Preparation of E. coli Spheroplasts

Take an E. coli culture.

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

Next, dilute the culture in media containing a bacterial division inhibitor and incubate.

The bacteria continue to grow without dividing, forming elongated filaments.

Transfer the culture to a fresh tube, then centrifuge to pellet the filaments and remove the supernatant.

Wash the pellet with an osmotic stabilizer to prevent spheroplast lysis. Remove the solution.

Add buffer, lysozyme, DNase, and a chelating agent sequentially. Incubate.

The chelating agent destabilizes the filament’s outer membrane.

Lysozyme degrades the exposed peptidoglycan layer, causing filaments to lose shape and form spherical structures called spheroplasts.

Additionally, DNase degrades contaminating DNA released from damaged filaments.

Add the osmotic stabilizer again, mix, and incubate.

Transfer the suspension to a tube containing buffer and centrifuge.

Remove excess supernatant and resuspend the spheroplasts in buffer for downstream analysis.

To set up an overnight culture, use a sterile micropipette tip to transfer a single bacterial colony from a solid agar culture plate to a 14 milliliter culture tube, containi