Thermoanaerobacter brockii subsp. brockii (Zeikus et al.) Lee et al.
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ATCC highly recommends that appropriate personal protective equipment is always used when handling vials. For cultures that require storage in liquid nitrogen, it is important to note that some vials may leak when submersed in liquid nitrogen and will slowly fill with liquid nitrogen. Upon thawing, the conversion of the liquid nitrogen back to its gas phase may result in the vial exploding or blowing off its cap with dangerous force creating flying debris. Unless necessary, ATCC recommends that these cultures be stored in the vapor phase of liquid nitrogen rather than submersed in liquid nitrogen.
2. If needed exchange the gas in the test tube for 80% N2 20% CO2.
3. If the medium is pink (see discussion about resazurin) add 0.1 ml Na2S.9H2O (1.5% sodium sulfide, stock solution) per 5 - 10 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, thaw the frozen vial at room temperature under a gentle stream of oxygen-free gas.
5. For inoculation, use an anaerobic (see c below) 1.0 ml syringe tipped with 22-gauge needle, withdraw the cell suspension from the vial and transfer it to the broth. Plate 0.1 ml of the inoculated culture onto a non-selective medium and incubate aerobically at 37oC. Use 0.1 ml of the inoculated culture to inoculate a nonselective aerobic broth and an additional tube of #1465 broth. Incubate the non-selective aerobic broth tubes at 37oC. Incubate the #1465 broth culture at 60oC.
6. Growth should be detected in the #1465 broth within 24 to 48 hours. There should be no growth detected on the aerobic plate or in the aerobic broth.
a. 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.
b. 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.
c. 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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Validation list no. 11. Int. J. Syst. Bacteriol. 33: 672-674, 1983.
Lee YE, et al. Taxonomic distinction of saccharolytic thermophilic anaerobes: description of Thermoanaerobacterium xylanolyticum gen. nov., sp. nov., and Thermoanaerobacterium saccharolyticum gen. nov., sp. nov.; reclassification of Thermoanaerobium brockii, Clostridium thermosulfurogenes, and Clostridium thermohydrosulfuricum E100-69 as Thermoanaerobacter brockii comb. nov., Thermoanaerobacterium thermosulfurigenes comb. nov., and Thermoanaerobacter thermohydrosulfuricus comb. nov., respectively; and transfer of Clostr. Int. J. Syst. Bacteriol. 43: 41-51, 1993.
Cayol JL, et al. Description of Thermoanaerobacter brockii subsp. lactiethylicus subsp. nov., isolated from a deep subsurface French oil well, a proposal to reclassify Thermoanaerobacter finnii as Thermoanaerobacter brockii subsp. finnii comb. nov., and an emended description of Thermoanaerobacter brockii. Int. J. Syst. Bacteriol. 45: 783-789, 1995.
thermal spring, Yellowstone National Park