Crocosphaera subtropica Mareš and Johansen

1. Open thawed vial.
2. Aseptically transfer the entire contents to a 5-6 mL tube of #616 broth. Additional test tubes can be inoculated by transferring 0.5 mL of the primary broth tube to these secondary tubes.
3. Use several drops of the primary broth tube to inoculate a #616 agar slant.
4. Incubate at 26°C for 7-14 days under 2000-3000 lux light.  It is helpful to incubate test tubes in a slanted position to increase gas exchange in broth and to enhance exposure to light.

Good growth, indicated by increased pigmentation in the broth or on the slant, should occur after one to two weeks of incubation.  Examine cells microscopically to assure that they are intact and healthy.  At this time, additional test tubes or flasks can be inoculated.  A 5% inoculum is recommended (i.e. 5 mL of culture to 100 mL fresh medium).

To minimize change in a culture, it is recommended that a frozen seed stock be established from early passage cells.  This may be accomplished by propagating the strain under ideal conditions, utilizing recommended medium, temperature and light.  Prepare a concentrated cell suspension, after good growth is achieved.  If grown in broth, pellet the cells by centrifugation.  Decant the supernatant and resuspend the pellet in fresh #616 broth using 1/10 or less of the original volume.  For slant cultures, wash cells off the agar surface with a minimal amount of #616 broth so that a concentrated cell suspension is attained.  Add 50% DMSO solution to the concentrated cell suspension so that the final concentration of DMSO in the suspension is 5%.  Dispense small aliquots (0.5 to 1 mL) of the suspension into small sterile vials.  Store the vials at -50°C or below.

When needed, remove vials from storage, thaw contents in a 37°C water bath and inoculate into recommended medium.  A minimum of 0.2 mL of the thawed stock should be used to inoculate 5 mL of broth or 1 agar slant.  Cultures may require up to a month to recover from being frozen.  Once grown, however, transfers will grow in a week.

Additional information on this culture is available on the ATCC^® web site at www.atcc.org.

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