Question

A student is performing a gram stain of a mixed culture of both

e. coli and s. aureus and he forgets to decolorize with ethanol. what should his slide look like?

Answer 1

If this happens, the crystal violet and the gram's iodine that has been stained in the smear wouldn't be washed out, it could make his slide or smear look like it has a mix color of violet, blue and orange, as well as having the safranin orange stained as the last. The decolorization of ethanol is important because it washes of unnecessary stain that is not essential to the smear, not using it will cause undesired results.

Answer 2

Both types of bacteria appear to be dark purple (stained with gentian violet), both will have a gram-positive appearance.

In the gram stain, two dyes are used: gentian violet and Fuschia pink, the two stains are separated by alcohol bleaching.

Alcohol bleaching (+ acetone) is the most important step in coloring. It must be fast in order to eliminate the rich lipid wall of the gram negative bacteria that has retained the staining, to then be colored by the pink Fuschia.

Beware, the abuse of alcohol will result in all Gram negative bacteria.