Whole Cell Nitrogenase Assay |
11-3-97 TSA |
| NOTE: no O2 and no N2 |
| 1. Fill a 40 ml Balch bottle with argon. |
| 2. Add 0.1 ml MeOH or less, then add 0.1 ml 2% Na2S. Let sulfide reduce O2 at least 10 minutes. |
| 3. Bring into anaerobic bag: 40 ml Balch bottle with MeOH and Na2S, and culture to be tested for nitrogenase activity. |
| 4. Add 10 ml of culture to the 40 ml Balch bottle with MeOH and Na2S (make sure syringe and needle have been in the hood for several days). |
| 5. Outside of anaerobic bag, add 1 ml of 10% acetylene or ethyne (see protocol) to get about 0.25% in culture. |
| 6. Shake at about 37°C. |
| 7. Measure ethylene (ethene) after 30 minutes and 1 hour (and overnight, if necessary) with the Perkin-Elmer GC. |
| 8. If ethylene doubles from 30 minutes to 1hour --------> good assay. |
| References |
| 1. Lobo, A.L. and Zinder, S.H. (1988) Applied and Environmental Microbiology 24, 1656-1661. |