In the Gram stain, why must fresh (young) bacterial cells be used to obtain reliable results?

• A. Older cells may have damaged cell walls or undergo autolysis, causing Gram-positive bacteria to stain Gram-negative (Gram-variable).
• B. Older cultures always have thicker peptidoglycan, making all bacteria retain crystal violet and appear Gram-positive.
• C. Fresh cells are required because the iodine mordant only binds to peptidoglycan that is less than 1 hour old.
• D. Fresh cells are required because crystal violet cannot penetrate cells that have been incubated overnight.

Answer: (A)

Verified step by step guidance

1. Understand the principle behind the Gram stain: it differentiates bacteria based on the structure of their cell walls, specifically the thickness and integrity of the peptidoglycan layer.
2. Recognize that Gram-positive bacteria have a thick peptidoglycan layer that retains the crystal violet-iodine complex, appearing purple, while Gram-negative bacteria have a thinner peptidoglycan layer and do not retain the stain after decolorization, appearing pink or red.
3. Consider the condition of bacterial cells in older cultures: over time, bacterial cell walls can become damaged or begin to break down through autolysis, which affects the integrity of the peptidoglycan layer.
4. Realize that damaged or lysed cell walls in older cells may fail to retain the crystal violet-iodine complex properly, causing Gram-positive bacteria to appear Gram-negative or Gram-variable.
5. Conclude that using fresh (young) bacterial cells ensures the cell walls are intact and healthy, providing reliable and accurate Gram stain results.