Hyperthermus butylicus Zillig et al.
700455 ™
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700455 ™
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2. If needed exchange the gas in the test tube for 80% H2 and 20% CO2. Do not overpressurize at this time.
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, thaw contents of the vial in a 37°C water bath with gentle agitation.
5. For inoculation, use an anaerobic 1.0 ml syringe (see below) tipped with 22 gauge needle. Withdraw entire contents of the thawed vial and transfer to the Balch tube containing Hyperthermus butylicus broth.
6. Plate 0.1 ml of the inoculated culture onto a non-selective medium and incubate aerobically at 37oC. Additional tubes of prepared anaerobic broth may be inoculated from the primary broth tube. After inoculation, pressurize the tubes to 1 bar overpressure with 80% H2 and 20% CO2 gas mixture. Incubate the inoculated tubes at 90 to 100oC.
7. Growth should be detected in the broth. There should be no growth detected on the aerobic plate.
ANAEROBIC CONDITIONS:
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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Zillig W, et al. Hyperthermus butylicus gen. nov., sp. nov., a hyperthermophilic, anaerobic, peptide-fermenting, facultatively H2S-generating archaebacterium. Int. J. Syst. Bacteriol. 41: 169-170, 1991.
type strain