Visualization of Virus-Infected Algae Using Fluorescence In Situ Hybridization

Take a membrane filter containing fixed green algal cells that lack a cell wall and are infected with a virus.

Embed the membrane in agarose to immobilize the cells.

Sequentially treat with ethanol and methanol to remove photosynthetic pigments that cause autofluorescence and to enhance membrane permeability for probe access.

Add a mild acid to inactivate endogenous peroxidases and reduce background signals.

Introduce a hybridization buffer containing peroxidase–labeled rRNA probes that hybridize to algal ribosomal RNA.

Wash, then add a fluorophore-tagged tyramide. The peroxidase converts it into reactive molecules that deposit near the probe, labeling the algae.

Wash, then add a hybridization buffer containing red fluorophore–labeled polynucleotide probes.

Incubate at high temperature to compromise the viral capsid integrity and denature the viral DNA.

Lower the temperature to allow probe hybridization, labeling the viral genome.

Wash again and observe under a fluorescence microscope.

The uninfected algal cells fluoresce green, while the infected cells display dual fluorescence.