Composition of reagent 1:
  • Sodium salicylate 120 g/L
  • Sodium citrate 120 g/L
  • Sodium nitroprusside * 2 H20 0.925 g/L
Composition of reagent 2:
  • Sodium hydroxide 32 g/L
  • Dichloroisocyanuric acid Sodium salt * 2 H2O 2 g/L
  1. Centrifuge 1 mL of culture in an Eppendorf tube at 14’000 for 12 min. 
  2. Dilute the supernatant with dest. water to reach an ammonium concentration in a measurement range of 0 to 50 mg/L NH4+ (3 mM). (i.e. 50x – 100x dilution in case of E. coli HCD fermentation at late stage) 
  3. Mix 100 μL sample with 200 μL reagent 1 and 200 μL reagent 2 in a 4.5 mL photometric cell with 1 cm thickness. (intensification of green colour with increasing ammonium content) 
  4. After 15 minutes reaction, the mixture is diluted with 3 mL distilled water and measure absorption photometrically at 658 nm.

External standard calibration is used to determine NH4+ concentration in samples. NH4Cl or (NH4)2SO4 can be used standard references preparation (std 1, 5 mg/L; std 2, 15 mg/L; std 3, 30 mg/L; std 4, 50 mg/L).

Ammonium dihydrogen phosphate  
µM NH4+ Absorption at 658nm
43.47 0.0267
130.4 0.0768
260.08 0.1548
434.67 0.2634

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