During the Gram staining procedure, for how long should the bacterial smear be washed with alcohol (decolorizer) to ensure proper differentiation between Gram-positive and Gram-negative bacteria?

• A. Less than 1 second
• B. 5-10 minutes
• C. 1-2 minutes
• D. 10-20 seconds

Answer: (D)

Verified step by step guidance

1. Understand the purpose of the decolorization step in Gram staining: it differentiates Gram-positive bacteria (which retain the crystal violet stain) from Gram-negative bacteria (which lose the stain and take up the counterstain).
2. Recognize that the decolorizer (usually alcohol or acetone-alcohol) must be applied for a precise amount of time to avoid over- or under-decolorization, which can lead to incorrect results.
3. Recall that the typical recommended time for washing the bacterial smear with alcohol is short, generally around 10-20 seconds, to effectively remove the crystal violet from Gram-negative cells without affecting Gram-positive cells.
4. Note that washing for less than 1 second is usually insufficient to remove the stain from Gram-negative bacteria, while washing for 1-2 minutes or 5-10 minutes is excessively long and will over-decolorize, potentially removing stain from Gram-positive bacteria as well.
5. Therefore, the correct approach is to apply the alcohol decolorizer for approximately 10-20 seconds, carefully monitoring the process to ensure proper differentiation.