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LABORATORY PROCEDURES FOR PLANT CELL LINES 

REFERENCE NO.: PC/1998/2/3.5


TITLE: VIABILITY TEST WITH FLUORESCEIN DIACETATE (FDA)


INTRODUCTION

The test is usually performed when a new cell line arrives at the collection centre or when a cell line is recovered from freeze preservation. The test gives no quantitative measure of viability but is mainly used to estimate the number of viable cells. The test is performed either with callus or suspension material. In case of suspension material cells can be counted. In case of callus material the percentage of viable cells is estimated.

PROCEDURE

1. To prepare a FDA stock solution 5 mg of fluorescein diacetate is dissolved in 1 ml acetone

2. 2,5 ml cell culture medium is cooled on ice.

3. 50 ml of the FDA stock solution is added and this diluted FDA solution is stored on ice.

4.1. 1 ml of the diluted FDA solution is mixed with 1 ml cell suspension and incubated for 5 minutes at room temperature.

4.2. 1.5 ml of the diluted FDA solution is mixed with 100 - 200 mg callus material (fresh weight) and incubated for 5 minutes.

5. A small amount of the solution containing FDA stained cells (1 or 2 drops) is put onto a microscope slide and covered with a cover slip.

6. The greenish fluorescence of the cells is observed at 100x to 400x magnification under a microscope with fluorescence equipment (filter set 09 ZEISS Co., l ex = 450 - 490nm, l em = 520 nm).

7.1. In case of a suspension culture the number of viable cells is counted. When the number of viable cells is more than 30 % they are cultivated for 1 more week at standard conditions. If the number of viable cells is less than 30 % the culture is cultivated for 2 more weeks and the depositor is informed and asked to provide new material or further information (PC/1998/2/3 Appendix 2). If the number of viable cells less than 15 % the cell culture is considered to be non-viable and the depositor is informed and asked to provide new material (PC/1998/2/3 Appendix 3).

7.2. In the case of a callus culture a rough estimate is made about the number of viable cells. If the number of viable cells is estimated to be at least 30 % the cell culture is cultivated under standard conditions for 4 more weeks. If the estimated number of viable cells is less than 30 % the depositor is informed and asked for new material or further information (PC/1998/2/3 Appendix 2. If less than an estimated number of 10 % of living cells can be observed the depositor is asked to provide new material (PC/1998/2/3 Appendix 3).


Guidelines prepared for CABRI by DSMZ, 20 Jan. 1998
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Copyright CABRI, 1998

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