Culture maintenance procedures and transfer protocols

The aim of maintaining a culture collection is to balance the needs of sustaining healthy stock cultures whilst also reducing the growth rate to provide for longer transfer intervals and less labour costs.

In some situations such as culture supply to external clients or for experimental studies we optimize culture conditions to stimulate near maximum growth rates (one measure of culture vigour).  This section, however, concentrates on the maintenance of stock cultures.

Fig 1. Culture generations for a 3-week transfer schedule

At ANACC each strain is maintained for 3 generations in liquid media in conical flasks with cotton steristoppers (foil-wrapped in axenic cultures) that we denote as the daughter, parent and grandparent cultures (Fig 1).

Volumes typically are 20-40 mL of culture in 50 mL flasks, 75 mL culture in 125 mL flasks or 150 mL culture in 250 mL flasks.  Petriplates, test tubes, round flat bottom flasks or larger conical flask volumes and  agar medium may be used for some strains. Transfer intervals vary from 2 weeks to 6 months depending on species and strains, however most strains are transferred once every 3 to 6 weeks.

When dealing with several hundred strains, it is not feasible to grow every strain under optimal conditions for culture longevity or inter transfer period. Therefore strains are grouped into functional transfer units. A group of benthic diatom species at 20 0C and a group of prymnesiophytes at 10 0C that both will tolerate low light (5-10 μmol. photons m-2 s –1 ) can be transferred once every 6 weeks compared with a group of oceanic diatoms and estuarine dinoflagellates that need frequent transfers every 2-3 weeks.  Volumes transferred will also vary depending on the density of the culture but generally 0.5 – 2.0 mL is transferred from the 50mL flask cultures into new media. All transfers are carried out in a laminar flow hood (complying to Australian Standards 1807-1, 1807-5, 1807-6) under sterile conditions.

While many strains will grow from innoculum to harvest on low light we generally give the fresh daughter innoculum 1-2 weeks of optimal light intensity before decreasing it to maintenance levels. The lower maintenance levels are achieved by placing sheets of glassine paper or paper towelling beneath the cultures and surrounding the flasks with black cardboard cylinders. Note that some types of paper towelling can cause spectral shifts in the light quality that reaches the culture. Daughter and parent cultures are kept together but the grandparents are removed to a separate location in the event of a major room/cabinet malfunction (e.g. compressor failure and dramatic increase in temperature). After the next transfer is completed the grandparent cultures are autoclaved and removed for washing.