Question

Arrange the following steps in a correct sequence as per Gram's staining technique:
Treatment with 0.5% iodine solution (1), washing with water. (2) treatment with absolute alcohol/acetone (3), staining with weak alkaline solution of crystal violet (4)

• A. 4 $\to$1 $\to$2 $\to$3
• B. 3 $\to$2 $\to$1 $\to$4
• C. 3 $\to$1 $\to$2 $\to$4
• D. 4 $\to$2 $\to$3 $\to$1

Answer 1

Answer: (A)

4 $\to$1 $\to$2 $\to$3

Bacteria can be classified into two groups based upon reaction bacteria to Gram's stain (Christian Gram, 1884. Bacteria are first stained with weakly alkaline solution of crystal violet and then treated with 0.5% iodine solution. This is followed by washing with water and then with alcohol or acetone. Bacteria which retain blue or purple colour are known as Gram (+) bacteria (e.g Bacillus subtilis). Bacteria which do not retain stain and become colourless are called Gram (-) bacteria (e.g. Escherichia coli). Washing of the stain in Gram (- ve) bacteria is due to high lipid content of their cell wall which gets dissolved in organic solvents like acetone. So, the correct answer is '4 →1 →2 →3'.