Question

What effect do you suspect the following will have on your judgement of the Gram reaction of an organism?

1) using a greasy, oily, dirty slide

2) failing to drain the smear between staining steps

3) destaining with ethanol for one minute

Answer 1

Please find the answers below:

Gram staining is the method to stain bacteria which differentiates them on the basis of their cell-wall composition. In gram positive bacteria, the cell wall contains a thick peptidoglycan layer. A primary stain of crystal violet is applied and cells are fixed under slight heat. Iodine is then applied to retain the dye inside the cells and then ethanol mediated decolorization is performed. Finally, cells are counter-stained with safranine. Post drying the slides, Gram positive cells give purple color whereas Gram negative cells give reddish pink color.

Part 1) using a greasy, oily, dirty slide:

Answer: If a greasy or oily slide is used, the cells will not stick to the surface of glass slide and rapidly wash out of the system. If a dirty slide is used, a lot of background will appear on the microscopic image making the real image difficult to analyse.

Part 2) failing to drain the smear between staining steps

Answer: If the smear fails to drain between the staining steps, uneven staining might occurs. Also, due to lack of draining between the steps, over-staining can take place. Consequently, a false positive or negative result will be observed.

Part 3) destaining with ethanol for one minute

Answer: Too long decolorization with ethanol can result in removal of stain from both, Gram positive and negative bacteria. The peptidoglycans are particularly sensitive to ethanol and hence only slight and rapid decolorization must be performed. Too long decolorization might result in false positive or negative results.