The best method to evaluate the potential of siderophore production in rhizobacteria under in vitro conditions?
Qualitative, semi-quantitative, and quantitative evaluation...
The chrome Azul S agar (CAS agar) is used for siderophores detection, prepared as described by Barghouthi et al (1989). Single colonies of each isolate should be transferred to this medium and the plates should be placed for incubation at 30°C for 48 hours. The presence of an orange halo around the colonies is recorded as positive for siderophores production.
Preparation of CAS media:
All glassware should be washed with 6M HCl to expel any following components and rinsed with distilled water.
A. Blue Dye:
a. Solution 1: I. Dissolve 0.06 g of CAS (Loba Chemicals) in 50 ml of dd.
b. Solution 2: I. Dissolve 0.0027 g of FeCl3-6 H2O in 10 ml of 10 mM HCl.
c. Solution 3: I. Dissolve 0.073 g of HDTMA in 40 ml of ddH2O.
d. Blend Solution 1 with 9 ml of Solution 2. blend it again with Solution 3. a blue shading solution will form.
autoclave it and store in any plastic holder/bottle.
B. Mixture Solution:
a. Minimal Media 9 (MM9) Salt Solution Stock
I. dissolve 15 g KH2PO4, 25 g NaCl, and 50 g NH4Cl in 500 ml of ddH2O.
b. 20% Glucose Stock
I. Dissolve 20 g glucose in 100 ml of ddH2O.
c. NaOH Stock
I. Dissolve 25 g of NaOH in 150 ml ddH2O (pH =12).
d. Casamino Acid Solution
i. Dissolve 3 g of Casamino corrosive in 27 ml of ddH2O.
ii. It is then mixed with 3% 8-hydroxyquinoline in chloroform to evacuate any trace iron.
iii. Finally, it was filter sterilize.
C. CAS agar Preparation:
a. 100 ml of MM9 salt solution was added to 750 ml ofddH2O.
b. 32.24 g piperazine-N, N’-bis(2-ethanesulfonic acid) PIPES were dissolved.(PIPES will not dissolve below pH of 5). i. The pH was brought up to 6 and slowly PIPES was added while stirring. There was a drop in PIPES while dissolve. While stirring, slowly bring the pH is brought to 6.8 slowly. the pH of medium should not be exceed 6.8 as this will turn the solution green.
c. 15 g of Bacto agar was added.
d. It was then autoclaved and cooled to 50oC.
e. 30 ml of sterile Casamino acid solution and 10 ml of sterile 20% glucose solution was added to MM9/PIPES mixture.
f. 100 ml of Blue Dye solution was added slowly along the glass wall with enough agitation to mix thoroughly.
g. Pour the media into plates, aseptically.
- Badges
- Science topic
- Similar topics
- Phytochemistry
- Extracts