In a strain of Bacillus megaterium the Gram reaction was investigated by comparing its effect on intact bacilli with its effects on bacilli treated with lysozyme in several different ways. The lysozyme-treated bacteria varied from bacilli showing only polar separation of the cell wall from the protoplasm to protoplasts free from cell wall. The uptake of the primary dye (crystal violet) by intact bacteria was higher than that of all lysozymedepolymerized cell forms. Iodine uptake depended on the

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... previous uptake of crystal violet. Therefore it was lower for the lysozyme-depolymerized forms. Cell wall took up crystal violet and iodine in small quantity. The same dye iodine complex was formed in all cell structures (cell wall and protoplasm). Gram differentiation was obtained with 95 yo (v/v) ethanol in water, and this was found to extract much less of the dye iodine complex from intact bacteria than from the lysozyme-depolymerized forms. The latter lost about S0-90~0 of their net dye and iodine content, whereas intact bacilli lost only 20-45 %. This was due to the cell wall which formed a barrier to the ethanol extraction of the dye iodine complex, for organisms whose cell walls had been depolymerized or even dissolved by lysozpe during or after the iodine step of the Gram reaction lost much more dye and iodine than intact bacilli. Chemical integrity of the cell wall is a prerequisite for Gram positivity.