1. A practice session with the mouth-operated pipet system is essential prior to dealing with a large number of eggs. Use media alone and then a small number of eggs to learn the technique. Experiment with potential mistakes, such as blowing bubbles into the media, a common problem that can cause a time-consuming dispersal of eggs. Ensure that the pipet does not become loose in the holder.
2. After BSA (see Chapter 16), paraffin oil is the second most likely source of toxins that will kill eggs. Experiment with noninjected eggs to test that development to the blastocyst stage (4 d in culture, by which time a ball of cells containing a blastocoel is formed) is permitted in more than 50% of the original one-cell eggs. It may be necessary to test several different products and possibly batches. In a recent experiment (Ho, M. Y., Carter, D. A , and Murphy, D., unpublished) in which different oils were compared, we have found that egg survival in culture was excellent using Fluka paraffin oil (Cat. No: 76235).
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