1. Gram staining involves three processes: staining with a water-soluble dye called crystal violet, decolorization, and Counterstaining, usually with safanin

2. Dyes known to produce such effect include cryptocyanine, rhodamine-B, rose bengal, crystal violet, and dyes of the quinone family such as Benenzo-quinone.

3. Difficile, associated with gastrointestinal illness, is one of the Gram-positive Clostridium that will appear dark blue/violet having retained the primary stain (crystal violet) in their thick peptidoglycan layer.

4. Physiology No pigment is produced and growth occurs at 30, 35 and 45 °C. Isolates grow on MacConkey agar without crystal violet and in the presence of 5% NaCl, are negative for arylsulfatase activity at three days.

5. Cells of UMR1899 accumulated glutamate under pH stress, and were markedly hydrophobic and resistant to the effects of crystal violet, the latter traits raising the possibility that outer membrane composition and structure could also be a factor in pH tolerance.