Exercise 12.8 - Computation of Transfer Aliquots

LEVEL II

Materials

• Suspension culture of cells
  
• Media appropriate to culture
  
• Culture flasks
  
• Transfer pipettes
  
• Hemacytometer and microscope

Procedure

1. Obtain 3 transfer culture vessels. The vessels may already contain fresh culture media, or you may be asked to transfer your own.

2. Obtain a suspension culture and count the number of cells/ml of culture using the procedure listed in Exercise 12.7.

3. Compute the volumes of the suspension culture needed so that when added to 25 ml of fresh media, the final concentrations will be:
   1 X 10 cells/ml
   5 X 10 cells/ml
   1 X 10 cells/ml

   For example: If you have 25 ml of fresh media, you will need to add 25 X 10 cells to obtain a final concentration of 1 X 10 cells/ml If your suspension culture contains 5 X 10 cells/ml, you will need to transfer 5 X 10 ml or 0.5 ml of culture to the fresh media (25 x 10 cells divided by 5 x 10 cells/ml).

   Use the formula:

   		Number of cells to be transferred
   Aliquot to be transferred	=	-------------------------
   		Culture concentration

4. Seed three flasks each containing 25 ml of fresh media to a final concentration of 10 cells/ml, 5 X 10 cells/ml and 10 cells/ml.

5. Label and place your fresh cultures in the tissue culture incubator at 37° C.

   Lay the culture flasks on their sides in order to maximize the air exchange surface of the culture and to prevent the cells from drowning. An alternative would be to incorporate mechanical shaking of some type.

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