Chloroflexus aurantiacus Pierson and Castenholz

1. Open vial according to enclosed instructions.                            
2. Using a single tube of #920 broth (5 to 6 ml), withdraw approximately 0.5 to 1.0 ml with a Pasteur or 1.0 ml pipette.  Rehydrate the entire pellet. 
3. Aseptically transfer this aliquot back into the broth tube.  Mix well.
4. Use several drops of the suspension to inoculate a second tube of broth and a #920 slant. 
5. Incubate tubes at 50°C under 1000-2000 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 #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 #920 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^oC 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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