SideroTec AssayTM Reagents Provided

1. Dye reagent (R1): 1 x 12 ml amber bottle.

(For Research Use Only)
2. Catalyst (R2): 1 x 1.2 ml amber bottle.
Intended Use 3. Standard: 1 x 1 ml clear bottle.
4. Diluent: 1 x 10 ml clear bottle.
TM
The SideroTec Assay is a colorimetric test for use 5. Assay tray: 1 x 96 well plate.
in the detection of siderophores secreted by Note: Do not mix reagents from different batches
bacteria [1] or fungi [2], or for the assessment of of kits
synthetic iron chelators. The test can be used with
liquid culture either directly or following filtration Additional Material Required
to remove microorganisms. The test may also be
1. Sample.
used with other aqueous based liquids.
2. Plastic or Eppendorf® tubes for
Background preparation of reference curve.
3. Accurate pipettes and disposable pipette
Siderophores are generally low molecular weight to dispense 100 µl of sample or reaction
peptides which are specific for chelating iron in material.
low iron environments. Siderophores are 4. A repeat 8-channel pipette (50-200 µl) for
synthesised by a wide variety of both fungi and addition of reagent to a 96-well plate.
bacteria. While there are many different 5. Microtitre plate reader capable of
siderophores, in general they fall into three main measuring at 620-630 nm.
groups depending on the chemical nature of the 6. Timer.
reaction with iron: hydroxymates, catecholates
and “mixed-type” i.e. they have both reactive Storage: If not used within 1 month of receipt,
hydroxymate and catecholate groups. In general the standard should be stored frozen until
bacteria produce either form of siderophore, and needed.
in some cases both, where as fungi generate
The kit should be stored at room temperature
hydroxymate siderophores only. The SideroTec
TM when not in use. Once opened, the kit should be
Assay is a universal assay that will react with all
used within 1 month. The kit has a shelf-life of 12
classes of siderophore regardless of chemistry or
months from the date the kit was made. The
origin and so can be used for detection of a wider
expiry date is located on the outer label of the box.
range of iron-binding compounds.
TM Method
SideroTec Assay Principle
Sample Preparation
The assay is based on the colour change that
occurs as result of ferric iron transfer from the Samples may be aqueous based or be from more
reagent complex to siderophore present in a complex media such as cell culture. With cell
sample. The key reagent is a complex of a dye, culture, it is not necessary to remove cells from
iron, and a detergent. The dye complex is initially cell culture fluid to detect the presence or absence
blue but on removal of iron the dye changes colour of siderophore, as the reagent will change colour
to purple or pink colour depending on the amount even in the presence of cellular material. However,
of siderophore present in a sample. The test can the extent of colour change will depend on the
be used as a qualitative indicator for siderophore amount of siderophore secreted, so very low levels
detection or can be used as quantitative test to (less than 10 µg/ml) may not be detected where
estimate the level of siderophore present in a the cell suspension is turbid. In this case, cells
sample. In the absence of any siderophore, the should be removed either by filtration or by
reaction colour remains blue. 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 culture medium or other aqueous matrix).
Add 100 µl of each to wells. Proceed to
Catalyst (R2) should be diluted 1:10 in the Dye step 3.
reagent (R1). The volume of R1 and R2 solution 2. For quantitative assessment, add 100 µl
used will depend on the number of samples to be of sample or standards to the appropriate
tested and whether standard or controls are used. wells. For accuracy we recommend that
Table 1 gives an indication of the amount of each all samples and standards are run in
to be mixed depending on the number of test duplicate. However, single wells can be
strips used in the plate provided. run where a qualitative result is required
i.e. where it is only necessary to indicate
Table 1
the presence or absence of siderophore.
Number of Volume of R1 Volume of R2 3. Add 100 µl of pre-mixed R1 reagent/ R2
wells used (ml) ( µl) using an 8 channel pipette.
16 2 200 4. Incubate for 10 minutes at room
32 4 400 temperature.
48 6 600 5. Record results. Wells can be read visually,
64 8 800 photographically, or on a microplate
80 10 1000
reader at 630 nm.
96 12 1200
Interpretation of results
Standards For qualitative interpretation, either visual
recording or plate reading can be used. Reaction of
Standards are prepared by serial dilution of the
the dye complex with any iron-binding compound
stock material (100 µg/ml) provided in the kit. It is
present will result in both a colour change and a
recommended that standards are run in duplicate.
reduction in optical density relative to the
The standard should be prepared following
zero/blank control.
addition and subsequent serial dilution of the 100
µg/ml standard (Table 2). For quantitative interpretation, results should be
obtained by reading the microplate on a
Table 2
microplate reader. A standard curve should be
Tube Concentration Volume Volume Serial prepared by plotting optical density of standards
Number (µg/ml) of of Dilution
versus siderophore concentration (µg/ml).
Calibrator Diluent (µl)
(µl) (µl)
C1 100 500 - Alternatively results can be expressed as a
C2 50 - 250 250 C1 percentage of the zero standard/blank. The
C3 25 - 250 250 C2
C4 12.5 - 250 250 C3
standard curve is calculated by dividing standard
C5 6.25 - 250 250 C4 OD by the blank/zero standards multiplied by 100
C6 3.1 - 250 250 C5
C7 1.5 - 250 250 C6 i.e. (OD Standard / OD zero Standard) x 100
C8 0 - 250 -

Similarly, divide the sample OD by the OD reading

of the zero standard and multiply by 100.
Note: there is sufficient standard to run two
Extrapolation from the reference curve will give an
complete standard curves.
indication of the relative amount of siderophore in
TM
SideroTec Assay Procedure the sample. However, quantitative results should
be interpreted with a degree of caution; different
(Note: the assay procedure described is for a siderophores may react at a different rate with dye
manual assay format. However, it is possible to reagent.
run the assay as single reagent format on an
autoanalyzer but the settings should be set Samples with an OD lower than the 100 µg/ml
locally for each type of analyzer). standard should be diluted 1 in 5 in the diluent
provided and retested.
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
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 References
determination of siderophore concentration.
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.

TM
Figure 1. Typical SideroTec Assay calibration
curve.

Interference

Turbidity caused by microbial growth may

interfere with reading of a test. However, for
visual reading, colour change should be visible
even in the presence of microbes, but may be
dependent on the amount of siderophore
secreted.

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
Disclaimer
test.
Products available from the Company are intended for research
Some salts at high concentrations (e.g. phosphate use only. It is the responsibility of the user to ensure that they
ions) may interfere with the test depending on the possess the necessary technical skills to determine the
concentration used. appropriateness of these products for the proposed
application. The Company cannot accept responsibility for
Sensitivity 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 sensitivity of the test using Desferoxamine as
the Company be held responsible for the loss of profits, or for
reference material is approx 2 µg/ml indirect or consequential loss including, but not limited to,
personal injury arising from the use of these products.
Contact Details Customers are advised that the Company accepts no
responsibility for the non-delivery of goods or for damages in
For technical support, contact: Emergen Bio transit unless it can be proved to have resulted from its
negligence. Claims for goods damaged in transit should be
• e-mail: kieran.walshe@nuim.ie made within 3 working days of receipt. Any replacements are
• Tel: 00 353 87 2823555 made strictly as an act of goodwill, and should in no way
• Webpage: www.emergenbio.com construed as an acceptance of liability.

Emergen Bio operates at NUI Maynooth. The term "The

The SideroTec Assay is distributed by Accuplex Company" is intended to relate to both independent entities.
LTD, Co.Meath Ireland
© Emergen Bio 2011
Ref: SideroTec 2301/6