Relationship of cell wall staining to gram differentiation.

Bartholomew and Mittwer (1951) presented photographic evidence that the gram-positive staining area of bacterial cells could be fitted internally to the area demonstrated by the Dyar cell wall stain. Their conclusion was that the bacterial cell wall was not included in the grampositive area. Lamanna and Mallette (1954) explained Gram differentiation of intact cells on the basis that crystal violet stained and formed a dye-iodine lake in the cell walls of gram-positive cells, but did not stain and therefore did not form a dye-iodine lake in the cell walls of gram-negative cells. They reported also that if normally gram-positive cells were stained with crystal violet, and then were washed with tap water, the dye was removed from the cell wall and on subsequent completion of the Gram stain these cells were gram-negative. It was their conclusion that the cell wall of intact cells must be stained with the primary dye for the cells to result in gram-positive staining, and failure of the dye to be present in the cell wall was the explanation of all gram-negative staining. Although the evidence presented in their paper was primarily for yeast cells, they reported that similar observations had been made for several species of gram-positive and gram-negative bacteria. Hence, they believed that the above basis for Gram differentiation could be generalized for all cases of gram-positive and gram-negative staining. The present paper concerns the results of additional studies on the relationship of cell wall staining to Gram differentiation for bacterial cells. Particularly studied was the effect of a wash step following the primary dye on apparent cell size, and also the effect of this wash step on the results obtained when the Gram stain procedure was completed. It was hoped that an explanation could be found for the conflicting viewpoints as