Bacterial Spheroplasts
Bacterial Spheroplasts - Related Videos
04:30
Preparation of E. coli Spheroplasts
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2025
Source: Figueroa, D. M., et.al. Production and Visualization of Bacterial Spheroplasts and Protoplasts to Characterize Antimicrobial Peptide Localization. J. Vis. Exp. (2018)This video demonstrates the production of Escherichia coli spheroplasts by first inhibiting cell division to induce filamentation, followed by enzymatic digestion of the cell wall. The process includes controlled addition of a chelating agent, DNase, and an osmotic stabilizer to ensure proper spheroplast formation and...
04:02
Generation of Spheroplasts from Gram-Negative Bacteria
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2026
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,...
10:13
Production and Visualization of Bacterial Spheroplasts and Protoplasts to Characterize Antimicrobial Peptide Localization
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Cited by 13 •
2018
Here we present a protocol to produce gram-negative Escherichia coli (E. coli) spheroplasts and gram-positive Bacillus megaterium (B. megaterium) protoplasts to clearly visualize and rapidly characterize peptide-bacteria interactions. This provides a systematic method to define membrane localizing and translocating peptides.
12:28
Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments
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Cited by 8 •
2016
We provide a method to simultaneously screen a library of antibody fragments for binding affinity and cytoplasmic solubility by using the Escherichia coli twin-arginine translocation pathway, which has an inherent quality control mechanism for intracellular protein folding, to display the antibody fragments on the inner membrane.
05:21
Bacterial Transformation - Student Protocol
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2019
Bacterial Transformation ExpandIMPORTANT: In addition to wearing the appropriate personal protective equipment, be sure to take care to keep your face away from suspension cultures, and to avoid inhaling reagents. Do not touch your face while performing the experiment. Always wash your hands before and after every experiment. When you are ready to safely begin, check to make sure you have a tube or tubes of competent E. coli cells in your ice bucket. Then, transfer 50 µL from one of these tubes...
FAQs
The two reagents act on different envelope features. Lysozyme hydrolyzes peptidoglycan, while EDTA destabilizes the outer membrane of Gram-negative cells, making the wall structure more accessible to removal. Their combined action produces a weakened envelope rather than simply disrupting the plasma membrane, which allows investigators to examine how envelope components contribute to cell integrity.
Osmotic stabilizers are essential because the plasma membrane no longer receives normal mechanical support from the wall. They reduce the osmotic stress that would otherwise drive water movement and rupture the fragile cells. The stabilizing environment therefore affects spheroplast survival and determines whether experiments can preserve intact membrane-bound cells for further analysis.
With much of the rigid barrier weakened, effects involving the plasma membrane and envelope become easier to observe without the full wall masking them. At the same time, remaining envelope structure can support studies of wall regeneration. This makes spheroplasts a model for linking envelope architecture with membrane behavior and recovery.
Preparation requires coordinated treatment with lysozyme and EDTA in the presence of an osmotic stabilizer. The cells must then be protected from mechanical and osmotic stress, because their weakened envelope can rupture readily. Controlling these conditions is central to obtaining a population suitable for downstream transformation, membrane studies, or other experiments rather than uncontrolled lysis.
Their weakened envelope can make access to the cell interior more feasible for experimental manipulation. Consequently, spheroplasts support bacterial transformation and intracellular delivery research, where investigators need to introduce material into cells. The same vulnerability also makes them useful for controlled cell lysis, although successful work depends on maintaining conditions that prevent premature rupture.
Spheroplasts can serve as experimental partners for examining membrane fusion because their altered envelope exposes membrane behavior that is less constrained by an intact peptidoglycan layer. This application connects envelope remodeling with questions about membrane interactions and delivery. It also highlights why osmotic protection and gentle handling matter during fusion experiments.