Preparation and Staining of Specimens

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• Preparation and Staining of Specimens

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• Often specimens must be fixed and stained to
  enhance their visibility and to distinguish
  morphological properties

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Fixation

• Preservation of internal and external features of
  cells
• Cellular enzymes are inactivated
• Cell structures are hardened
• Organism dies AND adheres strongly to the glass
  slide

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• Two types of fixation
• Heat fixing flame heating bacterial film,
  preservation of morphology but NOT internal
  structures
• Chemical fixing chemical fixatives penetrate
  cells and preserves intracellular components
• Acetone
• Ethanol
• Acetic acid
• Mercuric chloride
• Formaldehyde
• Glutaraldehyde

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Dyes and Simple Staining

• Dyes contain chromophore groups and bind to
  cells by ionic, covalent and hydrophobic forces
• Basic dyes (positively charged) bind to
  negatively charged groups
• Work best at high pH
• Acidic dyes (negatively charged) bind to
  positively charged groups
• Work best at low pH

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• Simple staining
• One reagent
• Usually involves basic dyes
• Crystal violet
• Methylene blue
• Carbol fuchsin
• Differential staining
• Used to differentiate groups on bacteria
• Examples
• Gram stain
• Acid fast stain

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Gram Stain

• Gram-positive bacteria retain the crystal violet
• Gram-negative bacteria become colorless
• Developed by Christian Gram in 1884

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• Steps
• 1. Crystal violet, 30, rinse 5
• 2. Grams Iodine (mordant that strengthens the
  association of crystal violet with the cell
  wall), 1, rinse 5
• 3. Decolorization 95 ethanol or
  isopropanolacetone, 15-30, rinse 5
• 4. Counterstain Saffranin, 1, rinse 5,
  Gram-negative bacteria become pink/red,
  Gram-positive remain purple

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• Gram staining is the most widely used
  differential staining procedure because it
  divides bacterial species into two roughly equal
  groups - gram positive and gram negative

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Acid-Fast Staining

• Some organisms do not stain well with
  conventional dyes (e.g. Mycobacterium)
• Harsher treatment required
• Steps
• 1. Heating with basic fuchsin and phenol
  (Ziehl-Neelson technique) 5, cool, rinse 30
• 2. Decolorize Acid/Alcohol 10-30, rinse 30
• 3. Counterstain with methylene blue, 2 , rinse
  30
• Acid-fast cells remain red due to high mycolic
  acid content (lipid)
• Non-acid-fast cells blue (counterstain)

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• Acid-fast staining is a differential staining
  procedure that identifies two medically important
  species of bacteria -
• Mycobacterium tuberculosis, the causative agent
  of tuberculosis, and
• Mycobacterium leprae, the causative agent of
  leprosy (Hansens disease)

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• Staining Particular Structures

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• Negative staining
• Widely used to visualize diffuse capsules
  surrounding the bacteria those capsules are
  unstained by the procedure and appear colorless
  against a stained background
• Morphology of cells determined
• No heat treatment or harsh chemicals
• Film spread, stained and dried
• Heating would cause shrinkage distortion of
  cellular morphology

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• Deposits generated around cell or dark background
  observed
• Dyes (acidic stain does not penetrate cells)
• Nigrosin
• India ink
• Eosin blue

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• Spore staining
• Detects endospores generated by Bacillus and
  Clostridium
• Formed within the cell ? dormant
• Hardy in adverse conditions
• Various sizes, shapes and locations
• Steps (Schaffer-Fulton procedure)
• Heat malachite green
• Rinse
• Counterstain with saffranin

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• Capsule stain
• Gently no heat fixation or may get shrinkage
• Steps
• Grow culture in skim milk broth
• Crystal violet
• Copper sulfate counterstain

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• Flagella stain
• Flagella are very difficult to observe directly
  because of their size
• Thickness is increased using mordants
• Tannic acid
• Potassium alum
• Stained with Pararosaniline (Leifson method) or
  basic fuchsin (Gray method)