Thermotoga elfii Ravot et al.
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2. If needed exchange the gas in the test tube.
3. If the medium is pink (see discussion about resazurin) add 2.0 ml of reducing agent (3% cysteine, stock solution) per 100 ml of medium. Let the medium sit at room temperature for 10 to 20 minutes - until the resazurin becomes colorless - before inoculating.
4. When the Balch tube is ready to inoculate, open the vial according to enclosed instructions.
5. For inoculation, use a 1.0 ml syringe tipped with 22 gauge needle. Make the syringe anaerobic (see discussion below) and withdraw 0.5 ml of #1977 broth and use this to rehydrate the freeze-dried pellet using anaerobic techniques. Transfer the rehydrated cell suspension back to the tube of #1977 broth and incubate at 60-65oC. Plate 0.1 ml of the inoculated culture onto a non-selective medium and incubate aerobically at 37oC. Inoculate a nonselective anaerobic and aerobic broth and incubate at 60-65oC
6. Growth should be detected in the #1977 broth within 48 hours. There should be no growth detected on the aerobic plate. There should be no growth in the nonselective aerobic or anaerobic broth.
a. Balch tube refers to a special type of test tube that is designed to be pressurized and is suited for anaerobic work. The Balch test tubes can be purchased form Bellco glass (www.bellcoglass.com; stock no. 2048-00150).
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
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Ravot G, et al. Thermotoga elfii sp. nov., a novel thermophilic bacterium from an African oil-producing well. Int. J. Syst. Bacteriol. 45: 308-314, 1995. PubMed: 7537064