Methanohalophilus halophilus (Zhilina) Wilharm et al.
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2. If needed, exchange the gas in the test tube for 100% N2 .or 80% N2-20%CO2.
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. Thaw the frozen vial and using a gassed 1.0 ml syringe tipped with 22-gauge needle, withdraw the cell suspension from the vial and transfer to the Balch tube. Plate 0.1 ml of the inoculated culture onto a non-selective medium and incubate the plate aerobically at 37oC. Incubate broth tube at 37oC.
5. Growth should be detected in the broth within 2 to 3 days. No growth should be detected on the aerobic plate.
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.
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Wilharm T, et al. DNA-DNA hybridization of methylotrophic halophilic methanogenic bacteria and transfer of Methanococcus halophilusVP to the genus Methanohalophilus as Methanohalophilus halophilus comb. nov.. Int. J. Syst. Bacteriol. 41: 558-562, 1991.