JoVE Encyclopedia of Experiments
Microbiology
0 views • 2:33 min • February 26th, 2026
Begin with plates containing surface-attached bacteria and planktonic or free-floating bacteria.
Add amoeba cells to both samples.
Place an agarose pad containing Calcein-AM, a cell-permeable dye, over each sample.
Remove excess liquid, allow to dry, and incubate the plates.
In the plate with surface-attached bacteria, surface contact upregulates virulence genes, expressing toxins.
These toxins disrupt the amoeba membrane and change their shape.
Damaged amoeba membrane allows more calcein-AM to enter the cytoplasm, where intracellular esterases cleave them into fluorescent calcein, leading to its accumulation.
In contrast, planktonic bacteria, lacking surface contact, remain non-virulent, leaving amoeba membranes intact. This reduces Calcein-AM entry into the amoeba, limiting fluorescent calcein accumulation.
Healthy amoeba also engulf the non-virulent bacteria.
Under a fluorescent microscope, bright, rounded amoeba cells indicate virulent bacterial interaction, while dim, amorphous amoeba indicate nonvirulent bacterial interaction.
To assay virulence of surface attached bacteria cells, add 10 microliters of amoeba cells from the previously obtained surface-attached bacterial cells.
To isolat
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