ACC deaminase assay

Citations:
https://doi.org/10.1371/journal.pone.0269640
https://doi.org/10.1016/j.soilbio.2004.07.033

Minimal salt media

Organism is cultured in organism specific minimal media for 48h.
- Culturing can go longer if needed
Culture is centrifuged at 5000rpm for 10 min.
Supernatant is then discarded
Pellet is resuspended in 1mM phosphate buffer
Steps 2-4 are repeated three times.

Note: - For organisms that do not grow in liquid media, growth on solid media can be substituted. - After incubation collect culture from plate with swab and resuspend in 1mM phosphate buffer - Then proceed with washing +++

ACC assay
- 1 mL of washed microbial suspension was added to 4 mL of ACC-MM
- Incubate culture for ~3 days at 22C
- After incubation centrifuge culture and resuspend in 1 mL 0.1 M Tris-HCl buffer (pH 8.5)
- Add 100 uL of 0.5M ACC and 1 mL Tris-buffer.
- Incubate at room temperature for 30 mins with shaking at 150 rpm.
- After incubation, add:
- 150 uL 0.2% 2,4-dinitrophenylhydrazine reagent (in 2 N HCl) and 690 uL 0.56 N HCl
- Incubate for 30 min at room temperature
- Then add 1 mL 2N NaOH
- Observe and record color
- Change in color from yellow to brown is regarded as positive for ACC deaminase production.

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