Agriculture Reference
In-Depth Information
1.5.5.3 Qualitative and Quantitative Estimation of Siderophores
The PS bacterial strains are further tested for siderophore production using Chrome
Azurol S (CAS) agar medium following the method of Alexander and Zuberer
(
1991
). Chrome Azurol S (CAS) agar medium is prepared from four solutions as
(i)
Solution 1
, Fe-CAS indicator solution: A 10 ml of 1 mM FeCl
3
·6H
2
O [in 10 Mm
HCl} is mixed with 50 ml of an aqueous solution of CAS (1.21 mg/ml). The above
solution is then added to 40 ml of HDTMA (1.82 mg/ml) and cooled to 50
C.
(ii)
Solution 2
, buffer solution: A 30.24 g of PIPES is dissolved in 750 ml of a salt
solution containing 0.3 g KH
2
PO
4
, 0.5 g NaCl and 1 g NH
4
Cl, pH 6.8, with 50 %
KOH, and water is added to bring the volume to 800 ml. (iii)
Solution 3
: (in 70 ml
water) 2 g glucose, 2 g mannitol, 493 mg MgSO
4
.7H
2
O, 11 mg CaCl
2
, 1.17 mg
MnSO
4
.H
2
O, 1.4 mg H
3
BO
3
, 0.04 mg CuSO
4
·5H
2
O, 1.2 mg ZnSO
4
.7H
2
O and
1mgNa
2
MoO
4
·2H
2
O. Autoclaved, cooled to 50
C, then added to the buffer
solution along with 30-ml filter-sterilized 10 % (W:V) casamino acids (Solution
4). The indicator solution is added last with sufficient stirring to mix the ingredients
without forming bubbles. Chrome Azurol S agar plates are then prepared separately
and divided into equal sectors and spot inoculated with 10
lof10
8
cells/ml and
μ
2
C for 5 days. Development of yellow-orange halo around the
bacterial growth is considered as positive for siderophore synthesis. Each individual
experiment should be repeated three times to ensure the reproducibility of results.
The production of siderophore by the PSB strains are further detected quantitatively
using Modi medium (K
2
HPO
4
0.05 %; MgSO
4
0.04 %; NaCl 0.01 %; mannitol
1 %; glutamine 0.1 %; NH
4
NO
3
0.1 %). Modi medium is inoculated with 10
8
cells/
ml of PSB and incubated at 28
incubated at 28
2
C for 5 days. Catechol-type phenolates are
measured on ethyl acetate extracts of the culture supernatant using a modification of
the ferric chloride-ferrocyanide reagent of Hathway. Ethyl acetate extracts is
prepared by extracting 20 ml of supernatant twice with an equal volume of solvent
at pH 2. Hathway's reagent is prepared by adding 1 ml of 0.1 M ferric chloride in
0.1 N HCl to 100 ml of distilled water, and to this, 1 ml of 0.1 M potassium
ferrocyanide is added (Reeves et al.
1983
). For the assay, one volume of the reagent
is added to one volume of sample, and absorbance is determined at 560 nm for
salicylates with sodium salicylate as standard and at 700 nm for dihydroxy phenols
with 2, 3-dihydroxy benzoic acid (DHBA) as standard.
1.5.5.4 Assay of Hydrogen Cyanide and Ammonia
Hydrogen cyanide (HCN) production by PS cultures is detected by the method of
Bakker and Schipper (
1987
). For HCN production, PS bacterial strains are grown
on an HCN induction medium (g/l: tryptic soy broth 30; glycine 4.4; agar 15) for
3-4 days at 28
lof10
8
cells/ml is spread on
the Petri plates. A disc of Whatman filter paper no. 1 dipped in 0.5 % picric acid and
2%Na
2
CO
3
is placed at the lid of the Petri plates. The plates are then sealed with
2
C. For each bacterial strain, a 100
μ
