ROUTINE MAINTENANCE AND STORAGE OF CELL LINES

LABORATORY PROCEDURES FOR PLANT CELL LINES

REFERENCE NO.: PC/1998/3/3

TITLE: ROUTINE MAINTENANCE AND STORAGE OF CELL LINES - LIVING CELL LINES -

INTRODUCTION

When a cell line has passed the procedures PC/1998/2/2, PC/1998/2/3 and PC/1998/3/1 it is submitted to the routine maintenance procedures. Cell lines are maintained as calluses grown on petri dishes of 60 mm diameter and 15 mm height. Petri dishes are either maintained under continuous illumination (ca. 600) lux or in the dark. The petri dishes grown under continuous light are stored in a shelf. In the shelf four petri dishes are stored one upon the other. The petri dishes maintained in the dark are stored in quadratic trays, 10 by 10 = 100 cell lines in one tray. In the trays three petri dishes are stored one upon the other. The standard growth conditions for all cell lines are: 24 °C and 40 % rel. humidity.

All cell lines are transferred to fresh medium at least every four weeks. Cell lines transferred to fresh medium more often are marked specifically.

Two independent sets of the whole collection are maintained independently. Two different technicians care for each set of the collection independently.

Suspension cultures, which are not routinely maintained but only for special purposes are transferred like described in the procedure PC/1998/3/3.3.

PROCEDURE

1. Every morning all cultures are controlled by a quick check. Therefore the responsible person controls the petri dishes on top of the others of each cell line by a quick overview for normal growth. If there is any reason to assume that growth and/or phenotype of the checked callus is not normal, the two other calluses of this specific cell line are also checked.

2. By standard criteria (PC/1998/3/3.1) all cultures are sorted out which need an immediate transfer to fresh medium during the every day quick check, transported to the transfer room and transferred to fresh medium according to the transfer procedure guidelines (PC/1998/3/3.2).

3. For normal culture transfer the trays and transported to the transfer room. In case of cultures maintained in the light the petri dishes are taken out of the shelf and transported to the transfer room.

(Routinely the cultures are transferred to fresh medium according to a fixed time schedule every four weeks.)

4. For transfer to fresh medium the petri dishes of a cell line are taken out of their specific place (in the tray) and placed into the clean bench.

5. The specific callus to be transferred to fresh medium is sorted out and transferred to fresh medium according to the transfer procedure guidelines (PC/1998/3/3.1and PC/1998/3/3.2)

(Normally 3 petri dishes of each cell line are maintained, one old and two young petri dishes. For routine transfer the old petri dish is discarded, one of the younger petri dishes is left as safety reserve and the other of the younger petri dishes is used to inoculate two new petri dishes with callus)

6. After inoculation the petri dish is closed with the lid and lid and petri dish are tightly closed by careful wrapping with parafilm.

7. The name of the culture, the collection number, the medium (if necessary) and the date of transfer is written on the lid of the petri dish.

8. After this procedure the petri dishes are put back into their specific place in the tray resp. in the culture room.

9. At the end of a working day all cultures that have been transferred to fresh medium and the corresponding cultures kept as reserves are transported back into the culture room.

10. Normally once a week a more detailed growth check is performed including a comparison of the different cultures in the two collection sets concerning the criteria listed in (PC/1998/3/3.1).

Guidelines prepared for CABRI by DSMZ, 20 Jan. 1998
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