VGO 511: Veterinary Andrology and Reproductive Techniques (1+1)

EOSIN - NIGROSIN STAINING METHOD

(Materials required, Eosin-nigrosin staining method and calculation)

Materials required

Semen sample
Glass slides
Eosin stain (5%)
Nigrosin stain (10%)
Immersion oil
Phase contrast microscope

5% eosin (vital stain) and 10% nigrosin (background stain) are used for live and dead sperm estimation.

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Eosin – Nigrosin staining method

Preparation of 5% eosin stain

Chemical	Quantity
Eosin powder	5 gm
2.9% sodium citrate	100 ml

• Weigh the eosin powder, put in pestle and mortar.
• Prepare 2.9% sodium citrate solution, boil it.
• Add the boiling solution to stain and grind it well. Finally filter and store it at 4⁰C.

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Preparation of 10% nigrosin stain

Chemical	Quantity
Nigrosin powder	10 gm
2.9% sodium citrate	100 ml

• The preparation of the stain is as per the above method.
• After filtration the stain is stored in an air tight bottle at 4⁰C.

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Staining procedure (Click here to view video demonstration)

• A drop of eosin, four drops of nigrosin and a small drop of semen are placed on a clean, grease free slide.
• Mix the semen first with eosin and then immediately with nigrosin stain.
• The mixture is taken on the edge of a slide and pulled across the top of another slide leaving a smear (click here to view picture).
• Allow it to dry in air.
• 200 spermatozoa are counted under oil immersion at a magnification of 1000X in different areas of smear and classify them as normal, head abnormal, mid piece abnormal and tail abnormal sperms. (click here to view picture)

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Calculation

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