Question

Both crystal violet and safranin are basic stains and may be used to do simple stains on Gram-positive and Gram-negative cells. This being the case, explain how they end up staining Gram-positive and Gram-negative cells differently in the Gram stain.

Answer 1

Answer:

Gram positive cells retains purple stains of crystal violet, gram-negative cells takes up red or pink stain of safranin

Explanation:

Gram staining is a simple method of distinguishing between Gram-positive cells and Gram-negative cells. The method make use of the following steps

Heat fix cells on a glass slide and then:

1. Stain cell with crystal violet for 1 min

2. Rinse with water

3. Decolourize with alcohol for 30 seconds and wash with water

4. flood cells with iodine as mordant

5. Flush with water

6. Counterstain with safranin for 1 min and wash with water

7. View under the microscope

Gram-positive cell possesses a thick peptidoglycan in its cell wall which helps retain the purple colour of the crystal violet, While Gram-negative cells will loose out this colour when flushed with alcohol but takes up the red or pink colour when stained with safranin.

Answer 2

Answer:

Due to differences in the thickness of a peptidoglycan layer in the cell membrane between Gram positive and Gram negative bacteria, Gram positive bacteria (with a thicker peptidoglycan layer) retain crystal violet stain during the decolorization process, while Gram negative bacteria lose the crystal violet stain and are instead stained by the safranin in the final staining process.

Explanation:

The process involves three steps:

Cells are stained with crystal violet dye. Next, a Gram's iodine solution (iodine and potassium iodide) is added to form a complex between the crystal violet and iodine. This complex is a larger molecule than the original crystal violet stain and iodine and is insoluble in water.

A decolorizer such as ethyl alcohol or acetone is added to the sample, which dehydrates the peptidoglycan layer, shrinking and tightening it. The large crystal violet-iodine complex is not able to penetrate this tightened peptidoglycan layer, and is thus trapped in the cell in Gram positive bacteria. Conversely, the the outer membrane of Gram negative bacteria is degraded and the thinner peptidoglycan layer of Gram negative cells is unable to retain the crystal violet-iodine complex and the color is lost.

A counterstain, such as the weakly water soluble safranin, is added to the sample, staining it red. Since the safranin is lighter than crystal violet, it does not disrupt the purple coloration in Gram positive cells. However, the decolorized Gram negative cells are stained red.