Generation of Spheroplasts from Gram-Negative Bacteria

Take media containing a cell division-blocking antibiotic.

Inoculate it with rod-shaped, Gram-negative bacteria.

Incubate with shaking.

In the presence of the antibiotic, the bacteria continue to grow without dividing and form long filaments.

Collect the culture and centrifuge it at low temperature. Discard the supernatant and wash the filaments with a sucrose solution to prevent lysis.

Discard the sucrose solution.

Add a buffer followed by enzymes and EDTA. EDTA weakens the outer membrane, allowing lysozyme to enter and break down the peptidoglycan layer.

This removes the cell wall, allowing the bacterial cell to shrink. DNase I breaks the released chromosomal DNA from cells and prevents cell clumping.

Add a hypotonic solution with gentle swirling, allowing the cell to draw in water.

The flexible membranes enable the cell to take on a spherical shape called a spheroplast.

Transfer an aliquot containing the spheroplasts into two tubes with a chilled isotonic solution to stabilize the spheroplasts.

To prepare Gram-negative E.coli spheroplasts, dilute the overnight culture at a 1 to 100 ratio in 25 milliliters of TSB in a 250 milliliter flask, and return the bacteria to the shaking incubator fo