Syntrophococcus sucromutans Krumholz and Bryant
43584 ™
43584 ™
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2. If needed exchange the gas in the Balch tube for 80% N2 20% CO2.
3. If the medium is pink (see discussion about resazurin) add 2.0 ml of reducing agent (1.5% sodium sulfide, 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. Take an anaerobic
1.0 ml syringe (see discussion below) tipped with a 22 gauge needle and withdraw 0.5 ml of 2188 medium from the Balch tube and rehydrate the freeze dried pellet. Immediately place the rehydrated vial under a stream of sterile gas to maintain anaerobic conditions.
5. Using the same syringe transfer the rehydrated cell suspension back into a tube of #2189 broth. Plate 0.1 ml of the inoculated culture onto a non-selective agar medium and incubate aerobically at 37oC. Inoculate a nonselective anaerobic and aerobic broth. Transfer 0.5 ml of the rehydrated culture to a second tube of 2188 medium. Incubate the inoculated tubes at 37 oC.
6. Growth should be detected in the #2189 broth within 24 hours. No growth should be detected on the aerobic plate, or in the nonselective aerobic or anaerobic 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 the 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
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Validation list no. 21. Int. J. Syst. Bacteriol. 36: 489, 1986.
Krumholz LR, Bryant MP. Syntrophococcus sucromutans sp. nov. gen. nov. uses carbohydrates as electron donors and formate, methoxymonobenzenoids or Methanobrevibacter as eletron acceptor systems. Arch. Microbiol. 143: 313-318, 1986.
type strain
DSM, personal communication