This study by Scherrer (1963) investigated the Gram staining mechanism in Bacillus megaterium by comparing intact cells with lysozyme-treated variants, including protoplasts and forms showing polar cell wall separation. Crystal violet uptake was consistently higher in intact bacteria than in lysozyme-depolymerized forms. Iodine uptake depended on prior crystal violet uptake, forming the same dye-iodine complex in all cellular structures. The critical finding was that intact bacteria retained 55-80% of their dye-iodine complex when treated with 95% ethanol, whereas lysozyme-depolymerized forms lost 80-90%, demonstrating that cell wall chemical integrity—not merely dye content—determines Gram positivity. The intact cell wall acts as a barrier against ethanol extraction of the dye-iodine complex. When lysozyme was applied during or after Gram staining, it further enhanced dye extraction from already-treated cells. These results establish that structural integrity of the bacterial cell wall is essential for maintaining the Gram-positive reaction.

Key findings

• Intact Bacillus megaterium cells retain 55-80% of their dye-iodine complex during ethanol decolorization, while lysozyme-treated cells lose 80-90%, demonstrating the cell wall's barrier function
• Crystal violet uptake was consistently higher in intact bacteria than in lysozyme-depolymerized forms, indicating lysozyme altered dye absorption properties before complete cell wall dissolution
• The same dye-iodine complex forms in both cell wall and protoplasm, but the intact cell wall's chemical integrity—not its dye content—is essential for Gram positivity
• Cell wall chemical integrity acts as a barrier preventing ethanol extraction of the dye-iodine complex, explaining why Gram-positive bacteria remain stained after decolorization