Letters in Applied Microbiology 2003, 36, 177–181

Use of CAS-agar plate modified to study the effect of different

variables on the siderophore production by Aspergillus

A. Machuca1 and A.M.F. Milagres2

1
Departmento Forestal, Universidad de Concepción, Campus Los Ángeles, Chile, and 2Department of Biotechnology, Faculdade de
Engenharia Quı´mica de Lorena, Faenquil Lorena, SP, CP 116 CEP 12 600 000, Brazil

2002 / 276: received 3 September 2002, revised 9 December 2002 and accepted 16 December 2002

ABSTRACT
A . M A C H U C A A N D A . M . F . M I L A G R E S . 2003.
Aims: To evaluate the suitability of chrome azurol S (CAS) agar plate assay as a quantitative methodology for
siderophore production.
Methods and Results: Aspergillus species (A. flavus, A. niger, A. tamarii) were inoculated in the CAS-agar plates
and the siderophores production was determined and expressed as CAS-reaction rate (mm per day). All the species
showed positive CAS reaction with different rates depending on culture conditions and A. flavus showed the highest
CAS-reaction rate. The siderophore production in solid medium expressed as CAS-reaction rate was correlated
with siderophore production in liquid medium.
Conclusions: The use of CAS-agar plate assay was modified and the evaluation of CAS reaction in mm per day made
it possible to study and quantify the effect of several variables on the siderophore production by Aspergillus fungi.
Significance and Impact of the Study: We describe the CAS-agar plate assay as a quantitative methodology,
which make it possible to select and evaluate the siderophore production by several microorganisms (fungi and
bacteria) according to different culture conditions.

Keywords: chrome azurol S agar assay, siderophores, iron-chelators, Aspergillus fungi.

siderophore producers have received much attention because

INTRODUCTION
To sequester and solubilize ferric iron, many microorgan-
isms utilize an efficient system consisting of low-molecular
mass (<1000 Da) compounds with high iron affinity termed
‘siderophores’ (Guerinot et al. 1990; Neilands 1995).
According to the generally accepted definition, siderophores
are ferric-specific microbial iron-chelator compounds whose
biosynthesis is regulated by the availability of iron in the
surrounding medium and under conditions of high iron
concentrations, the production of these compounds is
repressed. Therefore, a certain variation can be expected
depending on the particular species under investigation
(Neilands 1984; Guerinot 1994). Studies of microorganism
Correspondence to: A.M.F. Milagres, Department of Biotechnology, Faculdade
de Engenharia Quı´mica de Lorena, Faenquil Lorena, SP, CP 116 CEP 12 600 000,
Brazil (e-mail: adriane@debiq.faenquil.br).
ª 2003 The Society for Applied Microbiology
of the clinical applications and potential utilization of these
chelators in agriculture (Neilands 1993, 1995). Recently,
siderophores have been involved in the early stages of the
lignocellulose depolymerization in wood cell wall by fungi
(Goodell et al. 1997).
Most species of the genus Aspergillus are known to produce
several hydroxamate-type siderophores and many reports on
the isolation and characterization of siderophores have been
published (Hider 1984; Dube et al. 2000). Moreover,
Aspergillus fungi are producers of organic acids such as citric
and oxalic acids which have been reported to act as
siderophores in other microorganisms (Guerinot et al.
1990; Carson et al. 1992; Winkelmann 1992; Dutton and
Evans 1996).
The most common detection method for siderophore
production is the universal assay of Schwyn and Neilands
(1987). This assay is based on a competition for iron between
178 A . M A C H U C A A N D A . M . F . M I L A G R E S
the ferric complex of the dye chrome azurol S (CAS) and a
siderophore. The CAS assay can be applied as a liquid test or
alternatively the dye can be incorporated in the agar medium
for detection of siderophore in solid medium. The CAS assay
of liquid supernatants of cultures has been stated to be
quantitative, however on the solid medium it is not possible
quantify the CAS reaction (Schwyn and Neilands 1987;
Raaska et al. 1993). The major works that employ CAS-agar
plate assay use the term strong or light and symbols (+, ) or
±) to evaluate the diameter of coloured halo around the
microbial colony (Ames-Gottfred et al. 1989; Amaro et al.
1990; Manninen and Mattila-Sandholm 1994).
The objective of this study was to evaluate the suitability
of CAS agar plate assay as a quantitative methodology for
siderophore production measuring the rate of colour change
of CAS medium (in millimetres) as a function of time (in
days) when microorganisms were grown in CAS agar plates.
By employing this methodology, it was possible to evaluate
the effect of the inoculum type, iron concentration and pH
of the media on the siderophore production by Aspergillus
species. Besides, we attempted to correlate the siderophore
production in solid medium with the siderophore produc-
tion in liquid medium using the modified CAS assay.
M A T E R I A LS A N D M E T H O D S
Fungal species
The fungal species used in the present study were Aspergillus
tamarii Kita URM 1035, A. flavus NRRL 3251 and A. niger
NRRL 337. Stock cultures of fungal species were main-
tained on 2% malt extract agar (MEA) plates at 5C.
CAS-agar plate assay modified
The universal CAS assay (Schwyn and Neilands 1987) was
modified to test the ability of fungal species to produce iron-
binding compounds of siderophore-type in solid medium
avoiding the growth inhibition caused by the toxicity of the
CAS-blue agar medium (Milagres et al. 1999). Petri dishes
(10 cm in diameter) were prepared with the MEA medium.
After solidifying, the medium was cut into halves, one of
which was replaced by CAS-blue agar. The halves contain-
ing culture medium (MEA) were inoculated with species
taken from stock cultures. The inoculum was placed as far as
possible from the borderline between the two media (except
to loop inoculum). The plates were incubated in the dark at
28C for 6 days.
Fungi growth rates were monitored daily and expressed as
the number of days required by the microorganism mycelia
to cover the halves of Petri plates containing MEA medium.
The CAS reaction was determined by measuring the
position or distance of the advancing colour-change front
ª 2003 The Society for Applied Microbiology, Letters in Applied Microbiology, 36, 177–181
(in millimetres) in the CAS-blue agar, starting from the
borderline between the two halves during all incubation
times. CAS-reaction rates were calculated from the slope of
the curve distance against incubation time, and expressed as
mm per day. The CAS-agar colour changed from blue to
orange or purple. The growth of species and siderophore
production (CAS reaction) were determined when the effect
of type of inoculum, pH and iron concentration in the MEA
medium were evaluated.
Distilled and deionized water was used for washing the
laboratory material and preparing the culture media. How-
ever, no particular treatment was carried out to eliminate the
iron present in the reagents as a contaminant.
Effect of type of inoculum
The study of the effect of type of inoculum was performed
with two types of inocula: monosporic and loop. Growth of
species and CAS reaction were evaluated daily and
compared with the inoculum block (0Æ5 cm diameter) type.
The blocks were obtained from stock plates which contained
MEA medium and fungal mycelium.
Effect of pH
To study the effect of culture pH on siderophore produc-
tion, MEA medium was buffered with sodium acetate 0Æ1 M
at pH 4Æ5 and 5Æ5. The plates were inoculated and incubated
as described above. Growth of species and CAS reaction
were evaluated daily and compared with the non-buffered
MEA medium (pH 5Æ4).
Effect of iron
To study the effect of iron on siderophore production by the
three Aspergillus species, MEA medium without and with
iron (2 and 4 mmol l)1) were prepared. The plates were
inoculated with one block of agar mycelium (0Æ5 cm
diameter) on MEA medium modified and incubated at
28C for 6 days. Growth of species and CAS reaction were
evaluated daily.
Fungal growth in liquid medium
Fungal species were grown in 100 ml of 2% liquid malt
extract in 250 ml Erlenmeyer flasks, which were inoculated
with one block of agar mycelium (0Æ5 cm diameter) obtained
from stock plates. Liquid malt extract media supplemented
with iron (2 and 4 mmol l)1) were also inoculated with
Aspergillus species. The flasks were incubated at 28C for
6 days in stationary conditions. The broths were harvested,
filtered through filter paper and utilized in the detection of
siderophores and pH determination. Results of siderophore
 USE OF CAS AGAR PLATE MODIFIED TO STUDY THE SIDEROPHORE PRODUCTION 179

production in liquid medium determined by CAS-liquid A. flavus

50
assay were compared with CAS reaction in solid medium.
The dry weight mycelium was determined at 80C.
40 y = 8·17143x – 15·7714
r = 0·99851

Distance (mm)
Siderophores detection in liquid medium 30
A 1Æ0 ml aliquot of supernatant of fungal liquid cultures was
mixed with 1Æ0 ml CAS assay solution prepared according to 20
Schwyn and Neilands (1987). A reference was prepared with
exactly the same medium used for growing the fungi, but 10
uninoculated. The sample (s) and reference (r) absorbances
at 630 nm were measured after 1 h of incubation at room 0
temperature. The percentage of iron-binding compounds of
the siderophore type were calculated by substracting the 2 3 4 5 6 7
sample absorbance values from the reference. Siderophore Time (days)
units are defined as [(Ar ) As/Ar)]Æ100 ¼ % siderophore
Fig. 1 Curve of advancement of the colour change front (mm) in the
units. Percentages of siderophores units less than 10 were
chrome azurol S (CAS)-blue agar against incubation times (days)
considered as negative and in this case no change in the blue
obtained through the CAS-agar plate modified assay. Aspergillus flavus
colour of CAS solution was observed. was inoculated (one block) on malt extract-agar medium (pH 5Æ4, not
buffered) and incubated at 28C for 6 days. The distance of
advancement was daily monitored
RESULTS
Measurements of siderophore production Table 1 Effect of the type of inoculum and the pH medium on the
on the CAS-agar plate siderophore production by Aspergillus fungi growing on CAS-agar
plates modified
In the modified methodology of CAS assay in solid medium,
the microorganisms were grown on one half of the plate CAS reaction rate (mm per day)
containing MEA medium and simultaneously the sidero- Aspergillus Aspergillus Aspergillus
phore production by fungi was detected on the other half Species flavus tamarii niger
containing CAS-blue agar. The use of this modified assay to
measure the siderophore production, made it possible to find Type of inoculum
a linear correlation between the advance of the front colour Loop 5Æ95 ± 0Æ67 4Æ80 ± 0Æ75 3Æ0 ± 0Æ8
Monosporic 5Æ90 ± 0Æ30 4Æ92 ± 0Æ05 5Æ65 ± 0Æ21
change in the CAS-blue agar and the incubation time, up to
Block 8Æ0 ± 0Æ5 4Æ75 ± 0Æ40 5Æ75 ± 0Æ57
7 days (Fig. 1). This linearity was obtained in all the
experiments with modified CAS-agar plate when the pH medium
different species were studied in the different conditions 4Æ5 7Æ90 ± 0Æ41 5Æ32 ± 0Æ45 5Æ30 ± 0Æ32
of the inoculum type, pH of the medium and iron 5Æ5 6Æ38 ± 0Æ09 3Æ10 ± 0Æ07 5Æ40 ± 0Æ12
5Æ4 (not buffered) 7Æ9 ± 0Æ7 4Æ80 ± 0Æ52 5Æ63 ± 0Æ33
concentration. Thus, the production of siderophores in
solid medium by Aspergillus fungi was evaluated as the CAS-
reaction rate and expressed in mm per day. reaction rate. Alternatively, when the lowest reaction was
obtained using loop inoculum of A. niger. In general, the
best type of inoculum for three species was block inoculum.
Effect of the type of inoculum
The CAS reactions produced by A. tamarii were similar for
The type of inoculum influencing the growth of the species all the types of inocula tested.
and different periods of time were necessary for them to
cover half of the plate (MEA): 2 days for the loop, 3 days for
Effect of pH
the blocks and 5 days for monosporic inocula. The CAS-
reaction rate obtained with the three Aspergillus species was The effects of different pH values on the siderophore
similar when monosporic inoculum was used, but when loop production both in buffered MEA medium (pH 5Æ5 and 4Æ5)
and block inocula were used differences among three species and in non-buffered MEA medium (pH 5Æ4) are shown in
were observed (Table 1). The most significant influences Table 1. As can be seen, in the experiments with A. niger,
were observed in the experiments with A. flavus using blocks the CAS-reaction rate was not affected by different pH
as inocula. In this case, A. flavus produced the highest CAS- conditions (about the same for all the pH values tested),
ª 2003 The Society for Applied Microbiology, Letters in Applied Microbiology, 36, 177–181
180 A . M A C H U C A A N D A . M . F . M I L A G R E S
whereas with A. tamarii and A. flavus a lower rate was
observed for pH 5Æ5. Again, A. flavus showed the higher
rates of CAS reaction.
Effect of iron in siderophore production
in solid medium
The ability of Aspergillus species to produce siderophore in
an MEA medium with and without the addition of iron was
tested. All the species showed a good growth under both
iron-sufficient and iron-limited conditions. The incubation
period necessary for each of the three species to cover half
plate was 3 days. The highest rate of CAS reaction was
observed for A. flavus growing in non-iron supplemented
medium (Fig. 2). However, with 2 mmol l)1 of iron, the
CAS-reaction rate decreased and with 4 mmol l)1 this
species did not show any CAS reaction. Cultivations of
A. niger and A. tamarii in media without iron resulted in
similar responses of CAS reaction, but in MEA medium
supplemented with iron, A. tamarii did not show any
reaction. The negative CAS reaction with high iron
concentrations (4 mmol l)1) indicated that A. tamarii and
A. flavus produced no siderophores. On the other hand,
A. niger produced CAS reaction even when a high iron
concentration was present at MEA medium.
Effect of iron in siderophore production
in liquid medium
To attempt a correlation between siderophore production in
solid and in liquid media, the Aspergilli fungi were
10
Siderophores (mm day–1)
8
6
4
2 obtained were different for both media. In liquid medium,
0
A. niger A. tamarii A. flavus
Fig. 2 Effect of iron concentration on the siderophore production by
Aspergillus fungi in solid medium. The production of siderophore was
determined as CAS-reaction rate (mm per day) using the CAS-agar
plate modified assay. The three species were inoculated (one block) on
malt extract-agar medium (pH 5Æ4, not buffered), without and with
iron (2 and 4 mmol l)1), and incubated at 28C for 6 days: , without
Fe(III); h, 2mmol 1)1 Fe(III); n, 4 mmol l)1 Fe(III)
ª 2003 The Society for Applied Microbiology, Letters in Applied Microbiology, 36, 177–181
100
80
Siderophores (%)
60
40
20
0
A. niger A. tamarii A. flavus
Fig. 3 Effect of the iron concentration on the siderophore production
by Aspergillus fungi in liquid medium. The production of siderophore
was determined as siderophore units (%) using the CAS-liquid assay.
The three species were inoculated (one block) on malt extract liquid
medium (pH 5Æ4, not buffered), without and with iron (2 and
4 mmol l)1), and incubated at 28C for 6 days in stationary
conditions: , without Fe(III); h, 2mmol 1)1 Fe(III); n, 4 mmol l)1
Fe(III)
inoculated in malt extract liquid in the same conditions
used for the solid medium (with and without iron).
In liquid medium, the siderophore production was
evaluated by the universal CAS-liquid assay (Schwyn and
Neilands 1987). Percentages of siderophore units less than
10 were considered as negative CAS reaction. The sider-
ophores production by Aspergillus fungi in liquid medium
not supplemented with iron showed the same profile as in
solid medium, with A. flavus as the higher siderophore
producer. In 2 mmol l)1 iron-supplemented cultures sider-
ophores were detected only in the A. niger extracts. In this
condition, A. tamarii and A. flavus did not produce
siderophores in sufficient amounts to be detected by CAS-
liquid assay (responses <10%) (Fig. 3). However, at
4 mmol l)1 iron concentration a strong repression on the
siderophore production by A. niger (12Æ8% siderophore
units), not observed in solid medium, was obtained. The
response of A. tamari in liquid medium was similar to that
obtained in solid medium, but with A. flavus the responses
the siderophore production by A. flavus was significantly
affected by 2 mmol l)1 iron, but in solid medium, only a
decrease was observed in this condition (Fig. 3). Thus, the
siderophores production by A. niger and A. flavus was more
affected by the iron addition in liquid than solid medium.
DISCUSSION
In an earlier work the CAS-agar plate assay was modified
to allow the growth of different microorganisms (fungi and
 USE OF CAS AGAR PLATE MODIFIED TO STUDY THE SIDEROPHORE PRODUCTION
bacteria) without problems of inhibition caused by the
toxicity of the detergent hexadecyltrimethyl-ammonium
bromide (HDTMA) used in the CAS reagent preparation
(Milagres et al. 1999). Now, we propose a form to
quantify the siderophore production on the CAS-agar
plate assay.
The manner used by us for expressing the CAS-reaction
rate in solid medium (mm per day) can be considered as a
quantitative measurement of siderophore production by
microorganisms, conversely as mentioned in literature that it
is not possible on the agar plate assay (Raaska et al. 1993).
Besides, this methodology allowed studying the effect of
different variables (e.g. iron concentration, inoculum-type
and pH of the medium) on the siderophore production by
Aspergillus fungi.
It is known that the biosynthesis of siderophores is
regulated by the iron content of the medium (Neilands
1993). The lack of regulations for high iron concentrations
as the one observed for A. niger suggested the presence of a
non-typical siderophore which also reacted with CAS
reagent. As A. niger is known as a good producer of citric
and oxalic acids (Roehr et al. 1992), probably these organic
acids could be reacting with CAS at high iron concentra-
tions. Organic acid excretion is induced by growth-limiting
concentrations of iron in several microorganisms, but the
production of these metabolic chelators is not repressed by
iron as occurring for typical siderophores (Schwyn and
Neilands 1987; Guerinot et al. 1990).
On the other hand, the results showed that is possible to
correlate the production of siderophores in solid medium
evaluated as CAS-reaction rate (mm per day) with the
production in liquid medium evaluated as siderophore units
(%). In general, the siderophore production on the both
media showed similar profile by the three species of
Aspergillus when cultivated in the absence or presence of iron.
ACKNOWLEDGEMENTS
The authors thanks Fundação de Amparo à Pesquisa do
Estado de São Paulo (FAPESP), Conselho Nacional de
Desenvolvimento Cientı́fico e Tecnológico (CNPq), Secre-
taria de Ciência e Tecnologia do Estado de São Paulo
(SCTDE/SP) and FUNDACION ANDES – Chile, for
financial support.
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