Christmas Tree stain
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Two chemicals are used together to stain sperm cells; Kernechtrot solution will stain the head of the sperm cell a two-tone reddish pink color and the picroindigocarmine solution will stain the tail of the sperm cell a bluish-green color.
Christmas Tree Stain for Sperm
- Kernechtrot-Picroindigocarmine Stain (Christmas Tree Stain
- 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
- In a beaker dissolve 5 g of aluminum sulfate in 100 ml of hot
- Immediately add 0.1 g of Nuclear Fast Red and
- 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.
- Dissolve 1 g of Indigocarmine dye in 300 ml of a commercially
- 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).
- 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.
-
* 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)
Christmas Tree Staining Procedure
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