I was processing the cells ( S. aureus ) for SEM. I have done 3 times on different day's with same protocol and same concentration of fixative, 2.5% glutaraldehyde. The first trail was done only for the untreated cells and the cells were very intact, round and smooth in SEM image. The 2nd time was done both for the untreated and treated cells, here the treated cells are more intact then the untreated cells ( not the expected result ). Third time was again done only for the untreated cells, here the cells become shrunken which is suppose to be round and intact as in first image.

What could be the possible reason for this problem? Please share your comment.