This article describes a method for preparing spheroplasts from Gram-negative bacteria, specifically E. coli, using a cell division-blocking antibiotic. The process involves several steps including incubation, centrifugation, and the use of specific reagents to weaken the bacterial cell wall.
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 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 two equal aliquots between two 15 milliliter conical tubes containing seven milliliters of four degrees 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 resuspend 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.
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