Question

One person has done multiple attempts at gram staining and concludes that she is decolorizing too long because both controls have pink cells. What might you suggest she try?

Answer 1

To address the over-decolorization issue in gram staining, the person should reduce the decolorization time, enabling gram-positive cells to retain the crystal violet dye and appear purple, while gram-negative cells appear pink after staining.

Step-by-step explanation:

If a person is experiencing issues with gram staining and observes that both controls show pink cells, it's likely they are over-decolorizing the sample. The decolorization process is critical in the gram staining procedure as it involves adding alcohol or an alcohol-acetone solution. This step selectively removes the crystal violet stain from cells with thinner peptidoglycan layers (gram-negative), resulting in them appearing pink after the addition of the counterstain safranin. In contrast, gram-positive cells with thicker peptidoglycan layers retain the crystal violet dye and appear purple.

For someone encountering over-decolorization issues, I would suggest reducing the decolorization time. By doing so, this allows the cells with thicker peptidoglycan layers ample time to retain the crystal violet dye without removing it excessively. Remember that the ideal timing might require some trial and error, so it is advisable to perform several test stains with varying decolorization times to determine the optimal duration that differentiates clearly between gram-positive and gram-negative bacteria.