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

The performance of Gram stain on any sample requires four basic steps, including applying the main color (crystal violet) to the thermoset smear, then adding the mordant (Gram iodine), using alcohol, acetone or alcohol and acetone the mixture, finally counterstained with.

Complete answer:
Washing with water: Because it may be contaminated if you put your hands in a previously used contaminated pool, clean tap water should be used. However, cleaning with non-potable water when necessary can still improve health. The temperature of the water does not seem to rise. Affects the removal of microorganisms; however, warm water may cause more skin irritation and cause greater damage to the environment

Treatment with absolute alcohol/acetone: Once the slides of the bacterial sample are prepared, first stain the sample with crystal violet. At this time, both gram-positive bacteria and gram-negative bacteria will appear purple. Usually, the purple crystal is applied to the glass slide for 30 seconds, and then the excess stain is rinsed off with water. Crystal violet can slightly adhere to the peptidoglycan layer, so not all major stains will be washed off by water.

Staining with weak alkaline solution of crystal violet: First stain the bacteria with a weak alkaline solution of crystal violet, and then treat it with a 0.5% iodine solution. Then wash with water, then with alcohol or acetone. Bacteria that retain the blue or purple color are called Gram (+) bacteria (such as Bacillus subtilis).

Hence, the correct answer is option (D)

Note: Rinse with water after 5 seconds. The exact time to stop is when the solvent is no longer staining when flowing through the slide. A further delay will cause excessive discoloration of the Gram-positive cells and will defeat the purpose of staining. Wash the solution with water.