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

Résumé

Source: Fernández-Fernández, R., et. al. Bacteriophage Removal from Infected Salmonella Cultures. J. Vis. Exp. (2024)

This video demonstrates the use of exogenous LPS (Lipopolysaccharide) as a receptor mimic to inactivate lytic bacteriophages in Salmonella cultures. Through sequential incubation, centrifugation, and washing, phages are eliminated, resulting in a viable, phage-free bacterial suspension for downstream applications.

Protocole

1. Bacteriophage removal from infected Salmonella enterica cultures
   1. Incubate 20 µL of the bacterial suspension in the presence of a commercial Salmonella enterica lipopolysaccharide (LPS) to a final concentration of 3.75 mg/mL in 1 mL of 0.22 µm-filtered Lennox broth (LB) for 2 h at 37 °C with shaking at 200 rpm.
      
      NOTE: Phages released from cells into the medium may bind to the commercial S. enterica LPS instead of bacterial LPS.
   2. After 2 h of incubation, transfer 1 mL of culture to a microcentrifuge tube and centrifuge the suspension for 2 min at 10,354 x g. Titration point 4: Use the supernatant as the fourth aliquot for titration.
   3. Wash pellet 3x with 1 mL of filtered LB medium.
   4. After washing, resuspend the pellet in 1 mL of 2x LB. Incubate 20 µL of this mix in 1 mL of 0.22 µm-filtered LB with 0.8 mg/mL of commercial LPS for 2 h at 37 °C with shaking at 200 rpm. Titration point 5: Use the rest of the bacterial suspension for the fifth titration.
   5. After 2 h of incubation, transfer 1 mL of culture to a microcentrifuge tube and pellet by centrifugation for 2 min at 10,354 x g. Titration point 6: Use the supernatant as the sixth aliquot for titration by pouring the supernatant into a new vial.
   6. Wash bacterial cells 3x with filtered LB. After washing, resuspend collected cells in 1 mL of 0.22 µm-filtered LB.

Matériaux

Liste des matériaux utilisés dans cet article

Nom	Entreprise	Numéro de catalogue	Commentaires
Centrifuge	Eppendorf		No special requirements
Flasks			No special requirements
Forceps			No special requirements
Glass tubes			No special requirements
Lipopolysaccharide from Salmonella enterica serotype Typhimurium	Sigma-Aldrich	L6511-25 mg	Dissolved in sterile water