Application of Environmental Scanning Electron Microscope-Nanomanipulation System on Spheroplast Yeast Cells Surface Observation

The preparation and observations of spheroplast W303 cells are described with Environmental Scanning Electron Microscope(ESEM). The spheroplasting conversion was successfully confirmed qualitatively, by the evaluation of the morphological changebetween the normal W303 cells and the spheroplast W303 cells, and quantitatively, by determining the spheroplast conversionpercentage based on the OD800absorbance data. From the optical microscope observations as expected, the normal cells had anoval shape whereas spheroplast cells resemble a spherical shape. This was also confirmed under four different mediums, that is,yeast peptone-dextrose (YPD), sterile water, sorbitol-EDTA-sodium citrate buffer (SCE), and sorbitol-Tris-Hcl-CaCl2(CaS). It wasalso observed that the SCE and CaS mediums had a higher number of spheroplast cells as compared to the YPD and sterile watermediums. The OD800absorbance data also showed that the whole W303 cells were fully converted to the spheroplast cells afterabout 15 minutes. The observations of the normal and the spheroplast W303 cells were then performed under an environmentalscanning electron microscope (ESEM). The normal cells showed a smooth cell surface whereas the spheroplast cells had a bleb-likesurface after the loss of its integrity when removing the cell wall