A Study of the Spheroplast Observations for W303 Single Cells under Environmental-SEM

In this paper, the preparation and observations of the spheroplast W303 single cells are described. Spheroplasting which is a process for removing a cell wall from a whole cell is often required for many applications. The successful of the spheroplasting process were confirmed both qualitatively, i.e. the evaluation of the morphological change between the whole W303 cells and the spheroplast W303 cells, and quantitatively, i.e. determination of the spheroplast conversion percentage based on the OD₈₀₀ absorbance data. It is expected that under optical microscope observations, whole cells appear an oval shape whereas spheroplast cells resemble a spherical shape. This expectation was proved in our experiment. We have compared the shape of the whole and the spheroplast of W303 cells inside four mediums, i.e. yeast peptone-dextrose (YPD), sterile water, sorbitol-EDTA-sodium citrate buffer (SCE) and sorbitol-Tris-Hcl-CaCl₂ (CaS). The whole W303 cells exhibited an oval shape inside all mediums. On the other hand, a spherical shape was clearly observed for spheroplast W303 cells inside all mediums where the SCE and CaS mediums have a higher number of observed spheroplast cells as compared to the YPD and sterile water mediums. The OD₈₀₀ absorbance data also revealed that the whole W303 cells were fully converted to the spheroplast cells after about 15 minutes of the spheroplasting process. The observations of the whole and the spheroplast W303 cells were then performed under an environmental scanning electron microscope (ESEM).