INTRODUCTION The Gram stain was developed in 1884 by the Danish bacteriologist Hans Christian Gram. It is one of the most useful staining procedures because it classifies bacteria into two large groups: 1) gram-positive and 2) gram-negative. PROCEDURE 1) A heat-fixed smear is flooded with a basic purple dye, usually crystal violet. Because the purple stain imparts its color to all cells, it is referred to as a primary stain . 2) After 1 minute, the crystal violet is drained off and washed with distilled water. The smear is then covered with Gram's iodine, a mordant or helper . When the iodine is washed off, both gram-positive and gram-negative bacteria appear dark violet or purple. 3) Next, the slide is washed with alcohol (95% ethanol) or an alcohol-acetone solution. This solution is a decolorizing agent which removes the purple from the cells of some species but not from others. When the procedure is carried out, the slide is held ...