Yeast Spheroplasting
Yeast Spheroplasting - Related Videos
04:30
Preparation of E. coli Spheroplasts
0 Views •
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
0 Views •
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,...
06:49
Isolating Nucleic Acids from Yeast
0 Views •
2023
One of the many advantages to using yeast as a model system is that large quantities of biomacromolecules, including nucleic acids (DNA and RNA), can be purified from the cultured cells. This video will address the steps required to carry out nucleic acid extraction. We will begin by briefly outlining the growth and harvest, and lysis of yeast cells, which are the initial steps common to the isolation of all biomacromolecules. Next, we will discuss two unique purification methods for the...
08:30
Yeast Transformation and Cloning
0 Views •
2023
S. cerevisiae are unicellular eukaryotes that are a commonly-used model organism in biological research. In the course of their work, yeast researchers rely upon the fundamental technique of transformation (the uptake of foreign DNA by the cell) to control gene expression, induce genetic deletions, express recombinant proteins, and label subcellular structures. This video provides an overview of how and why yeast transformation is carried out in the lab. The important features of yeast...
08:30
Yeast Maintenance
0 Views •
2023
Research performed in the yeast Saccharomyces cerevisiae has significantly improved our understanding of important cellular phenomona such as regulation of the cell cycle, aging, and cell death. The many benefits of working with S. cerevisiae include the facts that they are inexpensive to grow in the lab and that many ready-to-use strains are now commercially available. Nevertheless, proper maintenance of this organism is critical for successful experiments. This video will provide an overview...
FAQs
Lyticase and zymolyase act enzymatically on yeast cell-wall components, particularly glucans and mannoproteins. By hydrolyzing these materials, they weaken the wall in a controlled way while the plasma membrane remains the cell’s boundary. Enzyme exposure therefore determines how completely wall components are removed, which directly affects subsequent transformation, fusion, or membrane-focused experiments.
Sorbitol maintains osmotic stabilization after wall removal, when the plasma membrane no longer has rigid wall support. Without this protection, water movement and osmotic imbalance can cause fragile cells to burst. Thus, sorbitol is not merely a reaction additive: it preserves spheroplast integrity during preparation and handling, improving the chance that viable cells remain for downstream work.
The main variables are enzyme exposure, mechanical stress, and osmotic conditions. Excessive or poorly controlled enzyme treatment can compromise the resulting spheroplasts, while pipetting or other mechanical handling can damage their unprotected membranes. Maintaining appropriate osmotic stabilization and gentle handling supports viability and makes conversion more reproducible across experiments.
A basic workflow begins with enzymatic exposure of yeast to lyticase or zymolyase under sorbitol-based osmotic stabilization. The preparation then requires careful handling because the converted cells are fragile and sensitive to osmotic changes and mechanical stress. Preserving these conditions throughout the process helps retain intact, viable spheroplasts for the intended assay.
Yeast spheroplasting is useful when a rigid wall would obstruct access to the plasma membrane or interfere with cell entry and contact. The resulting cells can support DNA transformation and cell fusion, while their altered boundary also makes them suitable for membrane and organelle studies. Researchers can therefore select the technique for molecular delivery, cellular interaction, or structural analysis.
In biology, this technique provides a way to examine cell-wall assembly by focusing on cells after wall glucans and mannoproteins have been hydrolyzed. The resulting preparation helps researchers analyze how these wall components relate to cellular structure and integrity. This makes spheroplasting relevant to molecular biology as well as cellular studies of yeast architecture.