Ultracentrifugation-Based Isolation of Legionella pneumophila Outer Membrane Vesicles

Begin with a culture of Legionella pneumophila, a pathogenic Gram-negative bacterium that releases outer membrane vesicles.

These OMVs carry lipopolysaccharides and membrane proteins that contribute to bacterial virulence.

Centrifuge the culture to pellet intact bacteria.

Collect the supernatant containing OMVs and centrifuge again to pellet residual bacteria.

Filter the supernatant through a membrane to remove the remaining bacteria while retaining OMVs.

Perform a second filtration using a fresh membrane to remove bacterial debris.

Collect the bacteria-free filtrate and ultracentrifuge it to pellet the OMVs.

Discard the supernatant, resuspend the pellet in buffer, and ultracentrifuge again to remove loosely associated proteins and lipopolysaccharides.

Remove the supernatant and resuspend the purified OMVs in buffer.

Streak an aliquot of the OMVs onto blood agar and BCYE agar plates and incubate.

The lack of colony formation confirms the absence of bacterial contamination.

Store the OMVs at a low temperature for host-pathogen interaction studies.

Add the remaining liquid culture to 90 milliliters of fresh YEB medium and incubate the culture on a rotating shaker until it reaches an OD₆₀₀ of 3.0