Gram Staining Flashcards

Gram Staining

• Gram staining is a differential stain used to categorize bacteria into two groups: Gram-positive and Gram-negative.
• Differential staining uses multiple chemical stains to differentiate between bacteria.
• Gram staining uses four chemicals:
  • Crystal violet
  • Iodine
  • Decolorizing alcohol
  • Safranin
• The order of chemical usage is crucial for proper Gram staining.
• Staining differences are due to cell wall structure variations.

Gram-Positive Bacteria

• Have a thick peptidoglycan layer in their cell wall.
• Peptidoglycan provides strength and complexity to the cell wall.

Gram-Negative Bacteria

• Have a thin peptidoglycan layer.
• Possess an outer membrane of lipids, which Gram-positive bacteria lack.

Gram Staining Steps

1. Crystal Violet:
   • Penetrates the cell walls of both Gram-positive and Gram-negative bacteria.
   • All bacteria appear purple.
2. Iodine (Mordant):
   • Binds to crystal violet to form a large complex.
   • Iodine itself does not impart color.
3. Decolorizing Alcohol:
   • Dissolves the outer membrane of Gram-negative cells.
   • Allows crystal violet/iodine complexes to wash away, rendering Gram-negative bacteria colorless.
   • The thick peptidoglycan layer in Gram-positive bacteria prevents the complexes from washing out, so they stay purple.
4. Safranin (Counterstain):
   • Stains all bacterial cells red.
   • Only Gram-negative bacteria (which lost the purple color) appear red/pink.
   • Gram-positive bacteria are also stained by safranin, but the purple color is more intense.

Key Concept

• "3 P’s" of Gram staining: Positive, Purple, Peptidoglycan (Gram-positive organisms stain purple due to thick peptidoglycan).

Significance

• Gram staining assists in microbe identification.
• Knowing if a bacterium is Gram-positive or Gram-negative informs treatment decisions (e.g., antibiotic selection).
• Penicillin is more effective against Gram-positive bacteria.
• Ciprofloxacin ("cipro") is used against Gram-negative bacteria.

Acid-Fast Staining

• Another differential stain.
• Distinguishes microbes with mycolic acid in their cell walls (e.g., Mycobacterium) from those without (most Gram + and - microbes).
• Carbol fuschin (red) binds to mycolic acid in Acid-fast positive microbes.
• No mordant step.
• Acid-alcohol decolorizing removes carbol fuschin from microbes without mycolic acid.
• Methylene blue counterstain stains Acid-fast negative organisms blue.

Cell Wall Structures

• Gram + cells have a thicker peptidoglycan layer.
• Gram – cells have a thinner layer of peptidoglycan in addition to a second membrane called the outer membrane, which contains endotoxin as part of the LPS.

Procedure for Gram Staining Fixed Sample

1. Prepare 3 slides with a smear prep and fix the sample, each with a different species of bacteria.
2. Gram staining steps:
   • a. Place slide(s) on the staining tray
   • b. Cover the sample area with crystal violet for 60 seconds
   • c. Wash with water
   • d. Cover the sample area with iodine for 60 seconds
   • e. Wash with water
   • f. While holding the slide at an angle, apply 95% ethanol drop-wise until the runoff is clear/colorless
   • g. Place the slide once again on the staining tray rack, wash with water
   • h. Cover the sample area with safranin for 60 seconds
   • i. Wash with wate
3. Pat the slide(s) dry on bibulous paper
4. View the slide using oil immersion microscopy and complete the observation report

Troubleshooting

• Problem: Do not see any color (cells are there but colorless)
  • Explanation: The stain was not left on the sample long enough
• Problem: Do not see many colored cells
  • Explanation: Too little of the sample may have been used in the smear prep, or some of the sample was removed during rinsing/blotting
• Problem: Incorrect Gram stain results
  • Explanation: The ethanol rinse may have been too long, and it caused Gram + cells to decolorize; this problem can also occur if the culture is too old.