Gram Staining

Principle of Gram Staining:

• Gram staining differentiates bacteria based on the structure of their cell walls.
• Gram-positive bacteria retain the primary stain, while Gram-negative bacteria do not, due to differences in their cell wall composition.

Procedure of Gram Staining:

1. Preparation of Smear and Fixation: As in simple staining.
2. Primary Stain: Crystal violet is applied to the smear and left for about 1 minute.
3. Mordant: Iodine solution is added, which forms a complex with the crystal violet, and left for another minute.
4. Decolorization: The slide is washed with alcohol or acetone for a few seconds. This step differentiates Gram-positive from Gram-negative bacteria.
5. Counterstain: Safranin is applied for 1-2 minutes to stain the decolorized Gram-negative bacteria.

Observation:

• Gram-Positive Bacteria: Appear purple or blue because they retain the crystal violet-iodine complex.
• Gram-Negative Bacteria: Appear pink or red due to the uptake of the counterstain safranin after the decolorization step.

Advantages:

• Differentiates a broad range of bacteria.
• Guides antibiotic therapy as Gram-positive and Gram-negative bacteria often respond differently to treatments.

Limitations:

• Some bacteria (e.g., Mycobacterium, Listeria) may not fit neatly into Gram categories.
• Over-decolorization or under-decolorization can lead to misclassification.

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