Nostoc punctiforme (Kützing) Hariot

1.  Open thawed vial according to enclosed instructions or visit www.atcc.org for instructions.
2. Aseptically transfer the entire contents to a 5-6 mL tube of #819 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 #819 agar slant.
4. Incubate at 26°C for 14-28 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.

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 #819 broth using 1/10 or less of the original volume.  For slant cultures, wash cells off the agar surface with a minimal amount of #819 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.

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