Exercise 12.6 - Transfer of Eukaryote Suspension Cultures

Procedure

1. Obtain a culture of mouse fibroblast cells in suspension culture. This will be a simple culture with minimal requirements, and one selected for excellent growth characteristics. The transfer procedure will be similar to that for prokaryotes, with a few major changes. First, all transfers will be done in a tissue culture hood in order to maximize asepsis. Secondly, the cells will be transferred with siliconized pipettes rather than wire loops. The silicone prevents adherance of the cells to the glass wall of the pipette.

   A tissue culture hood is a device that has air moving in layers and under positive pressure. Since the air is filtered, it contains minimal numbers of bacteria or fungal spores, and since it is under a positive pressure, those particles that are present are blown out of the hood. The layering prevents airborne organisms from settling on the work surfaces.

2. Arrange the materials in front of you, easily accessible through the opening of the tissue culture hood. Ensure that any alcohols and wrapping paper are kept clear of the bunsen burner. Pre-sterilize the hood before use, and use disposable sterile gloves.

3. Loosen the cap of a tissue culture flask and the cap of a stock bottle of tissue culture media. 6

   Insert the tip of a sterile pipette into the stock bottle and remove 10 ml of media. Transfer the media to the tissue culture flask.

4. Open the top of the suspension culture and use a sterile 1.0 ml transfer pipette to remove a 1.0 ml sample of the culture. Transfer it to the fresh media in the culture flask. Secure all caps that have been loosened.

5. Place the new cultures in an incubator at 37° C.

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