Question

List three technical reasons that a normal gram-negative cell might stain as if it were gram-positive. For each, explain the reason that this result would be observed.

Answer 1

Gram positive and gram negative staining techniques

Gram staining differentiate between gram positive and gram negative bacteria according to the thickness of peptidoglycan layer in the cell membrane.`Gram positive bacteria retains crystal violet while gram negative not.After decolourization it will take the color of counter stain.

Technical reasons are there in which normal gram-negative cell might stain as gram-positive.

- Use of old cultures

Gram staining works better with cells from cultures that are in the exponential phase of growth. Otherwise the culture may contain dead or ruptured cells. This will give confusing results.

- Longer decolourization

there are two possibilities. 1) If decolourization process carried out for more than 30s 2) if Ethanol wash was not done properly (there is a chance of the smear is too thick)

This may decolourize gram positive cells and give it as gram negative cells.

- Time of heat fixation

If heating the smear for fixation is done for more time the cells may rupture and give false results.

- And also washing and drying of the smear should be done properly.

Otherwise the water will dilute stains and result in false results,