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Plant Cell Laboratory Experiment-Determination of cell viability by TTC
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Objective: To study the effect of hydrodynamic stress on cell viability of Linum album.

 

Introduction: Plant cells in culture are more sensitive to hydrodynamic stress than microbial cells due to its large size, bound rigid cellulose based wall and presence of very large vacuoles. Such characteristics impart the perceived sensitivity of plant cell suspension to the hydrodynamic stress, which is associated with aeration/ agitation, shaking, pumping and other operations used for the supply of oxygen and mixing in bioreactor.

 

Requirements:

1.   Callus of L. album

2.   Orbital shaker

3.   Suspension culture media (MS +CFT 500 mg/ml)

 

Protocol:

 

1.  Transfer about 5 g (fresh weight) of callus to the suspension medium using sterile spatula.

2.  Incubate the flasks on a gyrator shaker at 125 & 200 rpm.

3.  Take out samples (5 ml) at regular interval of 2 days. (0,2,4,6 and 8 days).

 

Preparation of Phosphate buffer:

 

Dissolve 3.403 g KH2PO4 in 400 ml water. Adjust the pH to 5.8 with KOH solution and bring the volume to 500ml (final conc.0.05M). 

 

Preparation of Triphenyltetrazolium Chloride (TTC):

 

Prepare 1% solution of TTC in 0.05 Phosphate buffer.

 

Determination of cell viability by TTC:

 

1.  Add 2, 3, 5 triphenyltetrazolium chloride (TTC) solution to the cells in 2:1 ratio.

2.  Incubate the cells for 18-22 h in dark at 25oC.

3.  Discard the supernatant and add 3 ml of 95% ethanol.

4.  Incubate for 15 minutes at room temperature.

5.  Extract red formazan product from cells with 3 ml 95% ethanol for total 30 min with brief heating up to 60 oC for 15 min.

6.  Centrifuge at 3000 rpm for 15 minutes.

7.  Collect the supernatant and measure the O.D at 485 nm.

8.  Calculate percent viability.

 

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