PHYTOCHEMICAL ANALYSIS, VOL.

2, 199-203 (1991)

A Bioautographic Agar Overlay Method for

the Detection of Antifungal Compounds from
Higher Plants

L. Rahalison, M. Hamburger and K. Hostettmann*

Institut de Pharmacognosie e t Phytochirnie, Ecole de Pharmacie, Universite de Lausanne, CH-1015 Lausanne-Dorigny,
Switzerland
M. Monod and E. Frenk
Laboratoire de Mycologie, Service de Dermatologie, Centre Hospitalier Universitaire Vaudois, C H U V ,
CH-1011 Lausanne, Switzerland

A simple bioautographic agar overlay assay using Candida albicans as the indicator organism for the detection
and activity-guided fractionation of antifungal compounds by thin layer chromatography has been developed.
Inhibition of fungal growth was assessed by the detection of dehydrogenase activity with thiazolyl blue
(methylthiazolyltetrazolium chloride; MTT). A series of clinically used antimycotic agents were tested in order to
determine the sensitivity of the assay. The compatibility of the agar overlay technique with chemically modified
silica gel (Diol and RP-18) plates and with various organic solvents was evaluated. The methodology is also
applicable to the search for antibacterial compounds, as shown with Bacillus subtilis as a test organism.

Keywords: Bioautography; thin layer chromatography; agar overlay; MTT; Candida albicans; Bacillus subtilis.

matographic (TLC) plate, (b) contact bioautography,

INTRODUCTION
The search for antifungal drugs has received fresh
attention mainly as a result of an increasing occurrence
of opportunistic systemic mycoses, associated primarily
with AIDS and the treatment with immunosuppressive
agents. The fungal infections observed (Musial et al.,
1988) in the immuno compromised host are mainly
disseminated candidiasis (Candida albicans and other
species), cryptococcosis (C. neoformuns) and aspergil-
losis (Aspergilfa flaous, A . fumigatus, A . niger). There
are currently only a few antifungal agents indicated for
the treatment of mycotic infections, namely the
polyene antibiotic amphotericin B, the nucleoside ana-
logue flucytosine and the synthetic azoles clotrimazole,
ketoconazole and miconazole. The treatment of
mycoses often requires a combination of these agents.
Significant adverse reactions have been observed, while
the success rate has been rather limited (Bork, 1983;
Hufford and Clark, 1988). In the past, efforts in the
study of antifungal plant constituents have been mainly
directed towards compounds active against plant patho-
genic fungi. That plant-derived constituents may offer
potential leads for novel agents against systemic
mycoses has been demonstrated by Hufford and Clark
(1988).
Bioautography can be considered as the most effi-
cacious assay for the detection of antimicrobial com-
pounds, because it allows the localization of the activity
even in a complex matrix and therefore permits a
target-directed isolation of the active constituents.
Bioautographic assays can be divided in three groups
(Rios el al., 1988): (a) direct bioautography, where the
microorganisms grow directly on the thin layer chro-
Author to whom correspondence should be addressed.
09S8-0344/91/050199-05 $0S.00
01991 by John Wiley & Sons,
i Ltd.
where the antimicrobial compounds are transferred
from the TLC plate to an inoculated agar plate through
direct contact and (c) agar overlay or immersion bioau-
tography, where a seeded agar medium is applied onto
the TLC plate. The latter technique can be considered
as a hybrid of direct and contact bioautography.
Direct bioautography for antibacterial (Lund and
Lyon, 1975; Hamburger and Cordell, 1987) and spore
producing fungi (Homans and Fuchs, 1970; Van der
Sluis et a f . , 1981; Gottstein et a f . , 1984) is very sensitive
and gives accurate localization of active compounds. Its
applicability is, however, limited to microorganisms
able to grow directly on the chromatographic layer. In
contact bioautography, the transfer process leads to
larger inhibition zones which decrease the sensitivity
and the ability to discriminate between active com-
pounds with similar Rfvalues.
We have been involved for several years in the
isolation of antifungal plant constituents using direct
bioautography with the plant pathogen Cladosporium
cucumerinum as test organism (Hostettmann and
Marston, 1990). We report here on a simple and rapid
agar overlay assay with Candida albicans, which has
been developed in the course of a research programme
aimed at the discovery of novel antimycotic agents from
higher plants.
EXPERIMENTAL
Strains of C . albicans were
Microorganisms and culture media.
obtained from clinical isolates of the Service de
Dermatologie, Centre Hospitalier Universitaire Vaudois
(CHUV), Lausanne, Switzerland. Bacillus subtilis ATCC
6633 was purchased from Sigma. Sabouraud liquid medium
Received 20 June 1991
Accepted 9 July I991
200 L. RAHALISON ET. AL.

(Diagnostics Pasteur, Marnes La Coquette, France) was used

~ ~

for broth culture of C. albicans. Yeasts were maintained on Table 1. Bioautography on differnt supports (Silica gel G60
Fzs4, HPTLC Diol F254 S and HPTLC RP-18 WFzS4
Sabouraud agar slants. Bacterial strains were lyophilized or
S). Minimum amount (pg) of reference compounds
maintained on Luria-Bertani (LB) agar slants (bacto-tryptone required for inhibition of growth of microorganisms.
10 g/L, yeast extract 5 g/L, NaCl 10 g/L, bacto-agar 15 g/L). Ca =C. albicans, Bs =B . subtilis
Cultures were grown in LB broth (same composition but
TLC Support
without bacto-agar). All media were autoclaved at 120°C for s102 Diol RP-18
20 min. In order to obtain an exponential growth phase of C. Referencecompounds Ca 3s Ca Bs Ca 8s
albicans, the Sabouraud broth (20 mL) was inoculated 5-6 h Ampicillin >loo 0.01 > l o o >loo > l o o 10
before the test. B. subtilis was grown in LB broth about 8 h Chloramphenicol > 100 0.001 > 100 0.001 > 100 0.001
before the test. The cultures were shaken at room tempera- Griseofulvin >loo 10 >loo >loo 10 >loo
ture on a rotatory shaker at 200 rpm. ltraconazole 0.001 1 >loo >loo 10 >loo
Miconazole 0.001 0.1 0.01 1 1 100

lnoculum for the assay. Malt agar (Oxoid) for C. albicans and
LB agar for B. subtilis were used as the solid media for the
overlays. The molten media were maintained in a water bath were incubated overnight at 30°C in polyethylene boxes lined
at 45°C. The optical density at 600nm (OD,) of the with moist chromatography paper. The bioautograms were
C. albicans culture was measured with a UV/VIS spectro- sprayed with an aqueous solution (2.5 mg/mL) of thiazolyl
photometer (an ODhooequal to 1 corresponds to approx. lo7 blue (methylthiazolyltetrazolium chloride; M7T) (Fluka),
cells/mL). The final concentration in the solid medium was and incubated for 4 h at 30°C. Clear inhibition zones were
approx. 10' cells/mL. The suspension of B. subtilis was observed against a purple background.
prepared at a final concentration of approx. 10* cells/mL
(ODM,= 1 for the bacteria). These suspensions were pre-
pared immediately before carrying out the test. RESULTS AND DISCUSSION
Samples tested. The following reference compounds were used
in the determination of the threshold of detection: itracona- Preliminary experiments with direct bioautography
zole and miconazole (Janssen, Beerse, Belgium), griseoful- gave unsatisfactory results, the yeast apparently being
vin, ampicillin and chloramphenicol (Sigma). Stock solutions unable to grow directly on the silica gel plate. Aiming
were prepared with H 2 0 , CHCI,, EtOH or MeOH. Roots of at conditions for good growth of C . albicans and the
Swartzia madagascariensis were collected near Ifakara, applicability of the procedure to other microorganisms,
Tanzania. The ground plant material was extracted at room the agar overlay technique was chosen in a modified
temperature successively with light petroleum, CHCI, and version of published protocols (Slusarenko et al., 1989).
MeOH. In an agar dilution assay, the CHCI, extract showed The principle of the adopted procedure is as follows.
good activity against C. albicans (growth inhibition at 50 pg/ The inoculated medium is rapidly poured onto the
mL). The CHCI, extract was submitted to column chromato- developed chromatogram. Owing to heating of the
graphy (CC) on silica gel (light petroleum: EtOAc 1:1) to TLC plate at 35"C, the medium solidifies as a thin layer
afford 11 fractions. (d1 mrn layer thickness). Overnight incubation in a
humid atmosphere permits growth of the yeast. Zones
Thin layer chromatography. Silica gel G60 F2s4glass backed of inhibition are visualized by the detection of dehydro-
plates, HPTLC Diol F2s4S plates and HPTLC RP-18 WF254S
plates (Merck) were used. Dilutions corresponding to
0.0001-10 pg of the reference compounds and to 1-100 pg of
plant extract and fractions were applied. The TLC were
developed with the following solvent systems. Itraconazole:
CHCI,:MeOH 7:3 (SOz), CHC1,:MeOH 98:2 (Diol),
MeOH:H,O 8:2 (RP-18); miconazole: CHCI,:MeOH 8:2
(SiOJ, CHCI,:MeOH 98:2 (Diol), MeOH:H,O 8:2 (RP-18);
griseofulvin: CHCI3:MeOH 8:2 (SiOz), CHCI,:MeOH 98:2
(Diol), MeCN :H 2 0 1: 1 (RP-18); ampicillin: CHCI, :MeOH
8:2 (Si02), MeOH:H,O 1:1 (RP-18); chloramphenicol:
CHCI,:MeOH 8:2 (Si02), CHCI3:PrOH 9: 1 (Diol),
MeCN: H 2 0 1:1 (RP-18). The CHCl3extract and fractions of
S. rnadugu.scariensis were separated with CHCI,: MeOH 97 :3
or CHC1,:MeOH 95:5 (Si02), CHCI,:MeOH 98:2 (Diol) and
MeOH:H,O 8:2 (RP-18). Chromatograms were dried with a
hair dryer for complete removal of solvents. All TLC plates
were run in duplicate, one of them being used as the refer-
ence chromatogram. UV active spots were detected at 254
and 366 nm. The reference chromatograms were stained with
Godin reagent.

Bioautography. Chromatograms were placed on a hot plate

maintained at 35°C. Approx. 1 0 m L of the inoculum was Figure 1. Bioautography of miconazole with C. albicans on
rapidly distributed over the TLC plate (10 x 10 cm) with a silica gel G60 F,, plate; 0.0001-1 kg of the reference compound
sterile pipette. After solidification of the medium, TLC plates applied.
 BIOAUTOGRAPHIC DETECTION OF ANTIFUNGAL COMPOUNDS
Figure 2. Bioautography of CHCI, extract (ext) and three fractions (numbered I, IV and IX, see Experimental
Section) of S. madagascariensis roots. Chromatographic conditions: silica gel G60 F2% plates, CHCI,: MeOH
97:3.Left: reference chromatogram stained with Godin reagent; right: bioautograrn with C. albicans.
genase activity with a tetrazolium salt. Active com-
pounds appear as clear spots against a coloured
background.
In order to optimize the conditions for growth of C’.
albicans and the detection of active compounds, several
parameters were varied, such as inoculum size, type of
TLC plate, application procedures for the overlay,
incubation period for growth and enzymatic reaction
and substrate for detection. The results can be summar-
ized as follows. Optimal concentration of the inoculum
was approx. lo5 cells/mL in the case of C. albicans.
Below that concentration, the growth of C. albicans
was not confluent and insufficient reduction of the
substrate resulted in bioautograms with poor contrast.
Glass-backed TLC plates were more appropriate for
the agar overlay technique than aluminium-backed
sheets. Spreading of the medium was significantly bet-
Figure 3. Bioautography of chloramphenicol with €?.subtilis on
silica gel G60 F,, plates; 0.001-1 kg of compound applied.
20 1
ter, and the glass-backed plates were also more suitable
for thermal control.
As with contact bioautography, the agar overlay
technique requires the transfer of the active compounds
from the stationary phase into the agar layer through a
diffusion process. It could be anticipated that thin and
homogeneous agar films would minimize the problems
involved with the diffusion process, notably differential
diffusion of the constituents, poorly defined inhibition
zones and low sensitivity. A rapid distribution of the
inoculum with a pipette onto a TLC plate maintained at
35°C sufficiently delayed the solidification of the agar
that layers of typically less than 1 mm thickness were
obtained. Overnight incubation at 30°C was found to be
sufficient. Prolonged incubation (up to 48 h) led to
confluent growth obscuring the inhibition zones. A
number of tetrazolium salts were evaluated as sub-
strates for the enzymatic reaction. While 2,3,5-
triphenyltetrazolium chloride (TTC) was a poor sub-
strate, both p-iodonitrotetrazolium chloride (INT) and
MTT were readily converted to the corresponding for-
mazan. For practical reasons (solubility and contrast of
the bioautograms), MTT was preferred. The aqueous
tetrazolium salt solution (2.5 mg/mL) was sprayed onto
the agar layer with a glass TLC plate sprayer. The
amount of MTT formazan produced by enzymatic reac-
tion increased over a period of 4 h. Incubation for 2 h,
however, was sufficient to produce bioautograms with
good contrast. Under the above-mentioned conditions,
the sensitivity and selectivity of the assay was assessed
with a series of clinically used antimycotic drugs. The
detection limits for five antimycotic and antimicrobial
drugs are listed in Table 1. The inhibitory activity
against B. subtilis was determined under comparable
conditions to those used for C. albicans (for details, see
Experimental Section).
On silica gel plates, the detection limit for micona-
zole, a synthetic imidazole used for the treatment of
systemic candidiasis, was 0.001 pg against C . albicans
(see Fig. 1) and 0.1 pg against B. subtilis. The still
relatively good antibacterial activity in our assay is in
accord with the broad spectrum of action reported for
the drug (Janssen and Van Bever, 1979; Van den
202 L. RAHALISON ET. A L .
Bossche et al., 1980). The detection limit for itracona-
zole, an imidazole derivative undergoing clinical evalu-
ation, was similar against C. albicans, but was higher by
a factor of 10 against B. subtilis. This correlates well
with the increased specificity reported for this new drug
(Odds et al., 1984). The high detection limit (> 100 pg)
for griseofulvin can be explained by the spectrum of
activity of this drug which is used specifically against
dermatophytes. When tested at 100 pg, the antibiotics
ampicillin and chloramphenicol were not detected with
C. albicans, while the detection limit with B. subtilis
was 100-1000 times lower. It appears that the assay
reflects rather well the spectrum of activity/selectivity
of these antifungal and antibacterial drugs and that the
sensitivity is sufficient to detect active compounds even
at low concentrations.
A CHCl, extract of S. madagascariensis (Legumino-
sae) was selected as an example for testing the applica-
bility of the assay to activity-guided fractionation. In a
dilution assay, this extract inhibited growth of C. afbi-
cans at 50 pg/mL and had a good activity against
Cladosporium cucumerinum. In the bioautogram, 5 pg
of crude extract were sufficient to observe two zones of
inhibition at R, 0.50 and 0.82 (silica gel, CHC1,:MeOH
97:3). Some 100 pg of extract were required in order to
obtain a good reference chromatogram. Subsequent
fractionation by CC on silica gel (light petroleum:
EtOAc 1: 1) afforded 11 fractions (I-XI). A bioauto-
gram of the crude extract and of the representative
fractions IV and IX are shown in Fig. 2. The zones of
inhibition are clearly defined, a prerequisite for accur-
ate localization of activity. Final purification and struc-
ture elucidation of the active principles are underway.
A major drawback with direct bioautography is the
limitation in the choice of stationary phases and eluents
that can be used (Hamburger and Cordell, 1987). In
particular, solvents of low volatility such as n-BuOH,
acids or bases such as HOAc, trifluoroacetic acid
(TFA) and ammonia cannot be removed completely
even after prolonged drying and inhibit growth of
bacteria. With the agar overlay technique, addition of
TFA (0.5%) of ammonia (2%) to the mobile phase did
not affect the growth of C. albicans. Solvent systems
containing diethylamine and n-BuOH, however, were
found to be unsuitable.
Commercially available TLC plates with chemically
modified silica gel (Diol, NH2, CN, RP-8, RP-18) have
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an inoculum of 10' cells/mL, the enzymatic transforma-
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inhibition zones are obtained (See Fig. 3). Thus, it is
possible to obtain preliminary information about the
degree of selectivity of an active compound within a
crude extract by preparing bioautograms with these two
or other microorganisms. We are currently using the
assay for the isolation of plant constituents with anti-
Candida activity.
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