One of your lab partners has followed the recommended procedure of running Gram-positive and Gram-negative control organisms on her Gram stain of an unknown species. Her choices of controls were Escherichia coli and Bacillus subtilis. She tries several times and each time concludes she is decolorizing too long because both controls have pink cells (one more than the other). What might you suggest she try and why?

Reduced in holding time of decolrization step and also used less Alcohol because decolrization step is important in Gram's staining.The decolorization step must be performed carefully. Otherwise over-decolorization may occur. This step is critical and must be timed correctly otherwise the CV stain will be removed from the Gram-positive cells. If the decolorizing agent is applied on the cell for too long time, the Gram-positive organisms to appear Gram-negative..

Explanation:

Gram' staining is a technique used in microbiology labs to differentiate between Gram's positive and negative

Gram-positive bacteria :Stain dark purple due to retaining the primary dye called CV in the cell wall.

:Gram-negative bacteria Stain red or pink due to retaining the counter staining dye called Safranin or neutral red.

There are four basic step in Gram" staining

1) Application of the Primary Stain to a Heat-Fixed Smear of Bacterial Culture

2)Addition of Gram's Iodine

3)Decolorization with 95% Ethyl Alcohol:Alcohol or acetone dissolves the lipid outer membrane of Gram-negative bacteria, thus leaving the peptidoglycan layer exposed and increases the porosity of the cell wall. The CV-I complex is then washed away from the thin peptidoglycan layer, leaving Gram-negative bacteria colorless.

On the other hand, alcohol has a dehydrating effect on the cell walls of Gram-positive bacteria that causes the pores of the cell wall to shrink. The CV-I complex gets tightly bound into the multi-layered, highly cross-linked Gram-positive cell wall thus staining the cells purple.

The decolorization step must be performed carefully. Otherwise over-decolorization may occur. This step is critical and must be timed correctly otherwise the CV stain will be removed from the Gram-positive cells. If the decolorizing agent is applied on the cell for too long time, the Gram-positive organisms to appear Gram-negative. Under-decolorization occurs when the alcohol is not left on long enough to wash out the CV-I complex from the Gram-negative cells, resulting in Gram-negative bacteria to appear Gram-positive.

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