Pseudomonas diminuta (ATCC 19146) has been typically used in the bacterial challenge test for validation of the sterilizing filtration process. Cell size is critical for determining the retention characteristics of membrane filters with pore-size of $0.2{mu}m$. The changes of cell sizes after osmotic shocks at 150, 260, 500, and 700 mosM were measured by a particle size analyzer and the changes of their buoyant densities were analyzed with a Percoll gradient. The results indicated that there were no significant differences when cells were cultured in 260 mosM medium and osmotically shocked at 500 and 700 mosM. However, the osmotically shocked cells at 150 mosM showed a 38% increase of the cell size compared to the cells at 260 mosM. From these study, we concluded that the worst case condition for validation of a sterilizing filter would be 500 mosM, not because of changes in the cell size, but due to decrease in cell viability under those conditions.