Symbiotic Fungi: Principles and Practice (Soil Biology)

398 K. Haselwandter and G. Winkelmann
25.3.1.1 Dye Solution
(a) 6.0 mg CAS in 5 ml distilled water
(b) 1 ml Fe(III)-solution (1 mM FeCl 3 6H 2O, 10 mM HCl)
(c) Mix solution a and b to give the CAS–Fe solution
(d) 7.29 mg hexadecyltrimethylammonium bromide (HDTMA) in 4 ml distilled
water
(e) Dye solution: mix d with c and sterilize
25.3.1.2 Preparation of Chrome Azurol S Agar Plates (100 ml Solid Medium)
(a) PIPES salts solution: prepare the appropriate basal salts medium (without
glucose and agar) and add 3.25 g PIPES which dissolves after adjusting to
pH 6.8 with NaOH (50%)
(b) Add 1.5 g agar to the PIPES salts solution and autoclave. After cooling to
50–60 C add sterile solutions of carbon source and other required supplements
such as vitamins or antibiotics.
(c) The dye solution is then added to the PIPES-salts solution containing agar,
carbon source and other nutrient medium constituents.
25.4 Separation of Ferric Hydroxamates by HPLC
Modern laboratory equipment allows to separate and identify most siderophores
by HPLC, using reversed phase columns (4.6 250 mm, Nucleosil C18, 5mm), a
gradient system containing acetonitrile/water (6–40%) plus 0.1% trifluoroacetic
acid (TFA) or formic acid, pH 2, or gradients of acetonitrile/10 mM ammonium
acetate, pH 3, and a detector wavelength of 220 nm or a hydroxamate specific
wavelength of 435 nm (Konetschny-Rapp et al. 1988; Fernandez and Winkelmann
2005). Small amounts of hydroxamates can be purified on a semi-preparative
reversed-phase column (8 250 mm, Nucleosil C18, 7mm) using a gradient of
acetonitrile and a fraction collector system.
Ferrichromes (Fig. 25.6) as well as coprogens and fusarinines (Fig. 25.7) are
well-separated under the conditions as indicated above, although discrimination
between ferrichrome, ferricrocin and ferrichrysin requires careful construction of
the gradient system. The more lipophilic siderophores ferrichrome A, ferrirubin,
ferrirhodin and triacetylfusarinine C (triacetylfusigen) are generally easy to separate,
and represent no problem for identification according to retention times. It is,
however, recommended to use reference compounds which can be added in a cochromatography
mode (spiking). Siderophores as reference compounds can be
ordered, for example, from EMC Microcollections GmbH, Tuebingen, Germany.