STAINING OF MICROORGANISM

• Stains


  Used to make minute organisms visible, display their structure in the microscope, reveal their chemical nature and influence their growth and development
• Different methods of staining

  • Simple staining
  • Differential staining
  • Demonstration of special structures by staining
• Simple staining


  Employed to study the morphology of bacteria in films with simple dyes, direct staining
• Types of simple staining

  • Positive staining
  • Negative relief staining
• Positive staining


  Stained microorganisms with unstained background, Methylene blue
• Negative relief staining


  Unstained environment with stained background, India ink
• Differential staining


  Used to show by special staining methods and reveal the physiochemical characteristics and differences among bacteria that aid in their description and classification, two or more dyes employed
• Components of differential staining

  • Primary stain
  • Mordant
  • Decolorizer
  • Counter stain
• Primary stain


  Dye that initially stains the bacterial cell
• Mordant


  Enhances the affinity of the primary stain to the organism
• Decolorizer


  Most critical stage, differentiates bacteria based on cell wall composition that reacts to the primary stain used
• Counter stain


  Stains the background or stains the organism not reactive to the primary stain
• Demonstration of special structures by staining

  • Spore staining
  • Capsule staining
  • Flagellar staining
  • Staining of metachromatic granules
• Bacterial smear preparation


  1. Heat slide to remove grease

  2. Sterilize wire loop

  3. Place specimen on slide

  4. Air dry

  5. Heat fix smear

  6. Stain

  7. Wash off

  8. Blot dry

  9. Examine under microscope
• Gram's staining


  Differential staining method used to demonstrate the physio-chemical differences among bacteria that aid in their description and classification
• Components of Gram's staining

  • Gentian violet (primary stain)
  • Gram's iodine (mordant)
  • Acetone alcohol (decolorizer)
  • Safranin (counter stain)
• Gram's iodine


  Chemically binds with the cell wall of the bacteria and increases the affinity of the crystal violet (primary stain)
• Acetone alcohol


  Decolorizing agent that distinguishes Gram (+) bacteria from Gram (-), Gram (-) contains lipids that are soluble and will dissolve in the alcohol
• Safranin


  Red dye that makes the Gram (-) organism appear pink or red after staining
• Gram (+) bacteria appear blue/violet, Gram (-) bacteria appear red/pink
• Reasons for Gram stain results deviating from expected

  • Gram (+) appears Gram (-)
  • Gram (-) appears Gram (+)
• Gram's rule: All cocci are Gram (+) except for Neisseria, Moraxella, Veillonella, Branhamella. All bacilli are Gram (-) except for Mycobacterium, Corynebacterium, Bacillus, Erysipelothrix, Lactobacillus, Listeria & Nocardia. All spirals are Gram (-)
• Acid fast staining / Ziehl-Neelsen


  Acid fast bacteria are those organisms which are not decolorized by acid once they have been stained, due to high content of lipodal substance known as mycolic acid or hydroxymethoxyl acids
• General rule: All bacteria are none or not acid fast except for Mycobacterium and Nocardia (slightly acid-fast)
• Components of Ziehl-Neelsen staining

  • Carbol fuchsin (primary stain)
  • Steaming (softens mycolic acid)
  • Acid alcohol (decolorizer)
  • Loeffler's methylene blue (counter stain)
• Modified acid-fast staining / Kinyoun method


  Differentiates acid-fast from non-acid fast organisms, no heating or steaming required but the concentration of phenol is increased to increase penetration on the bacteria's cell wall
• Components of Kinyoun method

  • Kinyoun carbol fuchsin (primary stain)
  • Acid alcohol (decolorizer)
  • Methylene blue or malachite green (counter stain)