Methods 42 (2007) 325–329

www.elsevier.com/locate/ymeth

Colorimetric broth microdilution method for the antifungal

screening of plant extracts against yeasts
a,*
Manjuan Liu , Veronique Seidel a, David R. Katerere b, Alexander I. Gray a

a
Natural Products Research Group, Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde,
27 Taylor Street, Glasgow, G4 0NR, UK
b
PROMEC Unit, MRC, P.O. Box 19070, Tygerberg 7500, Cape Town, South Africa

Accepted 18 February 2007

Abstract

Screening plant extracts for antifungal activity is increasing due to demand for new antifungal agents, but the testing methods present
many challenges. Standard broth microdilution methods for antifungal susceptibility testing of available antifungal agents are available
now, but these methods are optimised for single compounds instead of crude plant extracts. In this study we evaluated the standard
NCCLS method as well as a modification which uses spectrophotometric determination of the end-points with a plate reader. We also
evaluated another standard method, the EUCAST method, which is a similar microdilution assay to the NCCLS method, but uses a
larger inoculum size and a higher glucose concentration in the medium as well as spectrophotometric end-point determination. The
results showed that all three methods had some drawbacks for testing plant extracts and thus we modified the NCCLS broth microdi-
lution method by including a colorimetric indicator—resazurin for end-point determination. This modified method showed good repro-
ducibility and clear-cut end-point, plus the end-point determination needed no instruments. It enabled us to evaluate the activity of a
selection of extracts from six Combretaceous plants against three Candida spp. and thus provided pharmacological evidence for some
traditional uses of these plants while assisting the identification of the active ingredients.
 2007 Elsevier Inc. All rights reserved.

Keywords: Combretaceae; Candida; Broth microdilution; NCCLS; EUCAST; Resazurin; Natural products; Antifungal tests

1. Introduction most widely used in traditional herbal medicine in Africa

Opportunistic fungal infections, including life-threaten-
ing invasive mycoses, have increased in incidence over the
last two decades due to the prevalence of immune-sup-
pressing disease conditions e.g., HIV-AIDS, organ trans-
plantation and cancer. The search for new antifungal
agents from natural sources has intensified in response to
the limitations of currently available therapy and the emer-
gence of drug-resistant strains [1]. The family Combreta-
ceae consists of 20 genera of which Combretum and
Terminalia (about 250 species each) are the largest and
*
Corresponding author. Tel.: +44 141 548 2204.
E-mail address: manjuan.liu@strath.ac.uk (M. Liu).
and Asia [2]. Both Combretum and Terminalia species are
used to treat abdominal disorders, diarrhoea, venereal dis-
ease, wounds and burns [3]. The species of the Combreta-
ceae family chosen for the present study have been shown
to possess anti-Candida activity [4–8] and the main aim
of this study was to evaluate the suitability of a standard
antifungal susceptibility test for plant extracts.
Testing for antifungal activity of natural products, espe-
cially plant extracts, presents many challenges. The diver-
sity of testing methods and lack of clearly defined testing
conditions such as inoculum size and medium type can lead
to low reproducibility and difficulties in comparison with
the anti-Candida activities of the same plant species. Addi-
tionally, the trailing effect (residual fungal growth) of Can-
dida species makes the end-points less well defined in broth

1046-2023/$ - see front matter  2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.ymeth.2007.02.013
326 M. Liu et al. / Methods 42 (2007) 325–329
tests than that of tests for bacteria and can affect the reli-
ability of such tests.
At the same time, the standardisation of the in vitro
antifungal susceptibility testing has advanced greatly in
recent years. The National Committee for Clinical Labo-
ratory Standards (NCCLS) have set the benchmark
methodology by providing laboratory tested, reproduc-
ible, consensus peer-reviewed standards. NCCLS M27-
A2 standard for yeasts [9] provides a broth microdilution
test which could be a good screening method for plant
extracts with its high through-put potential, considerable
savings in media usage, and requirement of a small
quantity of sample. Some recent studies have substituted
the traditional agar diffusion method with the NCCLS
method for screening plant extracts [10–12]. However
the optimisation of test conditions is needed in order
to adapt this method to test plant extracts because it is
optimised for accepted antifungal agents, i. e. pure single
compounds.
In this study, we evaluated three end-point determina-
tion methods for the standard NCCLS method, which
are the visual determination using turbidity levels as stated
in the NCCLS M27-A2 protocol, the spectrophotometric
determination using a plate reader [13,14], and the colori-
metric determination using an oxidation-reduction indica-
tor resazurin. We also evaluated another standard
method, the EUCAST method, mainly used in European
countries [15], which is a similar microdilution assay to
the NCCLS method, but uses a larger inoculum size and
higher glucose concentration in the medium as well as a
spectrophotometric end-point determination.
2. Materials and methods
2.1. Plant collection
All plants were collected in Guruve, Zambezi Valley in
Northern Zimbabwe in May 1999. Voucher specimens
(Table 1) have been deposited at the herbarium of the Har-
are Botanical Garden.
2.2. Preparation of plant extracts
Dried and powdered plant materials (ca.20 g) were
extracted in a Soxhlet apparatus successively with n-hex-
ane, ethyl acetate (EtOAc) and methanol (MeOH) for ca.
Table 1
Combretum and Teminalia spp. used in this study
Plants
Combretum zeyheri Sond.
Combretum fragrans F. Hoffm.
Combretum elaeagnoides Klotzsch
Combretum kirkii M.A. Lawson
Terminalia brachystemma Welw. ex Hiern
Terminalia mollis M. Laws.
eight hours. Extracts were evaporated to dryness under
reduced pressured at 40 C. The yields are reported in
Table 2.
2.3. Microorganisms, broth medium, inoculum and sample
preparation
2.3.1. Microorganisms
The Candida species used in this study, Candida parapsi-
losis (ATCC 22019), Candida krusei (ATCC 6258) and
Candida albicans (ATCC 90028), were obtained from
LGC Promochem. Strains were cultured from frozen
stocks and maintained at 37 C on Sabouraud dextrose
agar (Sigma-Adrich, UK).
2.3.2. Preparation of broth medium
Broth medium (0.5 L) used for the NCCLS test was pre-
pared as follows: RPMI-1640 medium supplemented with
glutamine and phenol red, without bicarbonate (10.4 g),
3-(N-morpholino) propanesulfonic acid (MOPS) (34.53 g)
were dissolved in 400 ml distilled water, adjusting the pH
to 7.0 at 25 C with 1 mol/L sodium hydroxide. Additional
water was added to bring the medium to a final volume of
0.5 L, which was filter sterilized and stored at 4 C until
required. The medium was further supplemented with glu-
cose (18 g) to achieve a final concentration of 2% glucose
(w/v) for the EUCAST assay. All chemicals were obtained
from Sigma-Adrich, UK.
2.3.3. Preparation of inocula
For the assays, organisms were subcultured once onto
Sabouraud dextrose agar and incubated for 24 h at
37 C. Inocula were prepared by transferring several colo-
nies to sterile distilled water (5 ml). The suspensions were
mixed for 15 s to ensure homogeneity and subsequently
diluted to match the turbidity of a 0.5 McFarland standard
(i.e. OD = 0.12–0.15 at k = 530 nm, ThermoSpectronic
UV1 X, corresponding to 1–5 · 106 CFU/ml). Further
dilutions in sterile distilled water were made to obtain the
required working suspensions (1–5 · 105 CFU/ml and 1–
5 · 103 CFU/ml for the EUCAST and NCCLS assays,
respectively). Colorimetric readings of the results for the
NCCLS assay employed resazurin (Sigma-Adrich, UK)
as an indicator of cell growth. For this purpose, the work-
ing suspension (20 ml, 1–5 · 103 CFU/ml) was supple-
Voucher number Part used Code
SRGH-DRK-5/99-cze Leaves CZ
SRGH-DRK-5/99-cfr Heart wood CF
SRGH-DRK-5/99-cel Stem CE
SRGH-DRK-5/99-cki Leaves CK
SRGH-DRK-5/99-tbr Leaves TB
SRGH-DRK-5/99-tmo Leaves TM
 M. Liu et al. / Methods 42 (2007) 325–329 327

Table 2
Inhibitory activity of plant extracts against Candida spp. using the modified NCCLS method with colorimetric end-point determination
Code Extract Yield % MIC (mg/L)
C. krusei C.albicans C. parapsilosis
CZ MeOH 12.73 8 32 63
EtOAc 0.95 250 250 500
CF MeOH 8.52 16 32 63
EtOAc 0.28 >500 >500 >500
CE MeOH 2.73 >500 >500 >500
EtOAc 0.31 >500 >500 >500
CK MeOH 11.18 32 63 250
EtOAc 1.87 >500 >500 >500
TB MeOH NA 8 8 16
EtOAc NA 63 125 125
TM MeOH 9.25 >500 >500 >500
EtOAc 1.37 >500 >500 >500
Itraconazole 0.06 0.12 0.06

mented with 0.1ml sterilized solution of resazurin (20 mg/
ml in water).
2.3.4. Preparation of samples
Stock solutions of the plant extracts and the positive
control drug Itraconazole (Sigma-Adrich, UK) were pre-
pared in dimethyl sulphoxide (DMSO) at the concentra-
tions of 100 mg/ml and 1.6 mg/ml, respectively and
further diluted (1:50) in broth.
2.4. Preparation of plates
Microdilution susceptibility testing was performed in
flat-bottom 96-well clear plates containing broth medium
(0.1 ml) in each well. Sample solutions (0.1 ml) were subse-
quently serially diluted two-fold in the plates with the
broth, starting with the final concentration of 500 mg/L
for plant extracts and 8 mg/L for Itraconazole. The work-
ing inoculum suspension (0.1 ml) was added to give a final
inoculum concentration of 0.5–2.5 · 105 and 0.5–2.5 · 103
CFU/ml for the EUCAST and NCCLS assays, respec-
tively. Itraconazole was used as the standard antifungal
drug. Sterility and growth controls in the presence of
organic solvents employed in sample preparation were also
included. No inhibitory effects were observed in the pres-
ence of DMSO at the highest concentration used (0.5%
v/v). The plates were incubated at 37 C for 24 and 48 h
for the EUCAST and NCCLS assays, respectively.
2.5. Results determination
Visual readings: The amount of growth in the wells con-
taining the agent was compared visually with the growth in
the growth control wells. The concentration with a promi-
nent decrease in turbidity was determined as the MIC.
Spectrophotometric readings: a plate reader (Spectra
MAX190) was used to measure the amount of growth at
k = 530 nm following agitation by pipetting to ensure
homogeneity. The backgrounds for each sample and the
growth control were also measured. The percentage of
growth was calculated by the following equation:
OD530 Sample  OD530 Corresponding Background
% Growth ¼
OD530 Growth Control  OD530 Corresponding Background
 100
MIC50, an inhibition of growth equal to or greater than
50% of that of the growth control was recorded as end-
point.
Colorimetric readings: Colorimetric MIC end-points
were interpreted as the lowest sample concentration that
remained blue (indicating no growth) or the first dilution
that changed from blue to slightly purple (equivalent to
prominent growth inhibition).
All assays were repeated at least three times. Because
this assay is developed from the susceptibility test, which
is a qualitative test instead of quantitative test, no statisti-
cal analysis is involved. The final activity of the plant
extracts was presented by the highest MIC of the three tests
if they are different in ± one-fold dilution otherwise the
assay requires to be repeated further to ensure
reproducibility.
3. Results and discussions
3.1. Evaluation of different methods
The visual reading method of the NCCLS M27-A2 test
was both subjective and less reproducible due to the trail-
ing effect. When testing plant extracts, additional problems
were encountered. For example, water-insoluble constitu-
ents within the extracts formed a precipitate which could
be confused with the cells; the strong colour of most
extracts obscured the judgment of the turbidity level. Thus,
the data that resulted from this method were discounted.
The use of a spectrophotometer helps to obtain an
objective and rapid MIC reading. The reproducibility is
also good although some negative readings (percentage
growth) can be observed due to the interference of the
328 M. Liu et al. / Methods 42 (2007) 325–329

background (the colour of plant extracts) and also due to for the operator. A background plate was also necessary
the turbidity caused by the insoluble compounds. In order to obtain the absorbance contribution from the colour of
to facilitate the analysis and comparison of the results from the plant extracts or the turbidity of insoluble compounds.
NCCLS and EUCAST methods, line charts of extract con- The EUCAST method gave the poorest results. For
centration against percentage growth (the mean values plant extracts, the higher inoculum size and glucose supple-
from three tests) were presented and the standard devia- mentation which was used to optimize the growth of Can-
tions were shown as single direction error bars. Fig. 1a dida species did not show the claimed advantage of
shows the results obtained from the NCCLS method with simplifying end-point determination of MICs if combined
the spectrphotometric end-point determination. As shown with a spectrophotometric method [16]. The readings were
in the chart, the MIC50 can be obtained easily and the stan- not reproducible; some plant extracts had the percentage
dard deviation is acceptable for most concentrations. How- growth fluctuating by around 50%, making the determina-
ever, this method demanded an agitation of the plates to tion of MIC50 impossible, while some plant extracts
ensure homogeneity in the wells before reading, which showed a dose-dependent growth-promotion phenomenon
could affect the reproducibility and can be very tedious (Fig. 1b). The reasons behind this phenomenon are not

a 160 b 160
140 140

120 120
% of growth control
% of growth control

100 100

80 80

60 60

40 40

20 20

0 0
500 250 125 62.5 31.25 500 250 125 62.5 31.25
-20 -20
Concentration mg/L Concentration mg/L

CZM CZE CFM CZM CZE CFM

CFE CEM CEE CFE CEM CEE

c Concentration of plant extracts mg/L

500 250 125 62.5 31.25 15.63 7.81 3.91 1.95 0.98 G S

A
B
C
Plant
D
extracts
E
F
G
Positive H
control
8 4 2 1 0.50 0.25 0.12 0.06 0.03 0.02
Concentration of positive control mg/L
Fig. 1. (a) and (b) Results obtained for 6 plant extracts* against Candida krusei using (a) the NCCLS method and (b) the EUCAST method, with the
spectrophotometric end-point determination. (c) An example plate showing the results obtained from our modification of the NCCLS method with the
colorimetric end-point determination. G, growth control, S, sterility control. From row A to F were plant extracts: CZM, CZE, CFM, CFE, CEM, and
CEE. Row G was an untreated row. Row H was Itraconazole treated row. * CZM and CZE, Methanol and EtOAc extract of Combretum zeyheri, CFM
and CFE, Methanol and EtOAc extract of C. fragrans, CEM and CEE, Methanol and EtOAc extract of C. elaeagnoides.
 M. Liu et al. / Methods 42 (2007) 325–329
clear and we have not so far pursued the matter further.
The failure of the EUCAST method also highlighted the
important influence of inoculum size and medium type in
the antifungal test.
Good reproducibility and well-defined end-points were
achieved by the colorimetric method which also requires
less instrumentation and a simpler operating procedure.
The deep blue indicator resazurin, which is reduced to
bright pink resorufin by viable cells, dramatically reduced
the interference from the plant extracts including the col-
our and the precipitation. Fig. 1c provides an example of
a plate with results obtained from the NCCLS method
after modification with the colorimetric end-point determi-
nation. Resazurin did not affect the efficacy of Itraconazole
used here as the positive control and this widely used indi-
cator has no inhibitory activity against Candida spp. [17].
3.2. Anti-Candida activity of plant extracts
Table 2 lists the MIC of 12 extracts from six Combretum
and Terminalia species against three Candida species using
the NCCLS method with colorimetric end-point determi-
nation. In our pre-tests, all the hexane extracts showed
no activity, which could have been due to the poor solubil-
ity of non-polar ingredients in a water-based medium.
However, no further testing was carried out on the hexane
extracts. Among the six plants, Terminalia brachystemma,
which is traditionally used to treat schistosomiasis [18],
showed the highest activity against all three Candida spp.
This is in agreement with a previous study [6] and worthy
of further phytochemical investigation. Combretum zeyheri
was the second most potent plant in our study. It is tradi-
tionally used to treat diarrhoea and its stem and root have
shown substantial antimicrobial activity [3]. In agreement
with the 24 well nunc plate bioassay [5], the polar extract
of C. fragrans showed moderate activity in our test
although it had no inhibition zone in the agar diffusion test
by Masoko et al. [3]. T. mollis showed no activity in con-
trast to high activity reported by Masoko et al. [6] and
again in agreement with the 24 well nunc plate bioassay
[7]. Finally, C. eleaegnoides and C. kirkii, have no pub-
lished data available as far as we are aware. C. eleaegnoides
showed no activity while the methanol extract of C. kirkii
showed some activity especially against C. krusei and
C. albicans.
4. Concluding remarks
In this study, we successfully modified the standard anti-
fungal susceptibility test for the screening of plant extracts
329
for their anti-Candida activity using the dye resazurin. This
colorimetric broth microdilution assay showed the advan-
tage of good reproducibility and easy operation compared
with other available methods.
Acknowledgments
We thank Allan Drummond for technical support.
This work was supported in part by a scholarship from
the Faculty of Science, University of Strathclyde and by
personal support from Xiao Liu and Peiying Liu.
References
[1] D. Barret, Biochim. Biophys. Acta (BBA)-Mol. Basis Dis. 1587 (2002)
224–233.
[2] D.R. Katerere, A.I. Gray, R.J. Nash, R.D. Waigh, Phytochemistry 63
(2003) 81–88.
[3] M. Gelfand, S. Mavi, R.B. Drummond, B. Ndemera, The Traditional
Medical Practitioner in Zimbabwe, first ed., Mambo Press, Gweru,
1985.
[4] S.M. Carpano, E.D. Spegazzini, J.S. Rossi, M.T. Castro, S.L.
Debenedetti, Fitoterapia 74 (2003) 294–297.
[5] K. Batawila, K. Kokou, K. Koumaglo, M. Gbéassor, B. de Foucault,
Ph. Bouchet, K. Akpagana, Fitoterapia 76 (2005) 264–268.
[6] P. Masoko, J. Picard, J.N. Eloff, J. Ethnopharmacol. 99 (2005)
301–308.
[7] F. Baba-Moussa, K. Akpagana, P. Bouchet, J. Ethnopharmacol. 66
(1999) 335–338.
[8] P. Fyhrquist, L. Mwasumbi, C.A. Hæggström, H. Vuorela, R.
Hiltunen, P. Vuorela, J. Ethnopharmacol. 79 (2002) 169–177.
[9] National Committee for Clinical Laboratory Standards. Reference
method for broth dilution antifungal susceptibility testing of yeasts;
approved standard. NCCLS document M27-A2. National Committee
for Clinical Laboratory Standards, Pa. Wayne, 2002.
[10] M.L. Motsei, K.L. Lindsey, J. van Staden, A.K. Jäger, J. Ethno-
pharmacol. 86 (2003) 235–241.
[11] H. Bouamama, T. Noël, J. Villard, A. Benharref, M. Jana, J.
Ethnopharmacol. 104 (2006) 104–107.
[12] L.V. Buwa, J. van Staden, J. Ethnopharmacol. 103 (2006) 139–142.
[13] M.A. Pfaller, S.A. Messer, S. Coffmann, J. Clin. Microbiol. 33 (1995)
1094–1097.
[14] B.A. Arthington-Skaggs, W. Lee-Yang, M.A. Ciblak, J.P. Frade,
M.E. Brandt, R.A. Hajjeh, L.H. Harrison, A.N. Sofair, D.W.
Warnock, Antimicrob. Agents Chemother. 46 (2002) 2477–2481.
[15] European Committee on Antibiotic Susceptibility Method for deter-
mination of minimal inhibitory concentration (MIC) by broth
dilution of fermentative yeasts. Discussion document E. Dis. 7.1.
European Society of Clinical Microbiology and Infectious Diseases,
Taufkirchen, Germany, 2002.
[16] M. Cuenca-Estrella, W. Lee-Yang, M.A. Ciblak, B.A. Arthington-
Skaggs, E. Mellado, D.W. Warnock, J.L. Rodriguez-Tudela, Anti-
microb. Agents Chemother. 46 (2002) 3644–3647.
[17] R.N. Tiballi, X. He, L.T. Zarins, S.G. Revankar, C.A. Kauffman, J.
Clin. Microbiol. 33 (1995) 915–917.
[18] P. Mølgaard, S.B. Nielsen, D.E. Rasmussen, R.B. Drummond, N.
Makaza, J. Andreassen, J. Ethnopharmacol. 74 (2001) 257–264.