Effects of glutaraldehyde and glycerol on freeze-fractured Escherichia coli

Glutaraldehyde and glycerol are widely used in the freeze-fracture technique as sample pretreatments before rapid freezing. However, they can both introduce relevant structural changes and influence the visualization of the fracture faces and surfaces of membranes. A comparison of the results obtained on E. coli cells differently pretreated with glutaraldehyde and glycerol is presented. In particular the effect on the distribution and density of the intramembranous particles (IMP) is pointed out. Glycerol treatment at 310 K introduces an IMP redistribution, outlined by the appearance of several smooth areas on the fracture faces of the cytoplasmic membrane, which is prevented by glutaraldehyde prefixation at the same temperature. On the other hand, glutaraldehyde treatment at 310 K following glycerol incubation results in the disappearance of the smooth areas, suggesting a substantial change in the IMP distribution caused by the fixative. Cells shifted down to 277 K and treated with glycerol at this temperature before quick-freezing, present on the convex fracture face of the cytoplasmic membrane large smooth areas resulting from a lipid transition while the smaller areas observed at 310 K are not detectable. Glutaraldehyde treatment at 277 K seems also to be responsible for a redistribution of IMP since poorly delimited large smooth areas, containing several IMP, can be observed. In this paper the results of a statistical analysis are also reported, showing that the IMP density can be strongly influenced by pretreatments.