Fig. 1. Schematics of the steps involved in conducting the vitality test. (A) Fifty microliters of well-mixed semen is placed into a Boerner slide well. Equal volume of E-N stain is added. The sample is mixed well with a wooden stirrer, and left to sit for 30 seconds. (B) Immediately after that, ten microliters of the mixture is placed on the labeled frosted slide and a thin smear is made. Perform this step in duplicate. Once the smear is air-dried, a coverslip is placed on the mounting media. (C) A drop of immersion oil is placed on each of the mounted slides. One hundred spermatozoa are observed under bright-field microscopy with 1,000× magnification. This step is performed twice on each slide. The vital (unstained) and non-vital (stained) sperm are counted. Results are recorded in replicate and the average percentage of vital sperm is reported.