Ninhydrin

We determined the activity of F. tularensis using a color test with ninhydrin [26 (link)] based on ability of citrulline ureidase to degrade citrulline to ornithine, which, on reaction with ninhydrin reagent gives a pink coloration [27 (link)].
A 1.0-ml sample of bacterial suspension (10¹⁰ bacteria) in 0.1 M phosphate buffered saline (PBS, pH 6.5) was mixed with 1.0 ml of 0.7% (w/v) l-citrulline (Sigma Chemical Co., St. Louis, MO, USA) and incubated for 20 h at 30°C. An aliquot (0.01 ml) of the mixture was removed and added to 0.49 ml of distilled water, 1.0 ml of freshly prepared ninhydrin reagent (625 mg of ninhydrin [Sigma] in 10 ml of 6 M H₃PO₄ and 15 ml of glacial acetic acid), and 1.5 ml of acetic acid. Samples were boiled for 1 h, and pink colored samples were considered to have citrulline ureidase activity.

The concentration of surface amine groups was measured using a novel ninhydrin assay, allowing both visualisation and quantification. A clean glass coverslip was submerged in a 0.1 M silane solution for 30 min to apply the –NH2 coating as detailed above. A control untreated coverslip was also used. Subsequently, 1 mL ninhydrin solution (0.35 g of ninhydrin (Sigma, UK) dissolved in 100 mL of ethanol) was added to the materials (including the untreated material) and then heated to 90 °C in an oven for 5 min. The solution was removed from the coverslips and diluted 1:3 with ethanol and then absorbance at 600 nm measured against the untreated control blank. The change in colour of the solution from yellow to purple due to the presence of amine was quantified by reference to a standard curve (ranging from 20 to 120 μM). The coverslips were imaged and photographed using transmitted light microscopy after removal of the ninhydrin reagent to visualise the distribution of the amine groups across the surfaces as purple staining. The concentration of –NH₂ group deposition was calculated from the loss of amine concentration from the coating solution.