Try this:

6 ml  10mM HDTMA

1.5 ml FeCl3 in 10mM HCl

7.5 ml 2 mM CAS solution

20 ml  2.5 mM Piperazine buffer in H2O pH5

fill up to 100 ml with water

add 100 mg sulfosalicylic acid

this solution can be mixed with culture filtrate 1 :1

I tried the procedure described by Charest et al. 2005 and I got positive colonies in both solid (CAS-Agar) and liquid (AT) media. 

Please see : http://www.bashanfoundation.org/hani/hanihunicsubstances.pdf

CAS assay solution: SHELLEY M. PAYNE

2 mM CAS stock solution: 0.121 g CAS in 100 ml water

1 mM Fe stock solution: 1 mM FeCI3 • 6H20 in 10 mM HCI

Piperazine buffer: dissolve 4.307 g piperazine in 30 ml water. Add 6.75 ml concentrated HC1 to bring the pH to 5.6. pH is important.

HDTMA: dissolve 0.0219 g HDTMA in 50 ml water in a 100-ml mixing cylinder.

Mix 1.5 ml Fe solution with 7.5 ml CAS solution and add to the HDTMA in the mixing cylinder. Add piperazine solution to the mixing cylinder and bring volume up to 100 ml with water.

Shuttle solution: 0.2 M 5-Sulfosalicylic acid. Store in the dark. 

Add 0.5 ml CAS assay solution to 0.5 ml culture supernatant and mix. Then add 10/zl of shuttle solution and mix. Let stand a few minutes.  Measure the absorbance (A630) for loss of blue color.

Thank you! I tried the Shelley Payne instructions and had no luck. Also the reference that you sent Salma, I tried that, also no luck... I just can't figure out why i am not seeing a change! I ordered new a CAS bottle so maybe that could be the source? Its so frustrating! 

I am struggling with siderophores as well. For the color problem, check carefully the pH it has to be 6.8 or the color could be yellow, green, purple, dark red (at least on CAS-plates). For color change, check that you are using 2X CAS solution instead of 10X which is the regularly described composition as “CAS solution” on publications. If you use the 10X CAS in 1:1 proportion with your sample, the Fe exchange is verylow and you can´t see the color change… I am having trouble with absorbance at 630nm, as it is not working as everyone said it should...

I think Gunther's answer should help you.

See www.emergenbio.com

http://www.emergenbio.com/siderophore-products.asp

They sell a siderophore detection reagent. It was developed in my lab and works very well.

Rgds

Sean

Dear Sean:

Please have a look at our company which is selling siderophores as well!

http://www. siderophore.com

with regards

Guenther

You must ultra pure water exempt iron and never use any instrument made in metal

Good day, 

For the CAS Agar medium, which one of the CAS is better to purchase from SIGMA, the  dye content 50% or the indicator for metal titration?

Prepare the CAS solution by idisolving 0.121g CAS in 100ml Dwater.

Weigh 0.027g of iron three chloride hexahydrate and dissolve in 10ml of 10mMHCLor the sguttle solution,

Weight 0.219g of HDTMA in 50ml Dwaer in a 100ml volumetric flask

Mix 1.5ml of the iron soultion with 7.5ml of CAS solution and add to HDTMA in the 100ml vokumetric fask. In place of piperazine you can use MES 9.76g dissolved in 50ml Dwater and pH adjusted to 5.6 with 50%KOH. Add this buffer to the 100ml volumetric flask conntaining CAS dye and make up to 100ml mark. Autoclave the prepared CAS solutuion.prepare 0.2M Sulfosalicylic acid and add 10micro liter when adding equal volume of the CAS solution to the supernantant obtained after centrifugation. Thie shuttle solution should be prepared imedaitely before using it

See:

emergenbio.com for the commercial Siderotec Assay.

The SideroTec Assay kit from Emergen Bio is a real time saver and very cost effective. It is ready to use, takes less than 15 minutes to complete an assay and is stable at room temperature.

Hello,

i prepared the CAS solution,

the color was blue at first but after adjusting the pH

to 6.8 it turns yellow, is it normal to find that,

can I have the yellow color after autoclaving .

thanks

I have same problem as of Maissa Bourahla.....The culture medium becomes yellow after mixing CAS reagent. Ideally there should be blue colored plate

See:

emergenbio.com for the commercial Siderotec Assay. Really easy to use and results in 15 min.

https://emergenbio.com/siderophore-products

I am able to prepare CAS assay solution without shuttle, it is blue in colour. But when I added cas assay solution and 5-sulfosalicyclic acid with culture it changed to purple, even the solution changed to purple in presence of media containing 250uM DIP. Even the absorbance values for reference and test are negative

can someone please guide me ?

Aastha Kapoor

Are you adding the shuttle directly into the CAS liquid reagent prior to adding to culture supernatant or are you adding all three items together once you are ready to measure with a spec? They should all three be added separately prior to measuring. A purple color typically indicates the presence of catecholate-type siderophores if the assay is completed correctly. I believe catecholate siderophores peak at 495nm.

Maissa Bourahla the CAS reagent can turn more of a greenish-blue if the pH is too high and can turn yellow due to ferric hydroxide formation (that latter happens at a pH of above 7.

I added the CAS assay solution to the culture then added shuttle solution (in equal volume) and then incubated the tubes in dark for an hour. Then measured the absorbance at 630nm. After one hour incubation the tubes changed the color to purple.

shall I add shuttle solution and directly measure the absorbance. Is incubation creating the problem?