This study examines the relationship between cell structure and the Gram staining reaction in Bacillus subtilis during lysis. Using sequential staining techniques on the same bacterial cells, the researchers mapped the spatial relationship between the cell wall, an outer layer called ectoplasm, and the Gram-positive staining material. They found that the cell wall lies exterior to the Gram-positive staining area. During autolysis or enzymatic digestion, cells first lose magnesium ribonucleate and become Gram-negative while retaining normal size and their ectoplasm and cell wall staining areas; these cells can be reconverted to Gram-positive by exposure to magnesium ribonucleate. With continued digestion, the ectoplasm and cell wall staining areas disappear, cells become significantly smaller, and can no longer be reconverted to Gram-positive. The authors conclude that the ectoplasm layer is important for the Gram-positive property, though it is not itself responsible for Gram-positivity. Gram-negative bacteria possess a different type of ectoplasm that cannot bind magnesium ribonucleate.

Key findings

• The cell wall is located exterior to the Gram-positive staining material, which lies within the cell wall boundary
• Gram-positive to Gram-negative conversion occurs first through loss of magnesium ribonucleate, with cells retaining normal size and ectoplasm/cell wall staining areas that can be restored to Gram-positive state
• Loss of ectoplasm and cell wall staining areas during autolysis correlates with cell size reduction and inability to reconvert to Gram-positive state
• The ectoplasm layer is necessary for magnesium ribonucleate binding and Gram-positivity, but is not itself the Gram-positive material
• Gram-negative bacteria possess a structurally different ectoplasm that cannot bind magnesium ribonucleate, explaining their permanent Gram-negative staining