Nuclear Fast Red (Kernechtrot) Reagent

100ml

1

Shake Eosin-nigrosin vital reagent bottle well

2

Mix equal volumes of Eosin-nigrosin vital reagent and ejaculate sample (for example, mix 50 µL of Eosin and nigrosine reagent with
50 µL of ejaculate in Eppendorf test tube)

3

 Mix well

4

Let it set for 30 seconds at room temperature

5

Prepare a glass slide that will contain a thin smear of the stained sample

6

Smear the sample on the side

Note: smear the sample according to the standardized method used in the laboratory or according to the following instructions:

1. Transfer 20 µL of the stained ejaculate sample onto the marked glass slide using the pipette and form a line on the centRE of the slide

2. Cover the slide using another glass slide so the drop spreads evenly. Separate the slides by pulling them horizontally in opposite directions, thus
creating two test slides

7

Dry the sections

Note: if you wish to keep the sections, apply a suitable BioMount medium on the sample (such as BioMount DPX) and cover it with a glass slide

8

Study the sample under a microscopical magnification factor of 40x or under an immersion lens (100x magnification factor)

Note: image under immersion lens 100x magnification will very clearly show the difference between stained and non-stained sperm cells

9

Count 200 sperm cells; non-stained are classified as living sperm cells, while those stained red or pink are classified as dead
sperm cells. Sperm cell that only has its neck stained is classified as living.