Gram Stain Analysis Questions Students are expected to answer the following questions in the analysis section once they have completed the gram stain videos: 1. Predict the effect on Gram-positive and Gram-negative cells of the following mistakes made when performing a gram stain. Consider each mistake independently: a. Failure to add the iodine. b. Failure to apply the decolorizer. c. Failure to apply the safranin. d. Reversal of crystal violet and safranin stains. 2. Both crystal violet and safranin are basic stains and may be used to do simple stains on Gram-positive and Gram-negative cells. In light of this, explain how they end up staining Gram-positive and Gram-negative cells differently in the Gram Stain. 3. What is the mordant for the gram stain? What does the mordant do in a gram stain?
The Correct Answer and Explanation is:
1. Effects of Mistakes in Gram Staining:
a. Failure to Add Iodine:
The iodine acts as a mordant, forming a complex with crystal violet inside the cell. This crystal violet-iodine complex becomes trapped within the thicker peptidoglycan layer of Gram-positive cells, making them appear purple. If iodine is omitted, the crystal violet will not bind as effectively, and the Gram-positive cells may appear faint or incorrectly colored.
b. Failure to Apply the Decolorizer:
Decolorization differentiates between Gram-positive and Gram-negative cells. Gram-negative cells have a thinner peptidoglycan layer and a lipid-rich outer membrane. The decolorizer (usually alcohol or acetone) breaks down the outer membrane of Gram-negative cells, causing them to lose the crystal violet stain and take up the safranin, turning them pink. Without decolorization, Gram-negative cells would retain the crystal violet and appear purple, misleading the result.
c. Failure to Apply Safranin:
Safranin is the counterstain that colors Gram-negative cells pink after the decolorization step. If safranin is not applied, Gram-negative cells will remain colorless after decolorization. This could make it difficult to distinguish between Gram-positive and Gram-negative cells since only Gram-positive cells would appear purple.
d. Reversal of Crystal Violet and Safranin Stains:
If crystal violet is used as the counterstain and safranin as the primary stain, both cells will likely appear purple. This is because the crystal violet is the more intense stain and will dominate, even for Gram-negative cells. The cell wall structure will not affect the color outcome in this scenario, and the distinction between Gram-positive and Gram-negative cells will be lost.
2. Difference in Staining Between Gram-positive and Gram-negative Cells:
Both crystal violet and safranin are basic stains, meaning they have a positive charge and bind to the negatively charged components of the cell wall. However, the reason they stain Gram-positive and Gram-negative cells differently lies in the differences in cell wall structure:
- Gram-positive cells have a thick peptidoglycan layer, which holds the crystal violet stain after iodine mordant is applied. This thick layer traps the crystal violet-iodine complex inside the cell, which gives the Gram-positive cells their purple color.
- Gram-negative cells have a thinner peptidoglycan layer and an outer membrane. The crystal violet stain is washed out during decolorization because the outer membrane is disrupted, and the stain cannot bind as strongly. After decolorization, the Gram-negative cells are stained with safranin, giving them a pink color.
3. The Mordant and Its Role:
The mordant in the Gram stain is iodine. A mordant is a substance that enhances the binding of a dye to the cell structure. In the Gram stain, iodine forms a complex with the crystal violet stain inside the cell. This complex is too large to easily escape from the thick peptidoglycan layer in Gram-positive cells. In contrast, Gram-negative cells have a thinner peptidoglycan layer and an outer membrane that is disrupted during decolorization, allowing the crystal violet to wash out. The iodine helps retain the crystal violet in Gram-positive cells, making it crucial for the differentiation between Gram-positive and Gram-negative cells.
