Microscopy Gram staning

• Gram stain


  The most important differential stain used in bacteriology to divide bacterial cells into two major groups: gram-positive and gram-negative
• Gram-positive cells


  • Have a thick peptidoglycan layer in their cell walls
• Gram-negative cells


  • Have a thinner glycogen layer in their cell walls, surrounded by outer lipid-containing layers
• Gram staining procedure


  1. Apply crystal violet (primary stain)

  2. Apply Gram's iodine (mordant)

  3. Decolorize with 95% alcohol

  4. Counter-stain with safranin
• Gram staining is based on the difference in the chemical composition of the bacterial cell walls
• Peptidoglycan is a polysaccharide composed of N-acetyl glucosamine and N-acetyl muramic acid, with some organisms having short peptide chains cross-linking the layers
• Gram-positive cells can be decolorized by lysozyme or penicillin, causing them to stain gram-negative
• Decolorization with alcohol


  Easier for gram-negative cells due to their thinner and less cross-linked peptidoglycan layer, leaving them colorless or unstained
• Decolorization with alcohol


  Harder for gram-positive cells due to their thicker peptidoglycan layer, retaining the primary stain and appearing purple
• Proper slide preparation, timing of decolorization, and age of culture are critical for successful gram staining
• Gram staining is used as a first diagnostic test on body fluids, tissue biopsies, and cultures to determine the gram type, shape, and orientation of bacteria
• The results of gram staining aid in determining appropriate antibiotic treatment for the patient
• Gram staining can be used to diagnose leprosy or Hansen's disease in lung infections
• Gram-positive cells


  Tend to lose their ability to retain the primary stain, may appear purple or pink
• Gram staining procedure


  1. Crystal violet

  2. Gram's iodine

  3. 95% alcohol

  4. Safranin
• Acid-fast stains


  Medically important in diagnosing mycobacterium species which cause tuberculosis, leprosy and other infections
• Nocardia


  Causative agent for many lung infections, identified by acid-fast staining methods
• Spore staining (Schaeffer-Fulton method)

  1. Apply malachite green primary stain with heat

  2. Decolorize with water

  3. Counter-stain with safranin
• Spores


  • Resistant to heat, freezing, radiation, desiccation and chemical agents
  • Formed by bacteria like Clostridium and Bacillus when environmental conditions are unfavourable
• Capsule staining (Anthony method)

  1. Apply crystal violet primary stain

  2. Decolorize with 20% copper sulfate
• Capsule


  Gelatinous outer layer secreted by cells, protects bacteria against phagocytosis
• Capsulated bacteria are generally more virulent and capable of producing diseases
• Bacterial infection not responding to antibiotics

  Staining of isolated organisms to determine presence of capsule may be warranted
• Visualizing microorganisms would be very difficult if they are in the living state because they are so minute, transparent and practically colorless when suspended in an aqueous medium
• To study the properties of microorganisms and divide them into specific groups or for diagnostic purposes, we use biological stains and staining procedures in conjunction with light microscopes
• Learning objectives


  • Explain the chemical and theoretical basis of biological staining
  • Manipulate the techniques of smear preparation
  • Perform procedures for simple staining and negative staining
  • Describe the method for performing differential staining procedures such as Gram, acid-fast, capsule, and spore stains
• Stain


  An organic compound which contains a benzene ring, a chromophore, and an auxochrome group
• Chromogen


  A colored compound that is not a stain, but contains the benzene and chromophore
• Acidic stains


  Ionic stains where the chromogen portion exhibits a negative charge
• Basic stains


  Cationic stains where the chromogen portion exhibits a positive charge
• Basic stains are more commonly used for bacterial staining because the negative charge on the bacterial surface repels most acidic stains, preventing their penetration into the cell
• Staining techniques


  • Simple staining
  • Differential staining (Gram, acid-fast)
  • Flagella staining
  • Capsule staining
  • Nuclear staining
• A patient presented with a deep laceration on the left lateral thigh, with a greenish purulent discharge, signs of inflammation, and early stages of necrosis
• The patient has shown signs of hypersensitivity to most broad-spectrum antibiotics, precluding their initial use
• Vancomycin


  Antibiotic for Gram-positive infections
• Ciprofloxacin


  Antibiotic for Gram-negative infections
• The physician needs to quickly and easily determine which antibiotic would be the correct one to use for the patient
• Simple staining may not be enough to determine the antibiotic use
• Questions to ask the doctor before taking antibiotics

  • Do I really need antibiotics?
  • What are the risks?
  • Are there simpler or safer options?
  • How much do they cost?
  • How do I safely take antibiotics?
• Preparing bacterial smears


  • Prepare clean glass microscope slides
  • Label the slides
  • Prepare the smear (different for broth cultures and solid medium cultures)
  • Heat fix the smear