Antimicrobial properties of natural dyes

Coloration
against Gram-negative bacteria
Technology
Deepti Gupta,a,* Sudhir Kumar Khareb and Ankur Lahaa
aDepartment of Textile Technology, Indian Institute of Technology Delhi, Hauz Khas, New
Delhi-110016, India
Email: deepti@textile.iitd.ernet.in
bDepartment of Chemistry, Indian Institute of Technology Delhi, Hauz Khas, New Delhi-
110016, India
Received: 5 January 2004; Accepted: 14 April 2004 Society of Dyers and Colourists

There is increasing interest in adding value to textiles by the use of natural products. Many of the plants
from which natural dyes are obtained are, for example, also known to have medicinal properties. In the
current study, the antimicrobial properties of eleven natural dyes against three types of Gram-negative
bacteria were studied experimentally. Seven of the dyes showed activity against one or more of the
bacteria. The minimum inhibitory concentration for three selected dyes was determined. The results
demonstrate that certain dyes are able to reduce microbial growth almost completely in the case of
Escherichia coli and Proteus vulgaris. Selected dyes would therefore be valuable for the dyeing of sheets
and gowns for hospital use, and on articles which are less suitable for laundering such as mattresses
and upholstery. The dyes examined exhibited good wash fastness and the antibacterial effect is
therefore likely to be durable.

known to have therapeutic properties. Indeed, many plants

Introduction
There has been increasing interest in building antibacterial
properties into textiles. Consumers worldwide are looking
for clothing which provides greater comfort and remains
fresh and odour-free in use. The scale of the market is
demonstrated by the fact that it is expected that the US
demand for antimicrobial chemicals for textile use will
exceed 1400 tonnes by 2005.
Clothing and other textile materials can act as carriers
for microorganisms such as pathogenic or odour-generating
bacteria and moulds. Cotton textiles in contact with the
human body offer an ideal environment for microbial
growth, providing oxygen, water and warmth, as well as
nutrients from spillages and body exudates. The popularity
of apparel based on synthetic fibres has emphasised the
requirement for biostatic finishes since the limited
moisture transportation offered by these fabrics causes a
more obvious degree of perspiration wetness. This is
particularly important in sportswear. Antibacterial finishes
are also highly desirable for textiles in the medical field
as textile materials used in hospitals and hotels are liable
to promote cross-infection and transmit disease; in fact
hygiene problems with hospital textiles can interfere with
the recovery of patients.
A number of studies are mentioned in the literature in
which medicinal plants have been tested for activity against
bacteria, fungi and viruses with a view to exploring their
potential medicinal value [1–3]. Interest in plants having
antimicrobial properties has revived as a consequence of
problems currently arising from the wide use of antibiotics.
Traditional medicinal plants contain a variety of compounds
synthesise secondary antimicrobial metabolites as part of
their normal programme of growth and development, often
sequestering them in tissues which require protection
against microbial attack. Substances which are able to
inhibit or destroy pathogens without toxic impact on the host
cells are obvious candidates for use in the development of
new antimicrobial drugs [4].
A number of previous studies have described the
characterisation, purification and application of natural dyes
[5–8], and the present investigation has focused on the use
of natural dyestuffs as antibacterials. Many of the plants
traditionally used in dyeing are credited with medicinal
properties. For example, a commonly used natural dye,
pomegranate, is obtained from Punica granatum and is rich
in hydrolysable tannins, a class of compounds known to
exhibit a remarkable degree of antimicrobial activity [9]. A
number of other plant dyes sources are rich in naphtho-
quinones, including lawsone from henna, juglone from
walnut and lapachol from alkannet, and this group of
compounds is reported to display antibacterial, antifungal,
antiviral and antineoplasic activity [10–13].
No systematic study has previously been undertaken to
study the antimicrobial properties of products used as
natural dyes. In the current investigation the antimicrobial
activity of eleven commercial natural dyes against three
common infection-causing bacteria has been examined.
Should the dyes themselves be inherently antimicrobial,
therapeutic and protective textiles could be produced at
no additional cost. Since all the commercial dyes selected
had good wash fastness it is to be expected that the effect
will be durable.

Web ref: 20040404 © Color. Technol., 120 (2004) 167

20040404_j0404.pmd 167 7/27/2004, 12:06 PM

 Experimental
Materials
A plain weave cotton fabric of 107 g/m2 having 40 ends/
cm and 35 picks/cm was used. Commercial samples of
natural dye powders were obtained from Alps Industries
Ltd, Sahibabad, India. Specifications of the dyes are listed
in Table 1. A commercial antimicrobial agent, Fabshield
AEM 5700, was supplied by Rossari Biotech India Pvt Ltd
and was used as a reference. All other chemicals were of
laboratory reagent grade.
For microbiological testing, three Gram-negative
bacteria, Escherichia coli, Klebsiella pneumoniae and
Proteus vulgaris, known to be responsible for cross-
infections in the hospital environment, were procured from
Microbial Type Culture Collection (MTCC), Chandigarh,
India. Agar powder and peptone of analytical reagent purity
were used in the experimental studies.
Equipment
For dyeing, a Julabo SW22 shaking water bath with
automatic temperature control was used. For the
microbiological studies, glassware and culture media were
sterilised in an autoclave (Intech Systems, Delhi). An
incubator of both static and rotary shaker type (Orbitek Ltd)
was used for incubation and growth of the bacteria. An
electronic colony counter (Yorco Scientific Industries Pvt Ltd,
New Delhi) was used to count the colonies present per unit
volume of culture. Optical growth was measured using a
Specord 200 spectrophotometer (Analytik Jena Ltd,
Germany). Inoculation was carried out in a vertical chamber
equipped with laminar airflow, an ultraviolet lamp and a
burner (Yorco Scientific Industries Pvt Ltd, New Delhi).
Determination of antimicrobial activity of natural dyes
in solution
All materials except the dyes were sterilised using the
standard procedure (120 °C, 20 min, 15 psi) prior to use.
As some natural dyes are prone to decompose under these
conditions, these were sanitised at 55 °C for 3 h.
The bacteria from the mother culture, as obtained from
MTCC, were inoculated into a freshly prepared liquid
nutrient broth containing 5 g/l peptone, 3 g/l beef extract
at pH 6.8 ± 0.1, and incubated for 24 h. A solid culture
medium containing 5 g/l peptone, 3 g/l beef extract and
20 g/l agar at pH 6.8 ± 0.1 was poured into a petri dish and
100 µl of microbial culture spread over it. Sterilised filter
paper discs were placed over it and 10 µl of 1% dye
solution placed on each disc. The agar plates and dye discs
were incubated for 16 h at 37 °C.
The incubated plates were examined for a clear zone of
inhibition. The average width of the zone of inhibition on
either side of the dye discs was calculated using Eqn 1:
(T − D)
W= (1)
2
where W is the width of the zone of inhibition (mm), T is
the total diameter of the test specimen and clear zone (mm)
and D is the diameter of the test specimen itself (mm).
Determination of MIC of the dyes
The effect of dye concentration on the antimicrobial activity
of three selected dyes was assessed. The effect of dye
concentration (0.1–5% w/v) on the antimicrobial activity
of three selected dyes was assessed. Initially five
concentrations (0.1, 0.2, 0.4, 0.8 and 1% w/v) were used.
Some dyes showed activity at these concentrations while
others showed little or no activity. With the latter dyes,
higher concentrations (1–5% w/v) were used. Minimum
inhibitory concentration (MIC) was determined using ATCC
6538: SN 195 920. An inhibition zone > 2 mm indicated
good antimicrobial effect.
Determination of antimicrobial activity of dyed fabrics
Dyeings were carried out at 80 °C at neutral pH and a liquor
ratio of 1:30, and at a variety of dye concentrations (6, 12
and 15% owf). To study the effect of mordants, cotton was
dyed in combination with copper sulphate and ferrous
sulphate (2.0% owf). Fabric samples of defined weight
(0.06 g) and size (16.4 cm2) were sterilised, placed on the
liquid culture medium and 100 µl of bacterial culture
added. The medium was incubated at 37 °C for 16 h.
The microbial inhibition was determined by two
methods, firstly by counting the colony forming units (CFU)
and calculating the reduction rate (R) before and after
incubation, using Eqn 2:
B− A
R% = ×100 (2)
B

Table 1 Natural dyes used in this study

Dye Common name Botanical name Commercial namea Main colorant Wash fastnessb

1 Acacia Acacia nilotica Caspian Bark 3–4

2 Indigo Indigofera tinctoria Nile Indigo 3–5
3 Lac Kerria lacca Rhine M Laccaic acid 4–5
4 Kamala Mallotus philippinensis Basant Flavonoid 4
5 Pomegranate Punica granatum Pacific Tannin 4–5
6 Gall nuts Quercus infectoria Amber M Tannin 3–4
7 Cutch Acacia catechu Thar Tannin 4–5
8 Myrobolan Terminalia chebula Kango Tannin 4–5
9 Himalayan rhubarb Rheum emodi Desert Hydroxyanthraquinone 4–5
10 Indian Madder Rubia cordifolia Indus Hydroxyanthraquinone 5
11 Golden dock Rumex maritimus Sahara Hydroxyanthraquinone 4–5

a Manufactured by Alps Industries

b Tested by ISO 105/C:1982 (Test 2)

168 © Color. Technol., 120 (2004) Web ref: 20040404

20040404_j0404.pmd 168 7/27/2004, 12:06 PM

 where A is CFU/ml for the treated sample after 16 h 8
incubation time and B is CFU/ml for the untreated sample
after 16 h incubation time.
In the second method, the optical density of the 6

Zone of inhibition, mm
incubated liquid culture medium was recorded at 660 nm.
The greater the growth, the higher the turbidity, and the
optical density figure was therefore directly proportional 4
to the number of bacteria in the medium [14].
Scoured cotton was also treated with a commercial
Dye 4
antimicrobial agent. Treatment was carried out with 2
Dye 5
Fabshield (0.5% owf) at pH 4.5–6.0 at 45–50 °C for 20 min, Dye 6
followed by drying at 130–135 °C. Inoculation was carried
out and bacterial growth was measured. 0
0 0.2 0.4 0.6 0.8
Dye conc., %

Figure 1 Effect of dye concentration on activity against E. coli

Results and Discussion
6
Screening of natural dyes for antimicrobial activity
Solutions of the eleven natural dyes used in this study were
screened for activity against the three Gram-negative Zone of inhibition, mm
bacteria at 1% concentration. The results are reported in 4
Table 2.

Table 2 Antimicrobial activity shown by natural dyes 2

Bacteriaa

Dye E. coli K. pneumoniae P. vulgaris 0

0 1 2 3 4 5 6
Dye conc., %
1 – – –
2 – – – Figure 2 Effect of dye concentration on activity against K.
3 – + – pneumoniae; for key see figure 1
4 + + +
5 + + + 6
6 + + +
7 – + +
8 – + +
Zone of inhibition, mm

9 – + – 4
10 – – –
11 – – –

a ‘+’ = active; ‘–’ = inactive

2

It can be seen from the results in this table that dyes 4,

5 and 6 displayed activity against all three bacteria, 0
whereas dyes 7 and 8 had activity against only two. Dyes 3 0 1 2 3 4 5 6
and 9 were less effective and were active only against K. Dye conc., %

pneumoniae. The remaining four dyes, including indigo
(dye 2), exhibited no antibacterial activity at all. Overall,
seven dyes were active against K. pneumoniae, five against
P. vulgaris and three against E. coli.
Determination of MIC of dyes in solution
The antimicrobial activity of the three most active dyes
(dye 4, 5 and 6) was tested at five different concentrations
against each of the three microbes. The results are shown
in Figures 1–3. The minimum concentration of dye at which
an inhibition zone diameter > 2 mm was observed was
taken as the MIC of the dye against a specific microbe. The
minimum inhibitory concentration for all dyes against K.
pneumoniae was higher than that against E. coli.
Figure 3 Effect of dye concentration on activity against P.
vulgaris; for key see figure 1
It can be seen that dyes 4 and 6 were completely effective
against E. coli, even at the lowest dye concentration (0.1%),
whereas the MIC for dye 5 was as high as 0.5% (Figure 1).
This confirms that the chemistry of the dye has a significant
influence on its antimicrobial activity.
Figure 3 illustrates the activity of the three dyes against
the bacterium P. vulgaris, which is known to convert urea
to ammonia and to lead to unpleasant odours. Of the three,
dye 4 had the lowest MIC and showed no improvement in
antimicrobial activity as its concentration on the fabric
increased.

Web ref: 20040404 © Color. Technol., 120 (2004) 169

20040404_j0404.pmd 169 7/27/2004, 12:06 PM

 Table 3 Antimicrobial activity of treated and untreated cotton dyed (dye 6), both with and without
mordants (copper and ferrous sulphate)

Dye conc. Absorbance Number of Reduction

Bacterium Fabric sample (% owf) at 660 nm CFU (× 105/ml) in CFU (%)

E. coli Untreated 0 0.515 539

Fabshield-treateda 0 0.017 20 96.2
Dye only 6 0.443 466 13.5
Dye only 12 0.400 422 21.7
Dye only 15 0.033 14 97.4
Dye + CuSO4b 15 0.348 348 35.4
Dye + FeSO4b 15 0.490 512 5.0

P. vulgaris Untreated 0 0.498 899

Fabshield-treateda 0 0.028 14 98.4
Dye only 6 0.500 910 0.0
Dye only 12 0.491 885 1.5
Dye only 15 0.041 4 99.5
Dye + CuSO4b 15 0.463 869 38.4
Dye + FeSO4b 15 0.444 810 9.8

a 0.5% owf
b 2% owf

Determination of antimicrobial activity of dyed fabrics
Having studied the activity of the dyes in solution, the next
step was to assess their effectiveness on the fibre. A dye
bound to a textile fibre may be expected to show lower
activity than in solution since some functional groups are
modified by interaction with the fibre during the dyeing
process. In addition, many dyeing procedures for natural
dyes involve the use of metallic salts, or mordants, for
colour development. Therefore, this study also investigated
the influence of two common mordants, copper sulphate
and ferrous sulphate, on cotton fabric dyed with dye 6
against two bacteria, P. vulgaris and E. coli. A commercial
antimicrobial agent (Fabshield) was also studied for
comparison.
An accurate quantitative evaluation of antimicrobial
activity was possible on the fibre both by colony counting
and spectroscopic assessment. These methods were used
to evaluate the effectiveness of dye 6 against the two
bacteria and the results are listed in Table 3. The effect of
dye concentration on antimicrobial activity is illustrated
in Figure 4.
It can be seen that the antimicrobial activity of cotton
dyed with dye 6 increased with dye concentration. At 15%
owf there was a sharp increase in activity, with the dyed
fabric showing a 97.4% reduction in CFU against E. coli.
No activity was seen against P. vulgaris below 12% owf dye
concentration, but again there was very high activity at 15%
owf with a reduction in CFU of ca. 99.5%. The activity of
dye 6 on cotton was greater than that of the commercial
antimicrobial agent, as is evident from the results in Table 3.
There was a drastic reduction in activity when mordant
was used. The reduction in activity was sharper when
ferrous sulphate was used as the mordant (as can be seen
in Table 3). This could be the consequence of chelation of
the dye by the metal salt. The only functional groups
present on these dyes are hydroxy groups and it is well
known that hydroxy groups ortho to the chromophore
become involved in coordinate bond formation, for
example in the case of chrome mordant dyes. It is therefore
likely that the formation of coordinate bonds blocks the
groupings responsible for antibacterial activity.

100 Conclusions
Against E. coli Seven out of the eleven natural dyes examined showed
Against P. vulgaris
80 activity against one or more of the three bacteria studied.
Of these, dyes 4, 5 and 6 were found to be efficient biocides
after dyeing on cotton, particularly dye 6. The latter was
Reduction, %

60
highly effective against E. coli and P. vulgaris, reducing the
number of colonies by 99%, and in fact was more effective
40 than the commercial antimicrobial Fabshield. Since these
three dyes exhibit reasonably good wash fastness it may
20 be assumed that the antimicrobial effect will be durable
in practice. These initial results have opened up an entirely
new avenue of research. Further work could focus on the
0
0 4 8 12 16 antibacterial activity of dyes on a range of natural and
Dye conc., % owf synthetic fibres, leading to the identification of the
Figure 4 Antimicrobial activity of cotton fabric dyed with functional groups responsible and the mechanism involved.
various concentrations of dye 6 The influence of mordants is a further area of interest. In

170 © Color. Technol., 120 (2004) Web ref: 20040404

20040404_j0404.pmd 170 7/27/2004, 12:06 PM

 practice, there is an exciting opportunity for the use of
natural dyes for adding antibacterial properties to clothing
and to the textiles used in the medical and the hotel fields.
Acknowledgement
The authors are grateful to Rossari Biotech India Pvt Ltd,
who kindly provided the sample of Fabshield.
References
1. M M Cowan, Clin. Microbiol. Rev., 12 (1999) 564.
2. D S Arora and J Kaur, Int. J. Antimicrob. Agents, 12 (1999)
257.
3. S B Bharate, J. Med. Arom. Plant Sci., 25 (2) (2003) 427.
Web ref: 20040404
20040404_j0404.pmd 171
4. K Haralampidis, G Bryan, X Qi, K Papadopoulou, S Bakht, R
Melton and A Osbourn, Proc. Natl. Acad. Sci. USA (2001)
13431.
5. D Gupta, S Kumari and M L Gulrajani, Color. Technol., 117
(2001) 328.
6. D Gupta, S Kumari and M L Gulrajani, Color. Technol., 117
(2001) 333.
7. D Gupta, Colourage (Aug) (1999) 41.
8. D Gupta, Colourage (Oct) (1999) 17.
9. S A M Hussein, H H Barakat, I Merfort and M A M Nawwar,
Phytochem., 45 (1997) 819.
10. H Wagner, B Kreher, H Lotter, M O Hamburger and G A
Cordell, Helv. Chim. Acta, 72 (1989) 659.
11. H Gershon, J. Can. Microbiol, 21 (1975) 1317.
12. M H Lagrota, M D Wigg and A N Aguiar, Rev. Latinoam.
Microbiol., 28 (1986) 221.
13. A R Schuerch, Eur. J. Biochem., 84 (1978) 197.
14. H Gürleyük, V van Fleet-Stalder and T G Chasteen, App.
Organomet. Chem., 11 (1997) 471.
© Color. Technol., 120 (2004) 171
7/27/2004, 12:06 PM