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, containing two to three milliliters of three percent trypticase soy broth, TSB, for a 16 to 21 hour incubation at 37 degrees celsius with shaking. To prepare Gram-negative E. coli spheroplasts, dilute the overnight culture at one to 100 ratio, and 25 milliliter of TSB in a 250 milliliter flask, and return the bacteria to the shaking incubator for another 2.5 hours. When the solution has reached an optical density of 0.5 to 0.8 at 600 nanometers, dilute the culture at a one to ten ratio in fresh TSB in a new 250 milliliter flask for a second 2.5 hour incubation with a final effective concentration of 60 micrograms per milliliter of cephalexin.

To produce single cell filaments of about 50 to 150 micrometers in length, as visualized under a light microscope at a 1000x magnification. Centrifuge the bacterial solution to harvest the filaments and decant the supernatant. Wash the filaments with the gentle addition of one milliliter of 0.8 molar sucrose, taking care not to disturb the pellet.

After one minute discard the supernatant without disturbing the pellet, and add the indicated reagents in their respective order to the filaments. After ten minutes at room temperature, gradually add one milliliter of solution A over a period of one minute, while gently swirling the solution by hand. Incubate the solution for four minutes at room temperature, then split the filament suspension into to two equal aliquots between two 15 milliliter conical tubes containing seven milliliters of four degree Celsius solution B. Next, pellet the filaments by centrifugation, and use a serological pipette to carefully remove all but one to two milliliters of the supernatant without disturbing the pellets.

Use a p1000 micropipette to gently re suspend the pellets. Check a small aliquot of the sample for spheroplast formation by light microscopy. The spheroplast can then be stored at minus 20 degrees Celsius for up to a week or until they have gone through three freeze thaw cycles.