Plant and Animal Tissue Culture

Experiment 6

Principle

All the Animal cells can be grown in a liquid culture medium consisting of a mixture of vitamins, salts, glucose, amino acids and growth factors. Moreover, Calf serum is an easily available source of growth and attachment factors. Antibiotics are added to prevent the growth of bacteria. Under these conditions cells will grow at physiological pH (7.4) and at body temperature (37ºC) to form a monolayer on the culture vessels.

Materials required

Medium (DMEM, RPMI1640) Adult bovine serum, Membrane filter (Millipore 0.45µ)

Sterilize

Double distilled water 1000 ml

1 litre measuring cylinder

100 ml measuring cylinder

1 litre filtration flask Medium storage bottles Other Glasswares

Procedure

Sterilize the laminar air flow by UV irradiation for 45 minutes before using it.

1. Take 500ml of sterile double distilled water in a 1000 ml measuring cylinder.

2. Transfer the contents of the powdered medium into 1 litre measuring cylinder add 3.7 gms of

NaHCO3 in the absence of CO2 incubator.

3. Mix thoroughly to dissolve the powdered medium, and add penicillin /streptomycin/gentamycin.

4. Fill the cylinder with1 litre double distilled water mix and transfer to sterile 2 litre flask and mix.

Pinkish red color of the medium indicates normal pH range.

5. Assemble the filter sterilization set-up and carry out the filtration under negative pressure.

6. Prepare 400 ml of medium containing 10% Adult bovine serum using 100 ml measuring cylinder and store in a 500 ml sera lab bottle.

7. Transfer the remaining medium without serum into big glass bottles.

8. Store the medium in refrigerator, dispose the used membrane and immerse the used glassware in water for washing.

9. Different types of medium is used for various kind of Experiments.

Result:

check your medium sterility

Reference Material:

I. Freshney- Animal Cell Culture.