Chapter 3

• Staining Techniques

  • Vital Staining
  • Supravital Staining
  • Simple staining (Monochrome staining)
  • Negative staining (Background staining)
  • Impregnation staining
  • Differential staining
• Vital Staining


  Organisms are stained in living state
• Supravital Staining


  Organisms are called
• Four Types of Supravital Staining

  • Simple staining (Monochrome staining)
  • Negative staining (Background staining)
  • Impregnation staining
  • Differential staining
• Simple staining (Monochrome staining)


  Water solution of a simple basic dye (methylene blue)
• Negative staining (Background staining)


  The background is stained, and organisms appear as colorless objects against a dark background
• Impregnation staining


  Very thin specimens are rendered visible by increasing their thickness by impregnating silver on their surfaces
• Differential staining


  Differentiates two types of organisms (example: Gram stain and acid fast stain)
• Unstained Preparations

  • Wet Mount
  • Hanging Drop Preparation
• Wet Mount


  Specimen can be observed directly, e.g. urine or in an emulsified suspension, e.g. stool
• Hanging Drop Preparation

  Unstained preparation
• Stained Preparations

  • Gram Stain
  • Acid-Fast Stain (Ziehl-Neelsen Stain)
• Gram Stain


  Most common staining method, devised by histologist Christian Gram to stain bacteria in tissue, a differential staining method which differentiates organisms into Gram-positive and negative according to their Gram reaction
• Gram Stain Bacteria

  • Gram-positive bacteria
  • Gram-negative bacteria
• Gram-positive bacteria


  Resist decolorization and retain the color of the primary stain and appear violet (Clostridia, Corynebacteria & Bacillus spp. – Gram + bacilli ; Pneumococci, streptococci and staphylococci – Gram + cocci)
• Gram-negative bacteria


  Decolorized by alcohol and take counter stain and appear red (Gonococci & Menigococci – Gram negative cocci ; E.coli, Salmonella, Shigella, V. cholerae, etc. – Gram negative bacilli)
• Acid-Fast Stain (Ziehl-Neelsen Stain)


  Organisms which are not easily stained by ordinary staining methods, but once stained resist decolorization by acids