single sperm freezing protocol

Protocol for Freezing Single Sperm

1. Collect all the individual sperm that you have found into a 2µl drop of Sperm Washing Medium or Hepes that is in an ICSI dish or the dish used for the search.
2. When you have finished collecting all the individual sperm that you want to freeze, prepare a mixture of 100 µl of Sperm Washing with 100 µl of Sperm Freezing Medium that does NOT contain egg yolk.
3. On the device prepare between 1 and 3 drops of the mixture (according to how many you need) with a volume of 0.5-1 µl.
4. Using the micromanipulator move the individual sperm from the original dish into the drops on the device.
5. Insert the device into the tube that has the patient details written on it.
6. Put the tube into liquid nitrogen vapour.
7. Freeze the tube according to normal protocol.

Protocol for Defrosting

1. Take the tube out of the freezer.
2. Remove the device from the tube as fast as possible.
3. Cover the drops on the device with oil that has been pre-heated to 37C. (before the device has been defrosted)
4. Move the device to the micromanipulator and collect the individual sperm.
5. Put the sperm into a 2µl of Sperm Washing medium that has been put in an ICSI dish.
6. Inject the sperm into the egg as per normal protocol.