Microscope image of semen cells on a purple background that was made by staining with eosin-nigrosin to test percent viability.

Introduction

In the realm of reproductive biology, assessing sperm vitality is a cornerstone of fertility testing. The eosin-nigrosin staining technique stands out as a simple, rapid, and reliable method for determining the percentage of live and dead sperm in a semen sample. This blog post offers scientists and technical users a guide to this procedure, exploring its history, mechanism, applications, and practical tips for optimal results in semen analysis.

A Brief History

The eosin-nigrosin staining technique was first developed by Blom (1950) for evaluating bull sperm. Initially a two-step process, it has evolved into a streamlined one-step method, enhancing its accessibility for laboratory technicians. Its ability to differentiate live from dead cells based on membrane integrity has made it a staple in both veterinary and human reproductive biology labs worldwide. A study by Bjorndahl et al. (2003) validated its reliability for human sperm vitality assessment, aligning it with World Health Organization (WHO) standards (WHO laboratory manual for the examination and processing of human semen, 2021). In fertility testing, eosin-nigrosin can identify when sperm motility is below 40%, and differentiate between asthenozoospermia (asthenospermia) in which sperm are unable to move but are still alive versus necrozoospermia (necrospermia), in which there is a significant percentage of dead sperm (Cooper et al., 2010; Gatimel et al., 2017; Lecomte et al., 1999).

How It Works

The eosin-nigrosin staining technique relies on two dyes: eosin and nigrosin. Eosin, a red dye, penetrates the compromised membranes of dead sperm cells, staining them pink or dark pink. Live sperm, with intact membranes, exclude the dye and remain unstained, appearing white or colorless. Nigrosin, a black dye, serves as a negative stain, creating a dark background that enhances contrast and makes sperm visualization easier under a bright-field microscope. This live-dead staining mechanism is both efficient and effective, providing clear results in minutes (Agarwal et al., 2016)

Additional Applications

While the primary application of eosin-nigrosin is in semen analysis for fertility testing, its utility extends to other research-based applications. The technique can assess the viability of various cell types by leveraging the same principle of membrane integrity. For instance, it has been used to evaluate bacterial or yeast cell viability, though such applications are less common, used in research, and may require specific protocol adjustments. Note that while nigrosin alone is used for negative staining of fungal capsules, such as in Cryptococcus neoformans, the combined eosin-nigrosin stain is not standard for fungal viability testing.

Detailed Staining Protocol for Semen Viability

To perform the eosin-nigrosin staining technique, follow these steps for accurate sperm vitality assessment:

Prepare the slide: Pre-label a clean glass slide with patient identifiers to ensure accurate tracking.
Sample collection: Mix a fresh semen sample thoroughly and pipette 30-50 μL into a test tube or equivalent. Samples should be assessed within 30 minutes to 1 hour of collection to avoid degradation.
Add stain: Add 2-3 drops of eosin-nigrosin stain to the tube, close it, and gently swirl for 30 seconds to ensure even mixing.
Create smear: Place one drop of the mixture on the pre-labeled slide and prepare a smear by drawing a coverslip across the slide.
Dry the slide: Allow the smear to air dry completely to prevent artifacts.
Microscopic examination: Coverslip with a compatible mounting medium and examine under a 40x or 100x objective using a bright-field microscope.

Results Interpretation

Viable sperm will appear colorless or faintly pink, while non-viable sperm will have red or dark pink heads against a dark background. For reliable results, count at least 200 sperm cells, as supported by research indicating sufficient accuracy for clinical purposes (Bjorndahl et al., 2003).

Tips and Best Practices

To maximize the accuracy and reliability of the eosin-nigrosin staining technique, consider the following best practices:

Timeliness: Perform staining immediately after motility assessment to ensure sample freshness, as delays can compromise sperm viability.
Sample quality: Use fresh semen samples collected after 48 hours of abstinence, following laboratory or facility guidelines, to ensure accurate vitality data.
Slide preparation: Ensure slides are clean and free of debris to avoid misinterpretation of stained cells.
Stain concentration: Use the correct volume of sample and stain (e.g., 30-50 μL semen with 2-3 drops of stain) to achieve optimal staining intensity.
Drying: Allow smears to dry completely before cover slipping to prevent hypotonic shock artifacts, which can cause a “whipped-around” appearance in sperm tails.
Counting: Count at least 200 sperm cells to obtain a statistically reliable percentage of live and dead sperm, as validated by studies like Bjorndahl et al. (2003).
Interpretation caution: Partially stained sperm may indicate compromised membranes and should be classified as non-viable to avoid overestimating vitality.
Quality stain: Using a high-quality Eosin-Nigrosin stain, such as the one offered by Ethos Biosciences, ensures consistent and clear staining results.

Conclusion

The eosin-nigrosin staining technique is an indispensable tool in semen analysis, offering a quick and reliable method to assess sperm vitality. Its simplicity, coupled with its alignment with WHO standards, makes it accessible for laboratories of all sizes. By adhering to best practices and using high-quality stains, scientists can generate precise data critical for fertility evaluations. For those looking to enhance their lab’s capabilities, explore high-quality Eosin-Nigrosin stains at Ethos Bioscience.

Citations

Agarwal, A., Gupta, S., & Sharma, R. (2016). Eosin-Nigrosin Staining Procedure. In A. Agarwal, S. Gupta, & R. Sharma (Eds.), Andrological Evaluation of Male Infertility: A Laboratory Guide (pp. 73-77). Springer International Publishing. https://doi.org/10.1007/978-3-319-26797-5_8

Bjorndahl, L., Soderlund, I., & Kvist, U. (2003). Evaluation of the one-step eosin-nigrosin staining technique for human sperm vitality assessment. Hum Reprod, 18(4), 813-816. https://doi.org/10.1093/humrep/deg199

Blom, E. (1950). On the evaluation of bull semen with special reference to its employment for artificial insemination. A/S Carl Fr. Mortensen.

Cooper, T. G., Noonan, E., Von Eckardstein, S., Auger, J., Baker, H. G., Behre, H. M., Haugen, T. B., Kruger, T., Wang, C., & Mbizvo, M. T. (2010). World Health Organization reference values for human semen characteristics. Human reproduction update, 16(3), 231-245. https://doi.org/10.1093/humupd/dmp048

Gatimel, N., Moreau, J., Parinaud, J., & Léandri, R. D. (2017). Sperm morphology: assessment, pathophysiology, clinical relevance, and state of the art in 2017. Andrology, 5(5), 845-862. https://doi.org/https://doi.org/10.1111/andr.12389

Lecomte, P. J., Barthelemy, C., Nduwayo, L., & Hamamah, S. (1999). Necrospermia: Etiology and Management. In S. Hamamah, F. Olivennes, R. Mieusset, & R. Frydman (Eds.), Male Sterility and Motility Disorders: Etiological Factors and Treatment (pp. 65-78). Springer New York. https://doi.org/10.1007/978-1-4612-1522-6_6 WHO laboratory manual for the examination and processing of human semen. (2021). (6 ed.). World Health Organization.

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