MIMET-03517; No of Pages 6

Journal of Microbiological Methods xxx (2010) xxx–xxx

Contents lists available at ScienceDirect

Journal of Microbiological Methods

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j m i c m e t h

Optimal methods for evaluating antimicrobial activities from plant extracts

Mukhrizah Othman a, Hwei San Loh b, Christophe Wiart c, Teng Jin Khoo a, Kuan Hon Lim a, Kang Nee Ting c,⁎
a
School of Pharmacy, University of Nottingham Malaysia Campus, Jalan Broga, 43500, Semenyih, Selangor, Malaysia
b
School of Biosciences, University of Nottingham Malaysia Campus, Jalan Broga, 43500, Semenyih, Selangor, Malaysia
c
School of Biomedical Sciences, University of Nottingham Malaysia Campus, Jalan Broga, 43500, Semenyih, Selangor, Malaysia

a r t i c l e i n f o a b s t r a c t

Article history: The search for antimicrobial agents from plants has been a growing interest in the last few decades. However,
Received 14 September 2010 results generated from many of these studies cannot be directly compared due to the absence of
Received in revised form 22 October 2010 standardization in particular antimicrobial methods employed. The need for established methods with
Accepted 2 November 2010
consistent results for the evaluation of antimicrobial activities from plant extracts has been proposed by many
Available online xxxx
researchers. Nevertheless, there are still many studies reported in the literature describing different
Keywords:
methodologies. The aim of this study was to find optimal methods to give consistent quantitative
Agar-based assay antimicrobial results for studying plant extracts. Three different agar-based assays (pour plate disc diffusion
Antimicrobial (PPDD), streak plate disc diffusion (SPDD) and well-in agar (WA)) and one broth-based (turbidometric (TB))
Broth-based assay were used in this study. Extracts from two plant species (Duabanga grandiflora and Acalypha wilkesiana)
Plant extracts were tested on two bacterial species, namely Escherichia coli and Staphylococcus aureus. Amongst the agar-
Turbidometric based assays, PPDD produced the most reproducible results. TB was able to show the inhibitory effects of the
test samples on the growth kinetic of the bacteria including plant extracts with low polarity. We propose that
both agar- (i.e PPDD) and broth-based assays should be employed when assessing the antimicrobial activity of
plant crude extracts.
© 2010 Elsevier B.V. All rights reserved.

1. Introduction
The search for novel antimicrobial agents from higher plants has
been of great interest in the last few decades. This area of research
faces many inherent problems due to various methodologies used in
the preparation of plant extracts and antimicrobial assays. The
solubility property of plant metabolites extracted with solvents of
different polarity also appears to contribute to the outcome of the
antimicrobial assays employed. The Clinical and Laboratory Standards
Institute (CLSI) methods for the evaluation of antimicrobial com-
pounds are widely accepted by regulatory bodies worldwide (Das
et al., 2010). However, these methods were designed to test pure
antimicrobial molecules such as antibiotics and modifications are
needed before they can be used for testing of plant extracts (Hammer
et al., 1999).
The lack of standardized in vitro methods for testing antimicrobial
activities of plant crude extracts has led to variations in results
between research groups. This compromises direct comparisons of
results and has led to false conclusions on the efficacy of extracts as
antimicrobial agents (Sarker et al., 2007). Methods that have been
widely employed in the evaluation of antimicrobial activities include
⁎ Corresponding author. School of Biomedical Sciences, Faculty of Science, University
of Nottingham Malaysia Campus, Jalan Broga, 43500, Semenyih, Selangor, Malaysia.
Tel.: +60 3 89248209; fax: +60 3 89248018.
E-mail address: Kang-Nee.Ting@nottingham.edu.my (K.N. Ting).
disc diffusion, well diffusion and broth dilution. Many published
studies either incorporated two different methods or just one single
method and the most often employed has been the disc diffusion
method (Kiska, 1998). However, conclusions cannot be drawn solely
from results obtained through the disc diffusion method since it is
well established that this method is not suitable for testing non-polar
samples (Rios et al., 1988; Rios and Recio, 2005).
In most published studies of antimicrobial assays, the bacterial
concentration is estimated by using the 0.5 Macfarland standard,
which corresponds to approximately 1.5 × 108 colony forming units
(CFU)/ml (Kiska, 1998). This estimation requires the researcher to
visually assess the turbidity level in broth culture to be the equal to
that of the standard. This estimation is therefore highly subjective and
may lead to inconsistent results. To date, there are only a few
published studies comparing different methods of antimicrobial
testing on plant extracts (Rios et al., 1988; King et al., 2008). However,
a standardized reproducible method for evaluating the antimicrobial
activity of plant extracts has not been established even though the
number of publications reporting antimicrobial activities of plant
extracts has increased exponentially over the last 10 years (Rios and
Recio, 2005).
In this study we compared four different antimicrobial assays used
to screen plant extracts by using a standardized inoculum with the
aim of obtaining optimal methods that provide consistent and
reproducible data. We used three different agar-based assays (pour
plate disc diffusion (PPDD), streak plate disc diffusion (SPDD) and

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doi:10.1016/j.mimet.2010.11.008

Please cite this article as: Othman, M., et al., Optimal methods for evaluating antimicrobial activities from plant extracts, J. Microbiol.
Methods (2010), doi:10.1016/j.mimet.2010.11.008
2 M. Othman et al. / Journal of Microbiological Methods xxx (2010) xxx–xxx
well-in agar (WA)) and one broth-based (turbidometric (TB)) assay.
Six plant extracts derived from two plant species were used to
evaluate the consistency of each method in assessing antimicrobial
activity.
2. Materials and methods
2.1. General
Streptomycin, dimethyl sulfoxide and phosphate buffer saline
(Sigma, USA); hexane, ethyl acetate and ethanol (Systerm, Malaysia);
Mueller Hinton agar and broth (Hi-Media, India); Escherichia coli
(ATCC 8739) and Staphylococcus aureus (ATCC 11632).
2.2. Plant extraction
Collected plants were left to dry in an enclosed room (25–28 °C)
for approximately two weeks. Dried plant samples were ground using
a heavy duty grinder (Waring, USA). Extraction was carried out as
reported previously (Spigno and Marco, 2007) with some modifica-
tions. One gram of ground plant samples was macerated with eight ml
of organic solvent (1:8 ratio) at room temperature for 24 h. Plant
samples were then subjected to sequential extraction using hexane
(H), followed by ethyl acetate (EA) and finally with ethanol (E). The
filtrates were concentrated by removing the solvents under reduced
pressure at 40 °C using a rotary evaporator (Buchi, USA). Concentrat-
ed crude extracts were dried in desiccators for approximately one to
two weeks. The dried and semi-dried crude extracts were cold stored
at −30 °C. The voucher specimen numbers for Duabanga grandiflora
leaves and Acalypha wilkesiana whole aerial plant were labelled as
UNMC 37L and UNMC 9W, respectively.
2.3. Standardized bacterial colony numbers
In order to ensure that the same number of bacteria was always
used, a set of bacterial growth curves was established in the
laboratory using the method described by Cappuccino and Sherman
(2005) for each bacterial strain prior to the evaluation of antimicrobial
activities. From these curves, we determined the optical density (OD)
at 600 nm that corresponded to the desired number of colony forming
units (CFU).
2.4. Preparation of test samples and paper discs
One hundred milligrams of plant extracts was dissolved in 1 ml
dimethyl sulfoxide (DMSO) while 0.5 mg of streptomycin was dissolved
in 1 ml sterile deionised water. Ten microliters of 100 mg/ml plant
extract (equivalent to 1 mg dose), 10 μl of 0.5 mg/ml streptomycin
(each equivalent to 5 μg dose) and 10 μl DMSO were applied to 6 mm
sterile filter paper discs. The discs impregnated with extracts and DMSO
were prepared a day before each experiment while antibiotic discs were
prepared on the same day of experiment.
2.5. Antimicrobial testing methods
2.5.1. Pour plate disc diffusion (PPDD)
Pour plate disc diffusion assay was carried out according to the
method described by Kelmanson et al. (2000) with some modifica-
tions. Several colonies which were grown on nutrient agar plates were
selected and cultured in Mueller-Hinton Broth (MHB) until they
reached their specific OD at 600 nm to give a starting inoculum of
1 × 108 bacteria/ml. Mueller-Hinton Agar (MHA) plates were each
divided into quadrants and labelled accordingly. One hundred
microliters of inoculum, equivalent to 107 cfu , was mixed with 6 ml
of molten soft MHA (to ensure even distribution of bacteria) and
poured immediately onto the base layer of MHA. The plates were left
Please cite this article as: Othman, M., et al., Optimal methods for evaluating antimicrobial activities from plant extracts, J. Microbiol.
Methods (2010), doi:10.1016/j.mimet.2010.11.008
to solidify for 10 min. Impregnated discs were placed onto the top
layer of the MHA plates.
2.5.2. Streak plate disc diffusion (SPDD)
Streak plate disc diffusion assay was carried out according to the
method described by Samy et al. (2006) with some modifications.
Several colonies that were grown on nutrient agar plates were picked
and cultured in MHB until they reached their specific OD at 600 nm to
give a starting inoculum of 1 × 108 bacteria/ml. Mueller-Hinton Agar
plates were each divided into quadrants and labelled accordingly. One
hundred microliters of inoculum, equivalent to 107 cfu was then
pipetted onto the MHA. A sterile cotton swab was used to inoculate
the MHA plate by streaking over the surface with rotation to ensure
even distribution of the inoculum. Impregnated discs were placed
onto the top layer of the MHA plates.
2.5.3. Well-in agar (WA)
Well-in agar assay was carried out according to the method
described by Mathabe et al. (2006) with some modifications. Bacteria
colonies from plates were grown in MHB until they reached
their specific OD at 600 nm to give a starting inoculum of 1 × 108
bacteria/ml. Mueller-Hinton Agar plates were each divided into
quadrants and labelled accordingly. One hundred microliters of
inoculum, equivalent to 107 cfu, was mixed with 6 ml of molten soft
MHA (to ensure even distribution of bacteria) and poured immedi-
ately onto the base layer of MHA. The plates were left to solidify for
10 min. A sterilized 5 mm borer was used to make holes in the centre
of the divided areas. The bottom of the well was then sealed with
molten soft agar. Ten microliters of each of the test samples (plant
extracts, streptomycin or DMSO) was then pipetted into the holes.
2.5.4. Bacterial growth condition and result acquisition
Plates with bacteria and test samples were incubated at 37 °C for
16 to 18 h after which the inhibition diameter (ID) was measured
using a calliper. Each experiment was carried out on at least three
separate occasions.
2.5.5. Broth-based turbidometric assay (TB)
Turbidometric assay was carried out based on the method reported
by Noga et al. (1994) with some modifications. Bacteria were grown in
MHB until the number of bacteria reaches 2 × 108 bacteria/ml. Bacteria
were then washed once with 1X PBS and fresh MHB added. The number
of bacteria was then adjusted to 2 × 105 bacteria/ml with fresh MHB.
Forty microliters of inoculum, equivalent to 8 × 103 cfu, or MHB was
pipetted into each corresponding well of a 96-well plate. Then, 160 μl of
plant extract dissolved in DMSO, streptomycin, ampicillin and control
DMSO was pipetted into corresponding wells. The final volume in each
well was 200 μl. The concentrations used for plant extracts were in the
range of 0.01 to 3 mg/ml while the concentrations used for strepto-
mycin were in the range of 0.01 to 10 μg/ml. A blank for coloured
extract without inoculum was included to correct for the colour. The
optical density at 600 nm of each well was recorded using a microplate
reader every 30 min for 12 h. Results were expressed as the kinetic
growth and Survival Index (SI) was calculated as described by Bexfield
et al. (2008):
OD600 of test sample at corresponding time point
SI ¼ × 100:
OD600 at mid−log of control bacterial growth
Survival index is used to express the growth of bacteria within the
tested samples as a percentage of growth of the controls at the mid-
log phase. In order to rank the potency of plant extracts used, an
effective concentration that inhibits 50% of bacterial growth (EC50)
was calculated from SI values. The EC50 calculation was based on that
presented by Alexander et al. (1999). Each experiment was carried in
triplicates and on at least three separate occasions.
 M. Othman et al. / Journal of Microbiological Methods xxx (2010) xxx–xxx 3

3. Results 3.2. Broth-based TB assay

3.1. Agar-based antimicrobial assays Figs. 1–4 show the effects of streptomycin and the plant extracts of
D. grandiflora on E. coli growth using the broth-based assay. As shown
Inhibition of E. coli growth was observed with streptomycin and
most of the plant extracts tested (Table 1). The vehicle control, 1.3
Control

Bacterial Growth (increase in OD600nm)

DMSO, did not affect E. coli growth (data not shown in Table 1). Streptomycin 10µg/ml
Streptomycin 3µg/ml
Streptomycin at 5 μg inhibited the growth of E. coli in all three agar- 1.1 Streptomycin 1µg/ml
based PPDD, SPDD and WA assays with mean inhibition diameter Streptomycin 0.3µg/ml
Streptomycin 0.1µg/ml
(ID) ± standard deviation (SD) of 21.0 ± 0.0; 14.2 ± 0.7; and 11.8 ± 0.9 Streptomycin 0.03µg/ml
0.9 mm respectively. These inhibition diameters showed that the Streptomycin 0.01µg/ml
PPDD assay produced the most reproducible result with consistent 0.7
ID and SD = 0. The hexane extracts of both D. grandiflora leaves
(UNMC 37LH) and A. wilkesiana whole aerial plant (UNMC 9WH) did 0.5
not affect the growth of E. coli (data not listed in Table 1). However,
ethyl acetate (EA) and ethanol (E) extracts of both plants (UNMC 0.3
37LEA, UNMC 9WEA, UNMC 37LE and UNMC 9WE) showed
substantial inhibition. The PPDD assay gave the most consistent ID 0.1
with the smallest variation (SD values of 0 and 0.1 mm) compared to
the SPDD and WA assays where the SD values ranged from 0.5 to -0.1 0 1 2 3 4 5 6 7 8 9 10 11 12 13
Incubation time (h)
1.1 mm.
The growth of S. aureus was inhibited by streptomycin (see Fig. 1. Growth curve of E. coli in the presence of a range of streptomycin concentrations.
Table 2). The vehicle control, DMSO and hexane extracts of both Each experiment was carried in triplicates and on at least three separate occasions.
plants did not affect the growth of S. aureus (data not shown in Error bars showing standard deviation.
Table 2). When compared to the SPDD and WA assays, the PPDD assay
produced the most consistent ID (SD = 0). For most experiments
involving the plant extracts, the SPDD assay showed the largest ID 1.4
Vehicle control (1% DMSO)
Bacterial Growth (Increase in O.D600nm)

although the data produced from the PPDD assay exhibited the least UNMC 37L H 3mg/ml
1.2 UNMC 37L H 1mg/ml
variation. The SD values for SPDD and PPDD range from 0.5 to 1.8 mm UNMC 37L H 0.3mg/ml
and 0 to 0.2 mm, respectively (Table 2). On the other hand, the WA UNMC 37L H 0.1mg/ml
1 UNMC 37L H 0.03mg/ml
assay produced the smallest ID for A. wilkesiana ethyl acetate and UNMC 37L H 0.01mg/ml
ethanol extracts, while the extracts from D. grandiflora did not appear
0.8
to affect the growth of S. aureus.
0.6

0.4

Table 1 0.2
Antimicrobial activity of streptomycin and plant extracts (UNMC 37L and UNMC 9W)
against E. coli in three different antimicrobial assays. (n = 3).
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13
Methods Sample/Inhibition diameter (mm)
Incubation time (h)
Streptomycin UNMC 37L UNMC 9W

(5 μg) EA (1 mg) E (1 mg) EA (1 mg) E (1 mg) Fig. 2. Growth curve of E. coli in the presence of a range of D. grandiflora hexane extract
(UNMC 37LH) concentrations. Each experiment was carried in triplicates and on at least
PPDD 21.0 ± 0.0 16.0 ± 0.0 18.9 ± 0.1 17.1 ± 0.1 17.0 ± 0.0
three separate occasions. Error bars showing standard deviation.
SPDD 14.2 ± 0.7 13.8 ± 1.1 15.0 ± 1.0 19.5 ± 0.6 18.1 ± 0.6
WA 11.8 ± 0.9 16.1 ± 1.0 16.3 ± 0.6 16.8 ± 0.5 11.8 ± 0.9
Bacterial Growth (Increase in O.D600nm)

DMSO (10 μl) and hexane extracts from both plants did not affect the growth of E. coli; 1.4 Vehicle control (1% DMSO)
Values shown are mean ± standard deviation; PPDD: pour plate disc diffusion; SPDD: UNMC 37L EA 3mg/ml
streak plate disc diffusion; WA: well-in agar; EA: ethyl acetate; E: ethanol; disc size for 1.2 UNMC 37L EA 1mg/ml
PPDD and SPDD: 6 mm; well size for WA: 5 mm. UNMC 37L EA 0.3mg/ml
UNMC 37L EA 0.1mg/ml
1 UNMC 37L EA 0.03mg/ml
UNMC 37 EA 0.01mg/ml

0.8
Table 2
Antimicrobial activity of streptomycin and plant extracts (UNMC 37L and UNMC 9W)
0.6
against S. aureus in three different agar-based assays. (n = 3).

Methods Sample/Inhibition diameter (mm) 0.4

Streptomycin UNMC 37L UNMC 9W
0.2
(5 μg) EA (1 mg) E (1 mg) EA (1 mg) E (1 mg)

PPDD 17.0 ± 0.0 16.0 ± 0.0 19.8 ± 0.2 16.0 ± 0.0 17.0 ± 0.0 0
SPDD 13.2 ± 0.8 18.8 ± 1.8 21.3 ± 1.0 16.9 ± 1.4 20.9 ± 0.5 0 1 2 3 4 5 6 7 8 9 10 11 12 13
WA 13.7 ± 1.2 NI NI 13.4 ± 0.6 12.7 ± 1.2
Incubation time (h)
DMSO (10 μl) did not affect the growth of S. aureus; Values shown are mean ± standard
deviation; NI: no inhibition; PPDD: pour plate disc diffusion; SPDD: streak plate disc Fig. 3. Growth curve of E. coli in the presence of a range of D. grandiflora ethyl acetate
diffusion; WA: well-in agar; EA: ethyl acetate; E: ethanol; disc size for PPDD and SPDD: extract (UNMC 37LEA) concentrations. Each experiment was carried in triplicates in at
6 mm; well size for WA: 5 mm. least three separate occasions. Error bars showing standard deviation.

Please cite this article as: Othman, M., et al., Optimal methods for evaluating antimicrobial activities from plant extracts, J. Microbiol.
Methods (2010), doi:10.1016/j.mimet.2010.11.008
4 Bacterial Growth (Increase in O.D600nm) M. Othman et al. / Journal of Microbiological Methods xxx (2010) xxx–xxx

1.4 Vehicle control (1% DMSO) Table 5

UNMC 37L E 3mg/ml Survival index of E. coli and S. aureus in the presence of ethyl acetate extract of
1.2 UNMC 37L E 1mg/ml D. grandiflora leaves (UNMC 37LEA) (n = 3).
UNMC 37L E 0.3mg/ml
UNMC 37L E 0.1mg/ml Concentration (mg/ml) Survival index (%)
1 UNMC 37L E 0.03mg/ml
UNMC 37L E 0.01mg/ml E. coli S. aureus
0.8 3 1.34 ± 1.90 −0.24 ± 1.35
1 18.92 ± 3.36 −1.16 ± 0.70
0.6 0.3 26.73 ± 2.70 −1.18 ± 2.95
0.1 56.72 ± 2.39 19.63 ± 1.00
0.03 73.36 ± 2.86 49.42 ± 0.90
0.4
0.01 98.39 ± 0.92 82.28 ± 3.08

0.2

0 Table 6
0 1 2 3 4 5 6 7 8 9 10 11 12 13 Survival index of E. coli and S. aureus in the presence of ethanol extract of D. grandiflora
Incubation time (h) leaves (UNMC 37LE) (n = 3).

Concentration (mg/ml) Survival index (%)

Fig. 4. Growth curve of E. coli in the presence of a range of D. grandiflora ethanol extract
(UNMC 37LE) concentrations. Each experiment was carried in triplicates on at least E. coli S. aureus
three separate occasions. Error bars showing standard deviation.
3 4.76 ± 1.40 −9.20 ± 0.88
1 7.57 ± 0.46 −3.35 ± 2.49
0.3 22.99 ± 3.65 −1.86 ± 1.04
in Fig. 1, streptomycin totally inhibited the growth of E. coli at a 0.1 43.78 ± 4.51 14.15 ± 0.94
concentration of 10 μg/ml. The survival index (SI) showed the 0.03 55.58 ± 6.93 15.29 ± 1.80
concentration response of E. coli against streptomycin (Table 3). The 0.01 78.37 ± 1.41 32.32 ± 2.91
hexane extract of D. grandiflora (UNMC 37LH) inhibited E. coli growth
at a concentrations of 0.3–3 mg/ml (Fig. 2). The SI (Table 4) further
confirmed the inhibitory effect of UNMC 37LH. Interestingly, no
Table 7
inhibitory effect of the hexane extract was detected in any of the agar- The EC50 values calculated from survival index for E. coli and S. aureus in the presence of
based assays. Concentration-dependent inhibitory antimicrobial streptomycin and D. grandiflora leaves extracts (UNMC 37L) (n = 3).
effects were also observed with UNMC 37LEA and UNMC 37LE
Test sample Bacterial strain/EC50 of survival index (mg/ml)
(Figs. 3 and 4, respectively). SI for UNMC 37LEA (Table 5) and UNMC
E. coli S. aureus
37LE (Table 6) showed that both extracts inhibited growth of E. coli at
−3
a much lower concentration range (0.01–0.3 mg/ml) compared to Streptomycin 1.68 ± 0.14 × 10 1.44 ± 0.09 × 10−3
a
UNMC 37LH. The EC50 values calculated from SI for streptomycin, UNMC 37LH CD 0.64 ± 0.17
UNMC 37LEA 0.09 ± 0.08 0.02 ± 0.01
UNMC 37LEA and UNMC 37LE were 1.68 ± 0.14 μg/ml, 0.09 ± b
UNMC 37LE 0.10 ± 0.02 CD
0.08 mg/ml, and 0.10 ± 0.02 mg/ml respectively (Table 7). The EC50
Values shown are mean ± standard deviation; aCD: cannot be determined, estimated
value for UNMC 37LH could not be determined. However, it was
value N3 mg/ml; bCD: cannot be determined, estimated value b 0.01 mg/ml H: hexane;
estimated to be N3 mg/ml, which was the highest concentration EA: ethyl acetate; E: ethanol.
tested. Similar concentration-dependent inhibitions were also
observed with extracts from A. wilkesiana (results not shown).
The growth of S. aureus was inhibited by streptomycin in a
concentration-dependent manner (where total inhibition occurred at
Table 3 10 μg/ml [SI b 1%]) (Table 3). The growth of S. aureus was inhibited by
Survival index of E. coli and S. aureus in the presence of streptomycin (n = 3).
UNMC 37LH at a concentration range of 0.1–3 mg/ml (Table 4). These
Concentration (μg/ml) Survival index (%) inhibition effects were not observed in any of the agar-based assays.
E. coli S. aureus
However, UNMC 37LEA and UNMC 37LE were able to inhibit the
growth of S. aureus at a much lower concentration range (0.01–
10 −0.07 ± 0.74 0.85 ± 1.23
0.3 mg/ml) based on SI values (Tables 5 and 6) when compared to
3 25.57 ± 2.28 33.82 ± 5.14
1 71.63 ± 9.31 85.34 ± 6.78 UNMC 37LH. The EC50 values calculated from SI for streptomycin,
0.3 87.59 ± 6.60 94.23 ± 4.61 UNMC 37LH and UNMC 37LEA were 1.44 ± 0.09 μg/ml, 0.64 ±
0.1 92.91 ± 6.04 92.69 ± 3.01 0.17 mg/ml, and 0.02 ± 0.01 mg/ml respectively. The EC50 value for
0.03 91.44 ± 10.16 94.95 ± 1.04
UNMC 37LE could not be determined. However, it was estimated to be
0.01 94.03 ± 11.15 101.59 ± 5.36
b0.01 mg/ml, which was the lowest concentration tested. Similar
concentration-dependent inhibitions against the growth of S. aureus
were also observed with extracts from A. wilkesiana (results not
Table 4 shown).
Survival index of E. coli and S. aureus in the presence of hexane extract of D. grandiflora
leaves (UNMC 37LH) (n = 3).
4. Discussion
Concentration (mg/ml) Survival index (%)

E. coli S. aureus There has been much interest in recent years on compounds
derived from plants and herbs for their medicinal properties or
3 78.71 ± 2.68 11.86 ± 5.39
1 86.95 ± 0.85 33.82 ± 1.00
biological activities (Al-Dabbas et al., 2006). Throughout human
0.3 72.02 ± 1.55 63.41 ± 0.20 history, infectious diseases are known to have been treated with
0.1 99.59 ± 0.95 95.43 ± 0.78 herbal remedies (Ozturk and Ercisli, 2006). However, standardized
0.03 100.26 ± 0.86 100.64 ± 1.28 and reliable antimicrobial methods are needed to study the potential
0.01 100.44 ± 0.09 97.40 ± 2.50
antimicrobial properties of plant-derived phytochemicals.

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Methods (2010), doi:10.1016/j.mimet.2010.11.008
 M. Othman et al. / Journal of Microbiological Methods xxx (2010) xxx–xxx
It is important to establish the growth curve of bacterial strains in
order to accurately target the logarithmic phase (where cells are
reproducing at a uniform and rapid rate by binary fission) and
determine the starting number of bacteria (inoculum) to be used in
assays (Greenwood and Slack, 2000). Each bacterial strain has a
different intrinsic growth rate and therefore the time for reaching the
logarithmic phase will differ between bacterial strains. In developing
the antimicrobial testing methods (agar-based assays), the starting
number of bacteria required was 1 × 108 bacteria/ml (Kelmanson
et al., 2000; Mathabe et al., 2006, and Samy et al., 2006) while for TB
assay it was 2 × 108 bacteria/ml (Noga et al., 1994). In order to obtain
the appropriate number of bacteria for each type of experiment, the
growth curve for each bacterial strain was established earlier in our
laboratory (data not shown).
To date, there are only a few published studies comparing different
methods of antimicrobial testing on plant extracts (Rios et al., 1988;
King et al., 2008) and it is not known if any of the aforementioned
methods is considered as the most optimal assay to be employed for
assessing potential antimicrobial activity of plant extracts. All three
agar-based methods provide a quantitative assessment of an
organism's susceptibility to potential antimicrobial samples. During
incubation, it was assumed that the test sample diffuses out from the
disc into the agar medium, creating a circular concentration gradient
that decreases logarithmically with increasing distance from the disc.
As the tested sample diffuses out from the disc, the bacteria multiply
creating a lawn of visible growth on the agar except in area (zones)
around the disc where diffused molecules possessed properties to
inhibit bacterial growth (Kiska, 1998).
Of all four assays tested in our study, the PPDD was the most
straightforward to execute and the results were consistent with little
variation (low SD) between experiments. Bacteria in the PPDD assay
were distributed evenly when compared to the SPDD assay. The
distribution and bacterial density on the agar plate are important as
susceptibility testing can be affected by the number of inoculum; a
low inoculum number can result in false-susceptibility, while a high
inoculum number can result in false-resistance (Kiska, 1998). The
main concern with the SPDD method is the distribution of bacteria
since it is intuitive that the area first streaked will have higher
bacterial density. Therefore, in order to alleviate this problem, the
streaking should be performed several times on all areas on the agar
plate though this was found to be very time consuming. The WA
assay was found to produce undesirable results. Using this assay,
S. aureus growth was not affected by UNMC 37LEA and UNMC 37LE
whilst both plant extracts showed overt inhibitory effects when the
PPDD and SPDD assays were employed. It has previously been
reported that the WA method gave a low level of reproducibility
(Bagamboula et al., 2004). Moreover, the SPDD and WA assays were
found to be more laborious and time consuming when compared to
the PPDD assay.
The principle behind the TB assay is that the increasing number of
bacteria grown in broth will increase its turbidity (Noga et al., 1994).
Changes in bacterial number or broth turbidity can be measured using
a microplate reader at a desired wavelength (Noga et al., 1994) since
absorbance is positively correlated with cell numbers (Turcotte et al.,
2006). This method is considered a descriptive method because the
curves generated from this assay can illuminate aspects of test
sample–microbe interactions which are not evident in conventional
end-point agar-based assays (Patton et al., 2006). Turbidometric assay
provides insight into the effect of test samples on bacterial growth
kinetics, which is scarcely obtained with end-point agar-based assays
(Rufián-Henares and Morales, 2008). Moreover, agar-based assays
provide poor accuracy that limits the interpretation of results
(Turcotte et al., 2006). In addition, results obtained through agar-
based assays depend largely on human ability and judgment and the
desired precision cannot be obtained when the inhibition zone is
unclear or not perfectly circular (Parente and Hill, 1992). In contrast,
Please cite this article as: Othman, M., et al., Optimal methods for evaluating antimicrobial activities from plant extracts, J. Microbiol.
Methods (2010), doi:10.1016/j.mimet.2010.11.008
5
turbidometric assay can be carried out relatively easily with high
reproducibility (Greenwood, 1977).
Minimum inhibitory concentration (MIC) is a quantitative end-
point measurement most commonly used for evaluating the antimi-
crobial effect of antibiotics. However, MIC provides little information
on the kinetic changes in bacterial growth when an antimicrobial
agent is present (Li et al., 1993). MIC values provide information on
the concentration of antimicrobial agents that halts bacterial growth
under visible threshold after 16 to 18 h. On the other hand,
monitoring bacterial growth in the presence of antimicrobial agents
over a specified time period shows changes in bacterial growth rate as
early as 2 h since the start of incubation. This can be explained by the
intrinsic activity of antimicrobial agents and the interaction between
antibacterial effect and bacterial response (Greenwood, 1976a). Even
under controlled experimental conditions, factors controlling the
ultimate endpoint, as in the MIC assay, vary markedly between different
antibiotics, therefore causing variations in results (Greenwood,
1976b). Another advantage of using TB assay is that the SI can be
used to determine EC50 which can subsequently be used to compare the
potency of different samples tested.
Streptomycin and the crude extracts tested inhibited the growth of
E. coli and S. aureus in a concentration-dependent manner. Dimethyl
sulfoxide, used to dissolve the plant extracts did not exert any
inhibitory effect on the bacterial growth. When tested against both
bacterial strains, UNMC 37LH inhibited the bacterial growth in the
broth-based TB assay but such activities were not observed in any of
the agar-based assays. This could be due to the presence of some
hydrophobic molecules in the hexane extract which were in direct
contact with bacterial cells in broth (Rufián-Henares and Morales,
2008). Therefore, issues relating to the inability of hydrophobic
compounds to dissolve and diffuse into agar were eliminated. This
diffusion problem has also been reported by Bexfield et al. (2004),
where their tested sample was inactive using WA but an overt
antibacterial activity was observed with TB. In the TB assay, the
potency of plant extracts can be ranked by calculating the EC50 values
from the SI. In decreasing order of potency (based on EC50 values), the
antimicrobial activity against E. coli and S. aureus exhibited by the
various leaf extracts of D. grandiflora was: UNMC 37LE N UNMC
37LEA N UNMC 37LH. This activity mirrors the results obtained from
the PPDD and SPDD assays.
From our observations, it was found that both the non-polar and
polar crude extracts inhibited growth of both bacteria in the broth-
based assay. The TB assay used in this study measures the total count
of bacterial cells which include the living as well as dead cells.
However, the agar-based assay measures only the viable count of
bacteria (living cells alone).
In this study, it was found that the PPDD and TB assays were the
optimal methods for assessing the potential antibacterial activities of
plant extracts. In conclusion, we propose that both agar- and broth-
based assays are required for assessing the antimicrobial activity of
plant crude extracts if reliable results are to be obtained.
Acknowledgements
This study is funded by the Research Innovation Services,
University of Nottingham. The authors would like to thank Dr Daniel
R. Smith for proof reading the manuscript.
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