Isolation of a Single Giant Virus from a Mixed Viral Population Using Amoebae

Begin with amoebae cultured in a starvation medium containing antimicrobial agents to prevent contamination. Amoebae serve as susceptible hosts for giant viruses.

Add a diluted viral suspension containing a mixed population of two giant viruses.

Place the culture dish in a bag to prevent evaporation of the medium and incubate.

The viral dilution increases the chance that each amoeba encounters a single type of giant virus.

The amoebae engulf the viruses, which replicate intracellularly, causing infected cells to round and detach, producing cytopathic effects.

Monitor the culture under a light microscope to confirm cytopathic signs.

Then, transfer the dish onto an inverted microscope stage.

Position a microcapillary on a micromanipulator under the inverted microscope. The microcapillary contains biocompatible mineral oil for controlled cell aspiration.

Using the microcapillary, gently aspirate a single, round, infected cell and release it into a fresh amoeba culture. Incubate.

The viral progeny accumulated within the infected cell are released by exocytosis and spread to infect neighboring cells, expanding the single giant virus population.

Perform a serial dilution of the viral sample in sta