Skip to main content

Microbiology

Gram

Gram stain is a foundational technique in clinical microbiology. It’s based on the differences in cell wall structure between bacteria, allowing us to classify them as either Gram-positive (purple) or Gram-negative (pink/red). Mastering the Gram stain procedure, understanding its principle, and accurately interpreting the results are crucial for every clinical microbiologist. It helps guide initial identification and direct patient care

Principle of the Gram Stain

  • Cell Wall Differences: The Gram stain hinges on the differences in cell wall structure between Gram-positive and Gram-negative bacteria
    • Gram-positive bacteria: have a thick peptidoglycan layer
    • Gram-negative bacteria: have a thin peptidoglycan layer and an outer membrane containing lipopolysaccharide (LPS)
  • Mechanism
    1. Primary Stain (Crystal Violet): Both Gram-positive and Gram-negative cells initially take up the crystal violet, staining them purple
    2. Mordant (Gram’s Iodine): The iodine acts as a mordant, forming a crystal violet-iodine complex within the cell. This complex is larger than the crystal violet molecule alone
    3. Decolorization (Alcohol or Acetone): This is the critical step!
      • Gram-positive cells: The thick peptidoglycan layer becomes dehydrated by the alcohol, shrinking the pores and trapping the crystal violet-iodine complex inside. The cells remain purple
      • Gram-negative cells: The outer membrane is dissolved by the alcohol, and the thin peptidoglycan layer cannot retain the crystal violet-iodine complex. The crystal violet-iodine complex washes away, and the cells become colorless
    4. Counterstain (Safranin): The decolorized Gram-negative cells are stained with safranin, turning them pink/red. Gram-positive cells, already purple, are not affected by the safranin

Gram Stain Procedure

  1. Smear Preparation
    • From a solid medium: Place a small drop of sterile saline on a clean slide. Using a sterile loop, gently pick a small amount of the bacterial colony and emulsify it in the saline to create a thin, even smear
    • From a liquid medium: Place a loopful of the liquid culture directly onto the slide and spread it thinly
  2. Air Dry: Allow the smear to air dry completely. This is crucial! Wet smears can boil during heat-fixing, distorting the cells
  3. Heat Fixation: Pass the slide quickly through a flame 2-3 times. This kills the bacteria, adheres them to the slide, and makes them more permeable to the stains. Don’t overheat, or you’ll distort the morphology!
  4. Staining
    • Crystal Violet: Flood the smear with crystal violet for 1 minute. Rinse with water
    • Gram’s Iodine: Flood the smear with Gram’s iodine for 1 minute. Rinse with water
    • Decolorizer: This is the tricky step! Hold the slide at an angle and drip the decolorizer (alcohol or acetone) onto the smear until the runoff is clear. Rinse immediately with water. Over-decolorizing will cause Gram-positive cells to appear Gram-negative.
    • Safranin: Flood the smear with safranin for 1 minute. Rinse with water
  5. Blot Dry: Gently blot the slide dry with bibulous paper or a lint-free paper towel. Don’t rub!
  6. Microscopy: Observe under oil immersion (1000x magnification)

Interpretation

  • Gram-positive: Purple cells
  • Gram-negative: Pink/red cells
  • Morphology: Note the shape (cocci, bacilli, coccobacilli, etc.) and arrangement (pairs, chains, clusters, etc.) of the bacteria
  • Reporting
    • Example: “Gram-positive cocci in clusters” (suggestive of Staphylococcus)
    • Example: “Gram-negative bacilli” (further testing needed for identification)
  • Yeast: Gram stain will stain yeast, they will appear large and oval

Common Problems and Troubleshooting

  • False Gram-Positive
    • Over-decolorization
    • Old cultures (cell walls can break down)
    • Thick smears
    • Using iodine before crystal violet
  • False Gram-Negative
    • Under-decolorization
    • Using too much heat during fixation
    • Some bacteria are Gram-variable or don’t stain well with Gram stain (e.g., Mycoplasma)
    • Antibiotic exposure

Importance in the Clinical Lab

  • Initial Identification: The Gram stain is one of the first steps in identifying bacteria. It helps narrow down the possibilities and guides further testing
  • Direct Smears: Gram stains can be performed directly on patient specimens (e.g., sputum, CSF) to provide rapid information to the clinician
  • Quality Control: Gram stains are used to assess the purity of cultures and to confirm the morphology of organisms

Tips for Success

  • Practice, practice, practice!: The more you perform Gram stains, the better you’ll become at recognizing the subtle nuances and avoiding common pitfalls
  • Use fresh reagents.: Old or contaminated reagents can give unreliable results
  • Control Slides: Use known Gram-positive and Gram-negative control organisms to ensure your staining technique is correct
  • Proper technique!: Ensure proper timing for each step, and proper decolorization

Key Terms

  • Peptidoglycan: A mesh-like layer made of sugars and amino acids that forms the cell wall of bacteria. It’s much thicker in Gram-positive bacteria
  • Lipopolysaccharide (LPS): A component of the outer membrane of Gram-negative bacteria. It’s an endotoxin that can trigger an immune response
  • Crystal Violet: The primary stain used in the Gram stain procedure, initially staining all bacterial cells purple
  • Mordant: A substance that helps a stain bind to the target structure. In the Gram stain, Gram’s iodine acts as a mordant, forming a complex with crystal violet
  • Decolorizer: A solution (usually alcohol or acetone) used to remove the crystal violet-iodine complex from Gram-negative bacteria during the Gram stain procedure. This is the most critical step
  • Safranin: The counterstain used in the Gram stain procedure, staining Gram-negative bacteria pink or red after decolorization
  • Gram-positive: Bacteria that retain the crystal violet stain and appear purple under the microscope due to their thick peptidoglycan layer
  • Gram-negative: Bacteria that lose the crystal violet stain during decolorization and appear pink or red after counterstaining with safranin due to their thin peptidoglycan layer and outer membrane
  • Morphology: The shape and arrangement of bacterial cells as seen under the microscope (e.g., cocci, bacilli, clusters, chains)
  • Heat Fixation: The process of passing a slide through a flame to kill bacteria, adhere them to the slide, and make them more permeable to stains