SideroTec AssayTM (For Research Use Only) Intended Use TM
The SideroTec Assay is a colorimetric test for use in the detection of siderophores secreted by bacteria [1] or fungi [2], or for the assessment of synthetic iron chelators. The test can be used with liquid culture either directly or following filtration to remove microorganisms. The test may also be used with other aqueous based liquids.
Reagents Provided 1. Dye reagent (R1): 1 x 12 ml amber bottle. 2. Catalyst (R2): 1 x 1.2 ml amber bottle. 3. Standard: 1 x 1 ml clear bottle. 4. Diluent: 1 x 10 ml clear bottle. 5. Assay tray: 1 x 96 well plate. Note: Do not mix reagents from different batches of kits Additional Material Required 1. 2.
Background 3. Siderophores are generally low molecular weight peptides which are specific for chelating iron in low iron environments. Siderophores are synthesised by a wide variety of both fungi and bacteria. While there are many different siderophores, in general they fall into three main groups depending on the chemical nature of the reaction with iron: hydroxymates, catecholates and “mixed-type” i.e. they have both reactive hydroxymate and catecholate groups. In general bacteria produce either form of siderophore, and in some cases both, where as fungi generate hydroxymate siderophores only. The SideroTec TM Assay is a universal assay that will react with all classes of siderophore regardless of chemistry or origin and so can be used for detection of a wider range of iron-binding compounds. TM
SideroTec Assay
Principle
The assay is based on the colour change that occurs as result of ferric iron transfer from the reagent complex to siderophore present in a sample. The key reagent is a complex of a dye, iron, and a detergent. The dye complex is initially blue but on removal of iron the dye changes colour to purple or pink colour depending on the amount of siderophore present in a sample. The test can be used as a qualitative indicator for siderophore detection or can be used as quantitative test to estimate the level of siderophore present in a sample. In the absence of any siderophore, the reaction colour remains blue.
4. 5. 6.
Sample. Plastic or Eppendorf® tubes for preparation of reference curve. Accurate pipettes and disposable pipette to dispense 100 µl of sample or reaction material. A repeat 8-channel pipette (50-200 µl) for addition of reagent to a 96-well plate. Microtitre plate reader capable of measuring at 620-630 nm. Timer.
Storage: If not used within 1 month of receipt, the standard should be stored frozen until needed. The kit should be stored at room temperature when not in use. Once opened, the kit should be used within 1 month. The kit has a shelf-life of 12 months from the date the kit was made. The expiry date is located on the outer label of the box. Method Sample Preparation Samples may be aqueous based or be from more complex media such as cell culture. With cell culture, it is not necessary to remove cells from cell culture fluid to detect the presence or absence of siderophore, as the reagent will change colour even in the presence of cellular material. However, the extent of colour change will depend on the amount of siderophore secreted, so very low levels (less than 10 µg/ml) may not be detected where the cell suspension is turbid. In this case, cells should be removed either by filtration or by centrifugation of 1-2 ml of culture fluid in a minifuge prior to assay. For optimal use or for quantitative analysis, samples should be clear of particulate material.
Ref: SideroTec 2301/6
Reagent Preparation Catalyst (R2) should be diluted 1:10 in the Dye reagent (R1). The volume of R1 and R2 solution used will depend on the number of samples to be tested and whether standard or controls are used. Table 1 gives an indication of the amount of each to be mixed depending on the number of test strips used in the plate provided.
2.
Table 1 Number of wells used 16 32 48 64 80 96
Volume of R1 (ml) 2 4 6 8 10 12
Volume of R2 ( µl) 200 400 600 800 1000 1200
3. 4. 5.
culture medium or other aqueous matrix). Add 100 µl of each to wells. Proceed to step 3. For quantitative assessment, add 100 µl of sample or standards to the appropriate wells. For accuracy we recommend that all samples and standards are run in duplicate. However, single wells can be run where a qualitative result is required i.e. where it is only necessary to indicate the presence or absence of siderophore. Add 100 µl of pre-mixed R1 reagent/ R2 using an 8 channel pipette. Incubate for 10 minutes at room temperature. Record results. Wells can be read visually, photographically, or on a microplate reader at 630 nm.
Interpretation of results Standards Standards are prepared by serial dilution of the stock material (100 µg/ml) provided in the kit. It is recommended that standards are run in duplicate. The standard should be prepared following addition and subsequent serial dilution of the 100 µg/ml standard (Table 2). Table 2 Tube Number
Concentration (µg/ml)
C1 C2 C3 C4 C5 C6 C7 C8
100 50 25 12.5 6.25 3.1 1.5 0
Volume of Calibrator (µl) 500 -
Volume of Diluent (µl)
Serial Dilution (µl)
250 250 250 250 250 250 250
250 C1 250 C2 250 C3 250 C4 250 C5 250 C6 -
Note: there is sufficient standard to run two complete standard curves. SideroTec Assay
TM
Procedure
(Note: the assay procedure described is for a manual assay format. However, it is possible to run the assay as single reagent format on an autoanalyzer but the settings should be set locally for each type of analyzer). 1.
For qualitative assay it is sufficient to run samples and both positive (undiluted standard) and negative controls (e.g. cell
Ref: SideroTec 2301/6
For qualitative interpretation, either visual recording or plate reading can be used. Reaction of the dye complex with any iron-binding compound present will result in both a colour change and a reduction in optical density relative to the zero/blank control. For quantitative interpretation, results should be obtained by reading the microplate on a microplate reader. A standard curve should be prepared by plotting optical density of standards versus siderophore concentration (µg/ml). Alternatively results can be expressed as a percentage of the zero standard/blank. The standard curve is calculated by dividing standard OD by the blank/zero standards multiplied by 100 i.e. (OD Standard / OD zero Standard) x 100 Similarly, divide the sample OD by the OD reading of the zero standard and multiply by 100. Extrapolation from the reference curve will give an indication of the relative amount of siderophore in the sample. However, quantitative results should be interpreted with a degree of caution; different siderophores may react at a different rate with dye reagent. Samples with an OD lower than the 100 µg/ml standard should be diluted 1 in 5 in the diluent provided and retested.
Figure 1 is an example of a typical standard curve but it is only provided as an example of what is expected and should not be used for actual determination of siderophore concentration.
References 1. Balado, M., Osorio, C.R. and Lemos, M.L. (2006) A gene cluster involved in the biosynthesis of vanchrobactin, a chromosome-encoded siderophore produced by Vibrio anguillarum. Microbiology. 152(12): 3517-3528. 2. Reiber, K., Reeves, E., Neville, C., Winkler, R., Gebhardt, P., Kavanagh, K. and Doyle, S. (2005) The expression of selected non-ribosomal peptide synthetases in Aspergillus fumigatus is controlled by the availability of free iron. FEMS Microbiol. Lett. 248(1): 83-91.
Figure 1. Typical SideroTec Assay curve.
TM
calibration
Interference Turbidity caused by interfere with reading visual reading, colour even in the presence dependent on the secreted.
microbial growth may of a test. However, for change should be visible of microbes, but may be amount of siderophore
The test works best within pH range 6-8. Acidic (less than pH 5) and basic (greater than pH 9) can interfere with colour development. While most culture fluids are within this range, the pH of those outside may need to be adjusted before use in the test. Some salts at high concentrations (e.g. phosphate ions) may interfere with the test depending on the concentration used. Sensitivity The sensitivity of the test using Desferoxamine as reference material is approx 2 µg/ml Contact Details For technical support, contact: Emergen Bio • • •
e-mail:
[email protected] Tel: 00 353 87 2823555 Webpage: www.emergenbio.com
The SideroTec Assay is distributed by Accuplex LTD, Co.Meath Ireland
Disclaimer Products available from the Company are intended for research use only. It is the responsibility of the user to ensure that they possess the necessary technical skills to determine the appropriateness of these products for the proposed application. The Company cannot accept responsibility for results obtained from these products, as their efficacy depends on conditions of use and the variability of other materials used which are beyond the control of the Company. In no event will the Company be held responsible for the loss of profits, or for indirect or consequential loss including, but not limited to, personal injury arising from the use of these products. Customers are advised that the Company accepts no responsibility for the non-delivery of goods or for damages in transit unless it can be proved to have resulted from its negligence. Claims for goods damaged in transit should be made within 3 working days of receipt. Any replacements are made strictly as an act of goodwill, and should in no way construed as an acceptance of liability. Emergen Bio operates at NUI Maynooth. The term "The Company" is intended to relate to both independent entities. © Emergen Bio 2011
Ref: SideroTec 2301/6
