Technical Abstract: We have previously shown that Phloxine B, a color additive for foods drugs, and cosmetics, has an immediate antimicrobial effect against Gram-positive bacteria, but not Gram-negative bacteria when used at a lower concentration than that approved for human consumption by the FDA. The data presented here suggest that Phloxine B has a potential use as a nontoxic, faster (< 2 min), inexpensive (350 tests for < 1 cent material) and simpler alternative use to Gram staining. Using Phloxine B staining it was possible to differentiate among Gram-negative and Gram-positive bacteria by visual determination under normal room lighting, light microscopy, fluorescence microscopy and confocal microscopy. This work demonstrated that Phloxine B can be used as a differential versatile bacterial stain and establishes a correlation between the staining properties of the dye and its bactericidal effect.