Inactivation of Bacteriophages Using Exogenous Lipopolysaccharide to Generate Phage-Free Salmonella Cultures

Begin with a suspension containing Salmonella, a pathogenic bacterium.

The bacterial cells were previously exposed to a lytic bacteriophage that binds to lipopolysaccharides or LPS on the bacterial surface to initiate infection.

The suspension contains uninfected cells and a few infected cells.

Introduce an exogenous LPS that acts as a competitive receptor.

Incubate to allow the infected cells to lyse and release the progeny phages.

These phages bind irreversibly to the exogenous LPS, which inactivates the phages and prevents reinfection of uninfected cells.

Transfer the suspension to a tube and spin down the cells.

Discard the supernatant containing LPS–phage complexes.

Add a medium and spin down the cells to remove the residual phages and LPS.

Resuspend the cells in the medium and perform a second LPS treatment to ensure complete phage inactivation.

This method uses exogenous LPS to inactivate phages while preserving bacterial viability.

Resuspend the cells in 1 mL of 2X LB.
Next, mix commercial Salmonella enterica lipopolysaccharide or LPS with 20 µL of the bacterial suspension in 1 mL of filtered LB.
Incubate the mix for 2 hours at 37 degrees Celsius with shaking at 200 rpm to prevent bacteriophage re-infection.
After that, transfer 1 mL of culture to a microcentrifuge tube and centrifuge the suspension for 2 minutes at 10,354 g.
Wash the pellet three times with 1 mL of filtered LB medium.
After washing, resuspend the pellet in 1 mL of 2X LB.
Add 20 µL of this mix to 1 mL of filtered LB containing 0.8 mg/mL of commercial LPS and incubate the mixture at 37 degrees Celsius and 200 rpm for 2 hours.