FAQ1: What is the in-use stability?
Once opened, the reagents can be used until the expiry date (24 months after production) if stored and used correctly.

FAQ2: How long should we wait before we count cells after staining?
Read the slide immediately. Nigrosin is toxic for spermatozoa. If the slide is not read immediately, the counted value might be false positive.

FAQ3: Is a smear necessary?
No, it is better to put a drop on the microscope slide and cover this with a cover slip.

FAQ4: Is there a method for QC testing of VitalScreen that can be performed by users in the laboratory? 

A. Our standard in-house QC test

1. Count motility of the native sample
2. Mix 50µl sample with 2 drops of eosin (reagent 1) from new lot
3. After 30 seconds, add 3 drops of nigrosin (reagent 2) from new lot
4. Within 30 seconds, place 19.5µl of this mixture on a microscope slide, and cover it  with a cover slide
5. Immediately count at least 100 spermatozoa (red is dead, colourless/white is alive)
6. Repeat these steps with the old lot number
7. Compare the results of old and new lot number
8. Interpretation: % dead ≤ % immotile or % dead max 20% higher than % immotile and results shouldn’t differ more than 10% between old (reference) lot and new lot number

B. Ethanol test

Measure vitality of native sample

1. Mix 50µl sample with 2 drops of eosin (reagent 1)
2. After 30 seconds, add 3 drops of nigrosin (reagent 2)
3. Within 30 seconds, place 19,5µl mixture on a microscope slide and cover with cover slide
4. Immediately count at least 100 spermatozoa (red = dead; white = alive)

Add ethanol to the sample so that the end concentration of ethanol in the sample is 21%, all spermatozoa will be dead after 5 minutes.

Measure vitality of sample with ethanol

1. Mix 50µl sample/ethanol  with 2 drops of eosin (reagent 1)
2. After 30 seconds, add 3 drops of nigrosin (reagent 2)
3. Witin 30 seconds, place 19,5µl mixture on a microscope slide and cover with cover slide
4. Immediately count at least 100 spermatozoa (red = dead, white = alive)

There should be a clear difference between the native sample and the sample spiked with ethanol. The number of dead cells should be higher in the samples that were treated with ethanol

FAQ5: Is there a VitalScreen kit available with only eosin?
FertiPro only sells a two-step kit, with nigrosin. This is to ensure that living cells (which will not stain red) are clearly visible as nigrosin provides a dark background. However, the second step can be altered by lowering the amount of nigrosin added to the mix, which will result in a lighter background. Alternatively, the second step can also be skipped, but this can result in a harder reading of living amount of spermatozoa.