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. Gram-positive will be purple, Gram-negative will be colorless. d. Reversal of crystal violet and safranin stains. Would not be able to read. Both would appear purple, because during decolorization the safranin will come out of the cell, and iodine will bind to crystal violet only and hence give the first purple color. 2 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. 3 If you saw large, eukaryotic cells in the preparation made from your gumline, they were most likely your own epithelial cells. Are you Gram-positive or Gram-negative? (You can make a good guess about this even if you didn’t see your cells.) 4 One of your lab partners has followed the recommended procedure of running Gram-positive and Gram-negative control organisms on her Gram stain of an unknown species. Her choices of controls were Escherichia coli and Bacillus subtilis. She tries several times and each time concludes she is decolorizing too long because both controls have pink cells (one more than the other). What might you suggest she try and why? She must try the experiment with new cultures because old culture lose the ability to retain the crystal violet. 202 MICROBIOLOGY Laboratory Theory & Application
The Correct Answer and Explanation is:
. Effect of Mistakes on Gram-Positive and Gram-Negative Cells
a. Failure to add iodine: Iodine forms a complex with crystal violet, which is crucial for trapping the dye within the peptidoglycan layer of the bacterial cell wall. Without iodine, the crystal violet will not form this complex, leading to poor retention of the dye. Gram-positive cells will likely appear pink (as they are unable to retain the dye effectively), and Gram-negative cells may appear colorless, as they have a thinner peptidoglycan layer that does not hold onto the crystal violet without iodine.
b. Failure to apply the decolorizer: The decolorizer (usually ethanol or acetone) helps differentiate Gram-positive from Gram-negative cells. Without decolorization, both types of bacteria would retain the crystal violet stain. Gram-positive bacteria, which have thick peptidoglycan layers, would remain purple, but Gram-negative bacteria, which have a thinner peptidoglycan layer and an outer membrane, would also retain the crystal violet, making them appear purple as well. This would lead to false results, as the typical Gram-negative pink color would not appear.
c. Failure to apply safranin: Safranin is used as a counterstain to stain Gram-negative bacteria, which do not retain crystal violet after decolorization. If safranin is not applied, Gram-negative bacteria would remain colorless, and Gram-positive bacteria would remain purple. The absence of safranin would result in a false conclusion that only Gram-positive bacteria were present.
d. Reversal of crystal violet and safranin stains: If crystal violet is applied last and safranin first, both types of bacteria would retain the purple stain, as crystal violet is a stronger stain than safranin. Decolorization would remove the safranin from Gram-negative bacteria, but the initial crystal violet would persist, making both types of bacteria appear purple, making it impossible to distinguish between them.
2. Difference in Staining of Gram-Positive and Gram-Negative Cells
Both crystal violet and safranin are basic stains, meaning they are positively charged and can bind to negatively charged cellular components. However, the difference in staining between Gram-positive and Gram-negative bacteria lies in their cell wall structure.
- Gram-positive cells have a thick peptidoglycan layer that traps the crystal violet-iodine complex inside the cell. The complex is retained even after decolorization, allowing these cells to appear purple.
- Gram-negative cells, on the other hand, have a thinner peptidoglycan layer and an additional outer membrane. During decolorization, the outer membrane is disrupted, and the crystal violet-iodine complex leaks out. When safranin is applied, it stains the Gram-negative cells pink. Thus, the structural differences in the cell wall determine how the cells retain or lose the crystal violet dye, leading to the distinct purple and pink coloration.
3. Are Epithelial Cells Gram-Positive or Gram-Negative?
Human epithelial cells, being eukaryotic, do not follow the Gram stain procedure since Gram staining is used to differentiate bacterial species. However, if you are comparing your own epithelial cells with bacteria, you can assume that human cells are neither Gram-positive nor Gram-negative. But if you are considering the bacteria in your gumline, the bacteria could be either Gram-positive or Gram-negative depending on their specific characteristics.
4. Suggestions for Lab Partner on Control Organisms
If your lab partner is seeing pink cells in both the Escherichia coli (Gram-negative) and Bacillus subtilis (Gram-positive) controls, despite following the procedure correctly, the issue may lie with the age of the bacterial cultures. Older cultures of Gram-positive bacteria, like Bacillus subtilis, can lose their ability to retain the crystal violet stain because their cell walls weaken over time, making them appear pink even when they should be purple. The suggestion would be to use fresh cultures for both the Gram-negative and Gram-positive controls, as this ensures that the bacteria are still capable of retaining the crystal violet dye and will give reliable results.
In summary, maintaining the integrity of bacterial cultures is key to obtaining accurate results in a Gram stain procedure.
