| | Re: Eosin Staining Cells
Viability of spermatozoa : fifteen minutes after sample collection, sperm viability is measured using the eosin-Y staining test that is obtained mixing 40 μl of an aliquot of spermatozoa with 20 μl of a eosin supravital stain (dissolve 0.5 - 67 g of eosin Y [C.I. 45380] in aqueous solution of 0.9% NaCl stored in a dark glass bottle at 4 °C. Before use, the staining solution was brought to room temperature). The suspension was incubated for 30 sec at room temperature. Then, a 10 µl droplet was transferred with the pipette to a microscope slide where it was smeared and allowed to dry in air. The slides were then stained in Harris' hematoxylin solution for 2 minutes and then washed for 30 sec with running cold tap water in order to remove the unbound nuclear stain. The next step involved 4-6 dips in acidic-ethanol solution (stock solution: 300 ml ethanol absolute + 100 ml dH20 + 2 ml HCl fuming 37%) to remove the non-specific bound dye (destaining) and then washed with running cold tap water for at least 2 minutes. The smears were air dried and examined directly. For each sample, 200 spermatozoa were assessed with microscope at a magnification of 1000x under oil immersion with a high-resolution 100x bright field objective and blue filter. Only sperm that were blue coloured were classified as alive and those that showed red or any reddish purple colouration were classified as dead. In alternative 1-2 min. after mixing 10 µl spermatozoa suspension with 10 µl of eosin supravital stain cover the sample with a coverslip and examined under a light microscope at 400-1000x magnification. Eosin dye penetrating head membrane of dead cells, which then have pink heads while the live cells appear unstained for membrane-intact (viable). Counting of living sperms (unstained) and dead ones (stained) on one hundred cells were randomly chosen in order to register the number of unstained mobile and immobile cells and stained spermatozoa (died cells). From this, the percentage of spermatozoa viability is estimated.