School of Engineering and Science - Jacobs University

Material and Methods
gentle shaking in a waterbath at 25°C, 1 ml of the bacterial suspension was
rapidly filtered through a Whatman nitrocellulose filter (0.45-µm pore size), and
the filter was washed twice with 2 ml of fresh incubation medium. Each filter
was then wrapped in aluminum foil, and counts were determined in a Gamma
4000 counter (Beckman). The radioactivity in the remaining 1 ml of bacterial
suspension was counted directly to determine the total amount of radioactivity
present in the assay. Control assays without bacteria were performed to verify
the complete solubility of labelled iron through siderophore complexation.
4.5.2 Determination of siderophore concentration by quantitative CASassay
Quantification of siderophore activity was performed according to the CASassay
(Schwyn & Neilands, 1987). This photometric assay quantifies a colour
change of a blue coloured iron complex to red/orange after removal of the iron
by a siderophore. The indicator solution was prepared as follows:
1.5 ml of 1 mM FeCl 3+ in 10 mM HCl was mixed with 7.5 ml of a 2 mM
chrome azurol S (CAS) solution and slowely added to 6 ml of a 10 mM
hexadecyltrimethylammonium bromide (HDTMA) solution. The resulting mixture
was subsequently added to 80 ml of a buffer solution (4.307 g piperazine in
80 ml of water, neutralized by addition of 6.25ml of conz. HCl). This indicator
mixture was stored in the dark. Directly before application, 4 mM sulfosalicylic
acid, a substance that enhances siderophore/iron complexe formation. For the
quantification assay, the indicator solution was diluted ½ by addition of the
sample or a dilution of the sample. The mixture was incubated for 1 hour and
the siderophore-dependent colour change was determined at λ=630nm using a
spectrophotometer (Ultrospec 2100pro, Pharmacia Biotech, Freiburg).
Deferoxaminmesylate (DFOM) was applied as a quantification standard. The
CAS reaction was linear to a concentration of 0-20 µM DFOM.
42