Generating Protoplasts from the Gram-Positive Bacterium Bacillus megaterium

Take a culture of Bacillus megaterium, a gram-positive bacterium.

Dilute the culture in fresh media and incubate it with shaking to promote bacterial division.

Transfer the culture to a tube and centrifuge to pellet the bacteria.

Remove the supernatant and resuspend the pellet in media containing osmotic stabilizers to prevent protoplast lysis.

Transfer the resuspended solution to a flask, add lysozyme, and incubate with shaking.

During incubation, lysozyme degrades the thick peptidoglycan cell wall of the bacteria.

As the cell wall degrades, the bacteria change from a rod-shaped form to spherical bodies bounded only by the plasma membrane, forming protoplasts.

Visualize the suspension under a microscope.

The round-shaped bacteria confirm protoplast formation, while the swollen rod-shaped bacteria indicate incomplete digestion.

Transfer the solution to a tube and centrifuge to pellet the protoplasts.

Remove the supernatant and resuspend the protoplasts in fresh media containing osmotic stabilizers for further analysis.

To prepare Gram-positive B. megaterium protoplasts, dilute the overnight bacterial culture at a one to 1000 ratio in 100 milliliters of three percent TSB in a 250 milliliter fl