Staining

• Fixation


  The process by which the internal and external structures of cells and microorganisms are preserved and fixed in position
• Fixation


  • Inactivates enzymes that might disrupt cell morphology
  • Toughens cell structures so that they do not change during staining and observation
  • Kills and attaches microorganism firmly to the microscope slide
• Types of fixation


  • Heat-fix bacterial smears by gently flame heating an air-dried film of bacteria
  • Chemical fixation using fixatives like ethanol, acetic acid, mercuric chloride, formaldehyde, and glutaraldehyde
• Dyes


  • Have chromophore groups that give the dye its color
  • Can bind with cells by ionic, covalent, or hydrophobic bonding
• Types of dyes


  • Basic dyes (e.g. methylene blue, basic fuchsin, crystal violet, safranin, malachite green)
  • Acid dyes (e.g. eosin, rose bengal, acid fuchsin)
• Basic dyes


  Have positively charged groups and bind to negatively charged molecules like nucleic acids and many proteins
• Acid dyes


  Have negatively charged groups and bind to positively charged cell structures
• Simple staining


  Using a single staining agent
• Differential staining


  Divides bacteria into separate groups based on staining properties
• Gram stain


  Divides bacteria into gram-positive and gram-negative
• Gram staining procedure


  1. Stain with crystal violet (primary stain)

  2. Treat with iodine solution (mordant)

  3. Decolorize with ethanol or acetone (generates differential staining)

  4. Counterstain with safranin
• Acid-fast staining


  Stains a few species, particularly those in the genus Mycobacterium, that do not bind simple stains readily
• Acid-fast staining procedure


  1. Heat with a mixture of basic fuchsin and phenol (Ziehl-Neelsen method)

  2. Acid-alcohol wash does not decolorize acid-fast cells
• Negative staining


  Reveals the presence of diffuse capsules surrounding many bacteria
• Negative staining procedure


  1. Mix bacteria with India ink or Nigrosin dye and spread in a thin film

  2. Bacteria appear as lighter bodies in the blue-black background
• Endospore staining


  Stains the resistant dormant structure formed by bacteria in the genera Bacillus and Clostridium
• Endospore staining procedure


  1. Stain with malachite green (penetrates endospores)

  2. Counterstain the rest of the cell with safranin
• Flagella staining


  Increases the thickness of bacterial flagella to observe them with the light microscope
• Flagella staining procedure


  1. Coat flagella with mordants like tannic acid and potassium alum

  2. Stain with pararosaniline (Leifson method) or basic fuchsin (Gray method)