Generation of Spheroplasts from Gram-Negative Bacteria

Source: Figueroa, D. et. al. Production and Visualization of Bacterial Spheroplasts and Protoplasts to Characterize Antimicrobial Peptide Localization. J. Vis. Exp. (2018).This video demonstrates the preparation of spheroplasts from rod-shaped, gram-negative bacteria. Bacteria are grown in the presence of an antibiotic to form elongated filaments. These are washed with a hypertonic solution and treated with a lysis buffer to remove the outer cell wall. Hypotonic treatment forms spheroplasts, which are stabilized with a chilled isotonic solution and stored under cold conditions.