Analysis of Escherichia coli cell damage induced by HPCD using microscopies and fluorescent staining.

Cellular damage of Escherichia coli (E. coli) induced by high pressure carbon dioxide (HPCD) at 37-57°C and 10-30 MPa for 5-75 min was investigated using scanning electronic microscopy (SEM), transmission electronic microscopy (TEM), confocal laser scanning microscopy (CLSM), and fluorospectrophotometer (FSM). HPCD-induced alterations in the morphology and the intracellular organization of E. coli cells was more susceptible to HPCD. A vast majority of HPCD-treated E. coli cells with seemingly intact morphology sustained severe damage in their intracellular organization. CLSM suggested that initial disruption of the outer membrane and later permeabilization of the cytoplasmic membrane of HPCD-treated E. coli cells was a consecutive and progressive process. These results were confirmed by FSM with the probes PI and SYTO 9. The membrane fluidity of HPCD-treated E. coli cells decreased as suggested by increased fluorescence polarization using FSM with the probe 1,6-diphenyl-1,3,5-hexatriene (DPH). The temperatures of 37, 42 and 47°C alone showed no impact on the outer membrane and membrane fluidity of E. coli cells whereas 57°C alone had greater impact on them. Combined with HPCD, the temperatures of 37, 42 and 47°C disrupted the outer membrane of E. coli cells without damage to the cytoplasmic membrane and of 57°C damaged the cytoplasmic membrane, but all these temperatures decreased the membrane fluidity of E. coli cells. Higher temperature increased HPCD-induced outer membrane disruption and the cytoplasmic membrane damage and decreased the membrane fluidity.