Negative staining of freeze-fractured envelopes of Escherichia coli K12

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Abstract
Envelope fragments of E. coli K12 were produced by freeze-fracturing by hand and negatively stained after thawing. The outer leaflet of the plasma membrane disintegrated upon thawing whereas the outer leaflet of the outer membrane did not. Negative staining revealed the following structural features on the outer membrane fragments: grooves 4-6 nm wide, spherical particles 6-8 nm in diameter, black dots 3-8 nm in diameter. Treatment of cells with EDTA before freeze-fracturing caused dilatation of grooves into holes eventually leading to fragmentation of the outer membrane. A mutant strain deficient in 2 outer membrane proteins fractured exclusively through the outer membrane. The outer leaflets so obtained disintegrated upon thawing similarly as observed for the outer leaflet of the plasma membrane.