This article details the process of preparing protoplasts from Bacillus megaterium, a gram-positive bacterium. The method involves the degradation of the bacterial cell wall using lysozyme, resulting in spherical protoplasts.
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 flask for a 4.5 hour incubation at 37 degrees Celsius with shaking. When the liquid culture reaches an optical density of 0.9 to 1.0 at 600 nanometers, split the bacteria between two 50 milliliter conical tubes for their centrifugation, and use a syrological pipette to aspirate the supernatants. Resuspend the pellets in 2.5 milliliters of protoplast medium per tube, and pull the suspension into a single conical tube for culture in a 125 milliliter flask.
Add one milliliter of five milligrams per milliliter lysozyme to the culture for a one hour incubation at 37 degrees Celsius with shaking. Monitoring the growth of the protoplast under the light microscope and noting any irregularities, such as bacteria that appear as swollen rods instead of spheres. At the end of the incubation, transfer the suspension to a 15 milliliter conical tube for centrifugation, and re suspend the pellet in five milliliters of protoplast medium.
The protoplast can then be stored at minus 20 degrees Celsius for up to a week or until they have gone through three freeze thaw cycles.
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