Christmas Tree Stain for Sperm

• Kernechtrot-Picroindigocarmine Stain (Christmas Tree Stain (Reference 11)
  
  • Kernechtrot-Picroindigocarmine (KPIC) differential biological stain is used to assist in the microscopic identification of spermatozoa. The solutions for this procedure can either be purchased or prepared in-house.
  • Safety Considerations
    • Aluminum sulfate - Caution! Harmful if inhaled, in contact with skin, and if swallowed! Emits toxic fumes under fire conditions!
    • Nuclear fast red - Caution! Irritant! Emits toxic fumes under fire conditions!
    • Saturated picric acid solution - Caution! Toxic! Explosive when dry! Emits toxic fumes under fire conditions!
    • Indigocarmine dye - Caution! Harmful if swallowed! Emits toxic fumes under fire conditions!
  • Equipment

  • Flame or heat block

  • Materials
    • Fixative (optional)
  • Reagents
    • Kernechtrot staining solution (KS)
    • Picroindigocarmine staining solution (PICS)
    • Distilled water
    • 95% ethanol or methanol
  • Stock Solutions (In-house Preparation)
    • Equipment
      • Filtration apparatus
      • 500 ml glass beakers
      • Balance
      • Spatula
      • Glass rod
      • Plastic bottles
    • Materials
      • Filter paper
      • Weigh boats or weigh paper
    • Reagents
      • Aluminum sulfate
      • Nuclear Fast Red
      • Distilled water
      • Picroindigocarmine dye
      • Saturated picric acid solution
    • Kernechtrot Solution (KS)
      • • In a beaker dissolve 5 g of aluminum sulfate in 100 ml of hot distilled water.
      • • Immediately add 0.1 g of Nuclear Fast Red and stir with a glass rod.
      • • Allow to cool and filter through filter paper.
        • Storage
          • The Kernechtrot Solution is stable at room temperature for up to 6 months, but may need to be refiltered after standing.
          • Labeling
            • Label the bottle as KS with the expiration date and a lot number (the date of preparation followed by the initials of person preparing the solution).
    • Picroindigocarmine Solution (PICS)
      • • Dissolve 1 g of Indigocarmine dye in 300 ml of a commercially purchased saturated solution of picric acid.
      • • Filter and store.
        • Storage
          • The Picroindigocarmine Solution is stable at room temperature for up to 6 months, but may need to be refiltered after standing.
        • Labeling
          • Label the bottle as PICS with an expiration date and a lot number (the date of preparation followed by the initials of person preparing the solution).

           

• Christmas Tree Staining Procedure (Reference 11)
  
  • Prepare a thin smear of an extract of a suspected semen stain and allow to dry. Fix the smear with a quick flame or fixative, or by placing it on a heat block overnight.
  • Add a sufficient amount (2-5 drops) of red reagent to cover the stained portion of the microscope slide.
  • Let the slide stand at room temperature for at least 15 minutes.
  • Wash off of the slide with a gentle stream of distilled water and drain the slide.
  • Add a sufficient amount (2-5 drops) of green reagent to cover the stained portion of the slide.
  • Allow green reagent to stain the smear for 5-15 seconds.
  • Wash green reagent off of the slide with 95% ethanol or methanol.
  • Dry the slide at room temperature.