The Phenomenon of Gram-Positivity; Its Definition and Some Negative Evidence on the Causative Role of Sulfhydryl Groups

It has been accepted for many decades that a Gram-positive organism is one which retains the primary dye when stained by accepted Gram stain procedures. It has also been known that the iodine step is essential if Gram differentiation is to be obtained. If bacterial cells are treated in such a way that they will retain the primary dye following a Gram staining procedure, regardless of whether or not the iodine step is included, then the mechanism of this dye retention must differ from that which normally is responsible for a Gram-positive state. Similarly, when both the iodine and decolorization steps are omitted, the counter-stain should always replace the primary stain. If it does not, then the mechanism of dye retention would not be normal, and any such dye retention would not be related to the Gram phenomenon. In such cases one is not studying the Gram reaction, but is studying chemical affinities or physical states which produce visually similar but actually unrelated phenomena. Failure to appreciate this has resulted in papers appearing under the guise of studies of the Gram reaction which have little or no relationship to the Gram phenomenon. In the interest of consistency, these criteria of true Gram-positivity (the necessity of iodine for Gram-positivity with a normal Gram procedure, and the ability of the counterstain to replace the primary dye when both the iodine and decolorization steps are omitted) should be applied to both intact cells and cell-free substances, even though their mechanism of Gram-positivity may differ. The above criteria have been applied in a study of the sulfhydryl concept of the mecharism of Gram-positivity as proposed by Fischer and Larose. It was found that while the experimental work of Fischer and Larose was reproducible, the supposedly Gram-positive states produced did not possess the characteristics which would identify them as true Gram-positive states. Our results would not support the sulfhydryl concept concerning the mechanism of Gram-positivity.