Siderophore detection

Aim

Once our bacteria is transformed with the plasmid with the Fur Binding Site and Lac I and the two plasmids with Enterobactins genes, we need to check if our system really work. The production of siderophores can be detected visualy with Blue Agar Chrome Azurol S (CAS). Without siderophore in the medium, CAS and Hexadecyltrimethylammonium bromide (HDTMA)complexes with ferric iron, producing a blue color. When a bacteria strain produce siderophore, the medium color change from blue to orange.

Preparation

Blue Dye

Solution 2
Dissolve 0,0027 g of FeCl3-6H20 in 10 mL of 10 mM HCl.

Solution 3
Dissolve 0,073 g of HDTMA in 40 mL of distilled water.

Mix
Mix solution 1 with 9 mL of solution 2. Then mix with solution 3.
Solution should have a blue color.
Autoclave and store in a bottle.

Mixture solution

Minimal Media 9 (MM9) Salt Solution Stock
Dissolve 15 g of KH2PO4, 25 g of NaCl and 50 g of NH4Cl in 500 mL of distilled water.

NaOH Stock
Dissolve 25 g of NaOH in 150 mL of distilled water.
pH should be around 12.

20% Glucose Stock
Dissolve 20 g of glucose in 100 mL of distilled water.

Casamino Acid Solution
Dissolve 3 g of Casamino acid in 27 mL of distilled water.
Extract with 3% 8-hydroxyquinoline in chloroform to remove iron.
Filter with a 0,22 µm millipore.

CAS agar preparation

Add 100 mL of MM9 salt solution to 750 mL of distilled water.
Bring pH up to 6 and dissolve 32,24 g of piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES); PIPES will not dissolve between pH of 5.
Add 15 g of Bacto Agar.
Autoclave and the cool to 50°C.
Add 30 mL of sterile Casamino acid solution and 10 mL of sterile 20% glucose solution to MM9/PIPES mixture.
Slowly add 100 mL of Blue Dye solution along the glass wall while mixing thoroughly.

Bacteria