Exercise 12.6 - Transfer of Eukaryote Suspension Cultures
Fibroblast suspension culture
Tissue culture laminar flow hood
Media appropriate to culture line used
Disposable pipettes (10 ml and 1.0 ml)
Disposable culture flasks
- 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.
- 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
- 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
- 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.
- Place the new cultures in an incubator at 37° C.
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Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- firstname.lastname@example.org