Spironucleus sp.

1. If the HIHS has not already been added, add 1.5 mL to each tube.
2. Loosen caps one full turn and place tubes in an anaerobic jar.  Add a BD GasPak (one anaerobic system GasPak per anaerobic culture jar).  Close the vessel securely and incubate at 25°C for at least 48 hours.  If the GasPak is of the variety that makes use of a palladium catalyst, the catalyst should be replaced biweekly.
3. Thaw the frozen ampule in a 35°C water bath without agitation until all of the contents are liquid (about 2-3 minutes).
4. Aseptically and gently, lower a sterile Pasteur pipette from which the air has been expelled to the bottom of the liquid in the ampule and slowly aspirate the entire contents into the pipette.  Be careful to minimize agitation of the fluid and so not introduce air bubbles from the tip of the pipette.
5. Inoculate a fresh tube of previously-reduced ATCC medium 1671 by inserting the Pasteur pipette tip aseptically through the liquid overlay-air interface (avoid expulsion of air bubbles or culture) and moving the tip of the pipette to the base of the solid-overlay interface.  Expel the entire contents of the Pasteur pipette into the culture tube (again avoid expulsion of air bubbles), then tighten the cap immediately unless placing the tube directly into an anaerobic jar.
6. With the cap of the freshly inoculated test tube loosened one full turn, place it in an anaerobic jar containing a BD GasPak and incubate at 25°C.  

1. When the culture has reached or is near peak density, remove the growing culture without agitation from the anaerobic jar and immediately screw the tube cap(s) down tightly.
2. The strain grows at the bottom of the liquid overlay as a dense mass of cells.  Carefully introduce a sterile Pasteur pipette aseptically through the liquid overlay-air interface (avoid expulsion of air bubbles) and move the tip of the pipette to the cell mass at the base of the solid-overlay interface.  Aspirate approximately one third of the mass into the pipette.  After removing the pipette, tighten the cap immediately unless placing the tube directly into an anaerobic jar.
3. Inoculate a fresh tube of previously-reduced ATCC medium 1671 as described above.  Place the freshly inoculated tube into the anaerobic jar with the cap loosened one full turn, add a GasPak, and quickly seal the jar.  Incubate at 25°C.
4. Subculture every 7-14 days or as necessary.  An optimal transfer interval should be empirically determined by examining the culture on a daily basis until the growth cycle has stabilized.
   Note:  Addition of antibiotics to the culture may be necessary if the bacterial density in the culture begins to inhibit growth of Spironucleus.  The optimal antibiotic treatment regimen should be empirically determined using a test culture kept in parallel to the original to avoid inadvertently harming Spironucleus.

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