Thermotoga naphthophila Takahata et al.

2. If needed, exchange the gas in the test tube for 80% N₂ -20% CO₂ or 100% N_2.

3. If the medium is pink (see discussion about resazurin)  add 1 drop of reducing agent (1.5% sodium sulfide stock solution) for each one or two ml of medium.  Let the medium sit at room temperature for 10 to 20 minutes, until the resazurin becomes colorless, before inoculating.

4. Open the freeze-dried vial according to the enclosed instructions.  Take an anaerobic (anaerobic conditions D. below) 1.0 ml syringe tipped with 22 gauge needle and withdraw 0.5 ml of medium from the Balch tube and rehydrate the freeze dried pellet.  Immediately place the re-hydrated vial under a stream of sterile gas, 80N₂-20%CO₂ to maintain anaerobicity.

5. Using the same syringe, withdraw the cell suspension from the vial and transfer it to the Balch tube.   Inoculate a second pre-reduced tube of medium with 0.5 ml of the rehydrated culture.  Plate 0.1 ml of the inoculated culture onto a non-selective medium and incubate aerobically at 37^oC.  Incubate the broth tube at 70^oC.

6. Growth should be detected in the broth within 24 to 48 hours.  No growth should be detected on the aerobic plate or broth.

ANAEROBIC CONDITIONS:   :

A.         Balch tubes (available from Bellco Glass, Vineland, NJ; are specially designed for anaerobic work and use an aluminum crimp cap to hold a rubber stopper in place. Needles can easily be inserted through the stopper, and the tubes can be pressurized to 2 atm.  Alternatively, serum vials may be used, or screw cap tubes with butyl rubber stoppers, in the latter case the stopper may be removed and the tube placed under a cannula system that dispenses sterile, oxygen free gas for addition of reducing agents or inoculation.

B.         Resazurin is a commonly used redox indicator that is pink when the redox potential is above –50 mv, and colorless when the redox potential is below –110 mv. i.e. highly reducing.  Most strict anaerobes require this low redox potential for optimum growth.

C.         To obtain a fully reduced medium, it is necessary that the medium be anoxic and that a reducing agent be added.  Common reducing agents are sodium sulfide, cysteine, dithiothreitol, and titanium citrate.

D.         Syringes can be made anaerobic by one of two methods. 

                  1.     Displace the dead space in the syringe with a sterile

2.      Displace the dead space in the syringe with a

                reducing agent.

          Once growth has been established, the culture should be transferred every 1 or 2 days.  The culture will remain viable for 1 week if stored at room temperature and maintained under anaerobic conditions.

          When this freeze-dried culture is incubated at elevated temperatures (60^oC or above) the cryoprotective agent precipitates.  This precipitation does not interfere with growth of the organism. 

          Cells appear as short rod that occur singly and in pairs. When examined under phase microscopy the sheath can some times be detected.

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