Gram Staining Flashcards
Explain the structural differences between Gram-positive and Gram-negative bacteria that result in their different Gram stain appearances.
Gram-positive bacteria
- Has a thick peptidoglycan layer (90% of the cell wall) and no outer lipopolysaccharide membrane -> traps the crystal violet-iodine complex; makes them appear purple or blue.
Gram-negative bacteria
- has a thinner peptidoglycan layer (10% of the cell wall) and outer lipopolysaccharide membrane, which is disrupted during the decolorization step, allowing the safranin to stain them pink to red.
What role does iodine play in the Gram staining process, and why is it referred to as a mordant?
Iodine acts as a mordant by forming a complex with crystal violet, which strengthens the dye’s binding to the thick peptidoglycan layer in Gram-positive bacteria. This makes it harder to wash out during decolorization.
Describe the appearance of Gram-positive bacteria under the microscope after each step of the Gram staining procedure.
• Fixation: Clear.
• Crystal Violet: Purple.
• Iodine Treatment: Purple (enhanced binding).
• Decolorization: Remains purple.
• Safranin Counterstain: Purple (safranin has no visible effect).
Describe the appearance of Gram-negative bacteria under the microscope after each step of the Gram staining procedure.
- Fixation: Clear (no stain applied yet).
- Crystal Violet: Purple (both Gram-positive and Gram-negative bacteria initially take up the primary stain).
- Iodine Treatment: Purple (the iodine forms a complex with the crystal violet in both Gram-positive and Gram-negative bacteria).
- Decolorization: Colorless (the ethanol or acetone dissolves the outer lipid membrane of Gram-negative bacteria, removing the crystal violet-iodine complex).
- Safranin Counterstain: Pink or red (the colorless Gram-negative bacteria take up the safranin dye, making them appear pink under the microscope).
Identify the purpose of counterstaining with safranin and explain its effect on Gram-negative bacteria.
Safranin stains Gram-negative bacteria pink or red after the crystal violet-iodine complex has been removed. It provides a contrasting color to distinguish them from Gram-positive bacteria.
A laboratory technician accidentally skips the decolorization step during Gram staining. Predict how Gram-positive and Gram-negative bacteria will appear under the microscope and justify your answer.
Both Gram-positive and Gram-negative bacteria would appear purple, as the crystal violet-iodine complex would not be washed out of Gram-negative bacteria without decolorization.
If a newly discovered bacterium has a thick peptidoglycan layer but an outer lipopolysaccharide membrane, how would it stain in the Gram staining process? Explain.
It would likely stain pink (Gram-negative) because the outer lipopolysaccharide membrane would dissolve during decolorization, allowing the crystal violet to wash out and the safranin to counterstain it.
Why is the decolorization step critical in differentiating Gram-positive from Gram-negative bacteria?
Decolorization with ethanol or acetone dissolves the outer lipid membrane of Gram-negative bacteria, removing the crystal violet-iodine complex. In contrast, the thick peptidoglycan layer in Gram-positive bacteria retains the stain, enabling differentiation.
Gram-positive bacteria are generally more susceptible to penicillin compared to Gram-negative bacteria. Use the structural differences in their cell walls to explain this observation.
Penicillin targets the synthesis of peptidoglycan, which is abundant in Gram-positive bacteria. Gram-negative bacteria have an outer lipopolysaccharide membrane that shields their thinner peptidoglycan layer, reducing penicillin’s effectiveness.
What color do Gram-positive bacteria appear after Gram staining?
Purple or blue.
What color do Gram-negative bacteria appear after Gram staining?
Pink or red
Name the four steps of the Gram staining process in order.
Step 1: Applying a primary stain (crystal violet).
• Step 2: Adding a mordant (Gram’s iodine).
• Step 3: Decolorization with ethanol, acetone, or a mixture of both.
• Step 4: Counterstaining with safranin.
Which stain is used first in the Gram staining process?
Crystal violet.
What is the purpose of decolorization during Gram staining?
To remove the crystal violet-iodine complex from Gram-negative bacteria by dissolving their outer lipid membrane.
What role does safranin play in Gram staining?
It counterstains Gram-negative bacteria, making them appear pink or red.
