Question

Describe the various steps in Gram's staining procedure?

Answer 1

Hint: Gram staining is a procedure which is usually based upon the capability of the bacterial cell wall to preserve the crystal violet dye when treated with solvent.

Complete Answer:
Generally the cell wall of the Gram-positive bacteria exhibits a greater amount of peptidoglycan and lesser amount of lipids as compared to the gram-negative bacteria.

The various steps involved in Gram’s staining are detailed below:
Step - 1: The sample is adhered over the slide and is stained, under the aseptic conditions.
Step - 2: Initially the sample is stained with crystal violet dye.
Step - 3: Excess stain over the treated sample is removed under the slow water flow.
Step - 4: To the treated slide Gram's iodine is added.
Step - 5: Again after addition, excess of stain is removed under the slow water flow.
Step - 6: At last the counter stain (safranin) is added over the treated slide.
Step - 7: After this again the necessary amount of water is permitted to flow slowly to eliminate excess stains.
Step - 8: The treated slide/s is then observed under the compound microscope.
Step - 9: Finally, as per the observation, if crystal violet stain (violet color) is preserved, then the sample is recognized as Gram positive, while it does not appear violet and appears as pink then it is recognized as Gram negative.

Note: Usually, during the process of Gram’s staining three types of strains are used, named as crystal violet, Gram's iodine and safranin. Crystal violet dye is also called as primary stain and its main role is to impart its coloration to all the treated cells. Safranin is also called as counter strain and helps in specified identification of bacterial cell type.