SEMEN ON PANTIES - HISTORY OF SEMEN DETECTIONS

                            

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.