Gram Stain

DISCUSSION:

Gram stain

Cell wall reacts differently with different reagents. Gram staining is a common technique used to differentiate two large groups of bacteria, which are the gram positive and gram negative bacteria, based on their different cell wall constituents. Gram positive bacteria stain violet or purple blue due to the presence of a thick peptidoglycan layer in their cell walls. This thick peptidoglycan cell wall retains the crystal violet molecules. When iodine is applied, the crystal violet reacts with iodine to form crystal violet-iodine complex (CV-I complex). This complex are too large to escape through cell wall, as such it retains the purple colour. Alternatively, gram negative bacteria stain pink, which is attributed to a thinner peptidoglycan wall. When alcohol is applied, it disrupts the outer wall of gram negative cells due to the presence of lipopolysaccharide in the cell wall causing the primary stain to be washed away. At the end, gram negative cells take safranin stain which causes it to appear as pink under microscope.

In this experiment, gram staining started off with heat fixing a loopful of bacteria on the slide and flooding the smear with crystal violet stain. Crystal violet is a purple dye that sticks to the peptidoglycan layer of the cell walls of Vibrio parahaemolyticus. It was then rinsed off and iodine solution was added as a mordant to form large crystals with the crystal violet molecules. As mention above, these crystals were too large to escape through cell walls. Next, alcohol was then added for decolorization. The alcohol dissolved the lipopolysaccharides at the outer membranes of the bacterial cells. The removal of the lipid layer enhances the leaching of the primary stain from the cells into the surrounding solvent. At this moment the cells were colorless. The length of the decolorization is critical as prolonged exposure to the decolorizing agent will remove all the stain from both gram positive and gram negative...