A simple method for determining the outer membrane permeability of gram-negative bacteria in intact cells

The permeability of the intact outer membrane of gram-negative bacteria was determined by weighing the centrifuged pellets of cells treated with hypertonic solutes [10]. The present study refined the existing procedures by focusing on the factors that may affect the results. The study revealed the following findinds: (i) The weight of cells treated with outer membrane-impermeable hypertonic saccharide was significantly reduced and the reduction was proportional to the solute osmolarity. On the other hand, the weight of cells treated with outer membrane-permeable hypertonic saccharide was unchanges. (ii) The centrifugal force up to 165,000 × g for 60 s or NaCl up to 150 mM produced no detectable influence on the assay. (iii) It was possible to measure the outer membrane permeability even in the hypotonic test solutes using plasmolyzed cells. (iv) This method proved equally applicable for measuring the outer membrane permeabilities of both smooth and rough strains. (v) Application of this technique to several species of gram-negative bacteria confirmed the previously reported exclusion limits. All these data show that the weighing of centrifuged pellets of hypertonic solute-treated cells is a useful method for determining the outer membrane permeability of gram-negative bacteria.