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Bacterial Gram Stain Activity (continued)
6. Counterstain Step
• Flood slide with Safranin O. Allow to sit for 1 minute.
• Rinse gently with water.
7. View
• Examine at 400x and 1,000x under oil.
Expected Results:
Results may vary based on materials used, setup, procedure, and other factors, however, here are a few examples on what to expect:
• Gram-positive organisms will appear PURPLE
• Gram-negative organisms will appear PINK
• A mixed culture of Gram-positive and Gram-negative organisms will appear as a combination of PURPLE and PINK
Follow-up Teaching Notes:
• Gram-positive organisms appear purple because their cell membrane takes up the purple Crystal Violet dye during the
Primary Staining Step and keeps it throughout the procedure.
– Primary Stain Step: Crystal Violet binds to peptidoglycan in cells.
– Mordant/Fixative Step: Crystal violet and Iodine form a complex.
– Decolorization Step: While some Crystal Violet-Iodine complex is washed away, much remains because the alcohol
dehydrates and shrinks the cell membrane, locking in the crystal violet-iodine complex (keeping the cells purple).
– Counterstain Step: Safranin is applied and is taken up by the cells, but visually is overpowered by the color still present from
the Crystal Violet. Cells still appear purple.
• Gram-negative organisms appear pink because though they take up the Crystal Violet, they lose it during the decolorization
step and subsequently take up and display the pink color during the Counterstain step.
– Primary Stain Step: Crystal Violet binds to peptidoglycan in cells (they have much less peptidoglycan than Gram-positive
cells do).
– Mordant/Fixative Step: Crystal violet and Iodine form a complex
– Decolorization Step: Alcohol washes most of the Crystal Violet-Iodine complex away because there's not much
peptidoglycan to hold it. Cells become colorless again.
– Counterstain Step: Safranin is applied to the decolorized cells, so it is taken up and the cells appear pink.
Disposal/Clean-up:
• Dispose of chemicals according to local regulations.
• Discard your used slide in a sharps-container to avoid injury.
