Eosin nigrosin staining for sperm vitality: Full guide

Eosin Nigrosin staining is a method that determines the vitality of sperm in a sample where the initial motility is less than 25%. This technique is done right after checking the motility of the sperm in the same semen sample. Vitality assessment of sperm vitality is an essential element of semen analysis. It is necessary for samples where many sperm are immotile to distinguish between dead sperm and immotile live sperm.
Scientists have used eosin to mark dead cells and nigrosin as a background stain to increase the contrast since the 1950s for various mammalian species. 

Test principle

The Eosin part of the stain only colors dead sperm, making them appear dark pink, while live sperm remain white. Eosin can only enter cells when their outer membrane is damaged. This characteristic allows it to indicate dead cells, as it will only stain cells with damaged membranes. Live cells with intact membranes remain unstained. Eosin is an acidic dye that binds to basic compounds, such as specific proteins, and turns them dark red or pink.Nigrosin helps visualize the sperm by increasing the contrast between the sperm heads and the background. 

Specimen Collection

The doctor/ medic instructs the patient on the proper semen collection technique. The patient must collect the specimen in a sterile container and submit it to the laboratory within the specified time.

Eosin nigrosin staining Requirements

• Eosin Nigrosin Stain
• Disposable Pasteur pipettes
• Porcelain Boerner slide
• Wooden applicators sticks.
• Glass slides
• Coverslips
• Mounting media

Eosin nigrosin stain preparation

Nigrosin 10% Solution 

1. Mix 5 grams of Nigrosin with 50 milliliters of deionized water.
2. Heat the mixture gently until the Nigrosin dissolves.
3. Allow the solution to cool to room temperature, then filter it using filter paper. Note: This solution will remain stable for up to 3 months if stored at room temperature.

Eosin Y 1% Solution 

Here’s an easier way to understand and follow the procedure for preparing an Eosin Y solution:

1. Mix 0.5 grams of Eosin Y with 50 milliliters of deionized water.
2. Heat the mixture gently until the Eosin Y dissolves.
3. Allow the solution to cool down to room temperature, then pour it through a filter paper to remove any impurities. Note: This solution can be stored at room temperature and will remain stable for up to 3 months.

Eosin nigrosin staining procedure

1. Label two frosted slides with the accession number, patient’s name, medical record number, and date.
2. Place a drop of well-mixed semen on a Boerner slide.
3. Add two drops of 1% aqueous Eosin Y to the semen.
4. Mix the semen and Eosin Y using a wooden stirrer for 15 seconds.
5. Add two drops of 10% aqueous Nigrosin to the mixture.
6. Mix everything well using a wooden stirrer in the Boerner slide well.
7. Immediately transfer 10-20 μL of the mixture onto each labeled slide to create two thin smears. Allow the smears to air-dry.
8. Place a coverslip over each slide using Cytoseal mounting media.

Eosin nigrosin staining result interpretation

Here’s an explanation of how Nigrosin and Eosin Y are used to assess sperm vitality:

1. Nigrosin creates a dark background that makes it easier to see the sperm.
2. When viewed under a microscope, live sperms have white or light pink heads, while dead sperms have red or dark pink heads.
3. If only part of the neck region is stained and the rest of the head is not, it is called a ‘leaky neck membrane,’ but the sperm is still alive.
4. Record the percentage of viable/ live sperm on the patient’s worksheet.
5. File the slide in the appropriate box for storage.

In normal sperm samples, over 58% of the sperm should be alive (this is the 5th centile, with a 95% confidence interval of 55-63%). If a sample has less than 25% motility, the percentage of live sperm should equal or exceed that of motile sperm.

Quality Control

1. Run a monthly check on a control sample to ensure the reagents are still good. Check the motility of the sample before running the quality control.
2. When you make new reagents, run a quality control check before using them on a new patient sample.
3. Stain the sample with both the old and new reagents. Compare the percentage of live sperm in both sets of slides.
4. The percentage of live sperm should equal or exceed the motility of the sample used. The results from the old and new reagents should be within 10% of each other.
5. Repeat the test using a different sample if the results are not within the acceptable range.

Conclusion

The eosin nigrosin stain technique is simple and does not need negative phase contrast optics. Since it also includes fewer methodological steps to control, it is suitable for standardization and quality control management. It is, therefore, suitable for basic semen analysis after sperm vitality assessment.

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