What stains are used in the Gram stain procedure?

Gram Stain Components

The Gram stain procedure uses four essential reagents: crystal violet (primary stain), iodine solution (mordant), alcohol or acetone (decolorizer), and safranin or carbol fuchsin (counterstain). 1, 2

Primary Stain: Crystal Violet

• Crystal violet is applied first and stains all bacterial cells purple. 1, 2
• The dye binds to bacterial cell walls and becomes trapped within the peptidoglycan mesh, contrary to older beliefs that it penetrates the cytoplasm. 3
• Crystal violet alone cannot differentiate between bacterial types until subsequent steps are applied. 4

Mordant: Iodine Solution

• Iodine acts as a mordant by forming large crystal violet-iodine complexes within bacterial cells. 1, 2
• These complexes are too large to easily escape from thick peptidoglycan layers in Gram-positive bacteria. 2
• The iodine concentration in alcohol affects decolorization rates—low concentrations accelerate decolorization of Gram-positive bacteria, while 0.1% iodine delays extraction from Gram-positives but still allows decolorization of Gram-negatives. 4

Decolorizer: Alcohol or Acetone

• 95% alcohol or acetone differentially removes the crystal violet-iodine complex based on cell wall structure. 4, 1
• Gram-negative bacteria decolorize within 2 minutes due to their thin peptidoglycan layer and disrupted outer membrane. 4, 1
• Gram-positive bacteria resist decolorization for at least 3 minutes because their thick peptidoglycan wall (composed of peptidoglycan and secondary polymers) retains the dye complex. 4, 1
• The decolorization step is critical—over-decolorization causes false Gram-negative results, while under-decolorization causes false Gram-positive results. 1

Counterstain: Safranin or Carbol Fuchsin

• Safranin (or carbol fuchsin) is applied last to stain decolorized Gram-negative bacteria pink/red. 5, 1
• Aqueous or alcoholic solutions of safranin, neutral red, or fuchsin can serve as counterstains. 4
• Alcoholic safranin (0.25%) for 15 seconds effectively distinguishes Gram-positive (violet) from Gram-negative (pink) bacteria. 4
• Gram-positive bacteria remain purple because the crystal violet-iodine complex masks the counterstain. 1

Clinical Application Context

• The Gram stain is inadequate for detecting mycobacteria, which require fluorochrome or Ziehl-Neelsen staining methods instead. 5
• When evaluating respiratory specimens, Gram stain morphology (such as Gram-positive cocci in clusters suggesting Staphylococcus) provides rapid diagnostic information before culture results. 5
• Gram stain results should be correlated with culture findings, and any culture result must match the predominant organism on Gram stain to be considered valid. 5