• Cryotech Vitrification Kit 101
• Equilibration Solution (ES) : 1 vial (1.0mℓ)
• Vitrification Solution (VS) : 2 vials (1.0mℓ)
• Microscope
• Timer

• Tweezers
• Scissors
• Micropipette (300μℓ)
• Pasteur pipette (150μm inner diameter)

1. 1
   Using the micropipette, fill the Vitri Plate wells with 300μℓ ES and 300μℓ VS respectively (fig. 1). Immediately place the lid on the Vitri Plate.
2. 2
   Observe the oocyte/embryo closely and memorize the size and shape of the perivitelline space in it. This will be used to indicate the full recovery of the oocyte/embryo during the equilibration process.
3. 3
   Aspirate the oocyte/embryo using the tip of the Pasteur pipette.
4. 4
   Place the oocyte/embryo along with a small amount of medium on the surface of the ES (fig. 2).
5. 5
   Activate the timer to begin counting.
6. 6
   If the oocyte/embryo is healthy, it should shrink within the first 30 seconds or so. If it does not, there may be a problem with it; note this for your own future reference.
7. 7
   Once you’ve noted the shrinking of the oocyte/embryo, recall the original perivitelline space and wait for it to fully recover. This recovery process generally takes 6-15 minutes.
8. 8
   While waiting for recovery to take place, use your scissors to open the Cryotec packaging; make sure your cut is perfectly round to ensure smooth removal. Write the ID information of the oocyte/embryo on the Cryotec “handle” and place it in the groove found on the Vitri Plate (fig. 3). Make sure the logo is facing up and away from you.

Fig.1.Solution Preparation

Fig.2.ES Equilibration

1. 9
   Prepare fresh liquid nitrogen in the Cooling Rack and place the cover in it.
2. 10
   When the oocyte/embryo’s volume has fully recovered, ES equilibration is complete.