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Cells grown at temperatures 22-39oC showed average negative zeta potential (-26) mV that could be only slightly neutralized by octacationic dye. However, at 45oC we recorded two subpopulations of cells with almost neutral zeta potential varying about 0-(-7) mV and highly negative surface charge (-45) mV. The latter was attributed to cells with rough LPSs. Additional incubation of stressed cells at standard temperature led to restoration of their zeta potential (-26) mV and loss of the dye binding ability. The ratio of saturated fatty acids to unsaturated was about 30/70 at 22oC and changed with increasing temperature to 52/48. However, when the temperature changes from 45oC to 37oC, the ratio of saturated and unsaturated acids becomes the same as at 37oC. The results of the MALDI-TOF analysis showed temperature-dependent expression changes of the proteins responsible for the metabolism of the fatty acid. The outer membrane composition of the Pseudomonas aeruginosa PAO1 cells varies considerably with temperature. The described methods can be used in experimental and clinical microbiology to analyze the state of membranes and assess the effects of antibiotics and antiseptics.