cells that have lost their plasma membrane integrity emit purple or red fluorescence. Panels in the first and third rows were inspected using filters to help see blue/purple emissions of Hoechst 33342 and propidium iodide (no green emission of calcein was visible), whereas the filters used for the panels in the second row helped to see green/red emissions of calcein-AM and propidium iodide, respectively (no blue emission was visible). The control cells in Panels (A) and (G) exposed to the ethanol solvent only exhibited blue fluorescence in intact nuclei in cells with plasma membranes impermeable to propidium iodide. The intactness of the plasma membrane integrity is indicated by the absence of purple emission in Panels (A) and (G) and confirmed by the green emission of the control cells in Panel (D). Cells in Panel (B) exposed to the crude extract at concentrations of 13 µg mL⁻¹ exhibited fragmentation of blue nuclei in c. 50% of the cells, and a lack of purple emission indicated the intactness of the plasma membrane, confirmed by the green emission of the same cells pictured using the green/red filters, as shown in Panel (E). Details of the fragmented nuclei and nuclear fragments in cells with intact plasma membranes are shown in Panels (H) and (I) (no purple emissions visible). Panels (C) and (F) show cells exposed to 200 µg mL⁻¹ of the crude extract. The nuclei in 100% of the plasma-membrane-damaged necrotic cells stained with propidium iodide emitted purple and red fluorescence (no blue or green emissions visible).