Journul of EthnophaImacology, 23 (1988) 127- 149 127

Elsevier Scientific Publishers Ireland Ltd.

Review Paper

SCREENING METHODS FOR NATURAL PRODUCTS WITH

ANTIMICROBIAL ACTIVITY: A REVIEW OF THE LITERATURE

J.L. RIOS, M.C. RECIO’ and A. VILLAW

“Departamento de Fannacotogiu y Farmacotenia Laboratorio de Farmacognosiu y
Farmacodimzmia, Facultad de Farmacia, Universidad de Vale&q Avda. Blasco Ibafiez 18,46010
Vakncia and bDepartamento de FarmacologM, Facultad de Farmacia, Universidad &n&tense
de Madrid, Ciudad Universitaria, 28040 Mad% /Spain)
(Accepted May 111988)

Summary

Diffusion and dilution methods have been employed to study the

antimicrobial activity of medicinal plants. A number of modifications have been
made in the technique in order to obtain better results. Since some factors
(culture medium composition, microorganisms tested, extractive method, pH,
solubility of the sample in the culture medium, etc.1 can change results, it is dif-
ficult using these methods to standardize a procedure for the study of
antimicrobial plants. Bioautography is another method for studying
antimicrobial activity. With it, previously chromatographed principles are
diffused to the agar. The results can also change according to the method
employed. All the various techniques are reviewed here and, in order to unify
the different criteria and parameters, standard methods to study the
antimicrobial activity of medicinal plants are proposed.

Introduction

Research on the antimicrobial activity of medicinal plants has encountered

some problems because of the diversity of criteria and techniques employed
and the lipophilic properties of some samples. The water insolubility of essen-
tial oils and non-polar extracts makes it very difficult to use an aqueous medium
in the study of antimicrobial activity (Allegrini et al., 1973; Pellecuer et al.,
19761.
We have prepared a bibliography on the different techniques and methods

Cowespondence to: J.L. RYos

0378-8741/88/$08.40 0 1988Elsevier ScientificPublishersIreland Ltd.

Published and Printed in Ireland
employed in the antimicrobial study of medicinal plants and the principles
obtained from them. While the methods can be classified into only three groups
(diffusion, dilution and bioautographic methods), a great number of factors can
influence the results: the extraction method (Nadir et al., 19861,inocula volume
(Bauer et al., 1966; Hamburguer and Cordell, 19871,culture medium composition
(Bauer et al., 1966; W.H.O., 19771,pH (Leven et al., 1979; Gutkind et al., 1981;
Emeruwa, 19821and incubation temperature (Emeruwa, 19821.Recently, Jans-
sen et al. (19871 have reviewed the methods employed for the study of the
antimicrobial activity of essential oils and their analysis of the methods
employed is of interest. These authors deal with aspects such as the
microorganisms used and the volume of the assayed sample. All of these
experimental factors can modify the inhibition of in vitro microorganism
growth.
There is no standardized method for expressing the results of antimicrobial
screening (Naqvi et al., 1976; Ayafor et al., 1982; Singh et al., 19841. Some
authors use the diameter of inhibition halos and/or the minimal weight of
extract that inhibits the growth of a microorganism. However, there are no
reports on the activity of dry plants.
After reviewing the different methods used in the study of antimicrobial
activity, and drawing from our own work (Villar et al., 1983,1984,1985,1986a,b,
1987; Rios et al., 19871,we propose the best methods for the different extracts
or compounds described.

1. Principal diffusion methods

A technique which does not require homogeneous dispersion in water is the

agar-overlay method using a disk, hole or cylinder as reservoir. The reservoir
containing the sample to be tested is brought into contact with an inoculated
medium and, after incubation, the diameter of the clear zone around the reser-
voir (inhibition diameter) is measured. This method was originally designed to
monitor the amount of antibiotic substances in crude extracts. In order to lower
the detection limit, the inoculated system can be kept at a low temperature
before incubation, which favours diffusion through the culture medium and this
increases the inhibition diameter. This technique can also be used for obtaining
biograms.
In Table 1, we summarize the different methods and the applications and
modifications suggested by several authors in the antimicrobial study of
vegetal samples.

2. Dilution methods

Dilution techniques are those which require a homogeneous dispersion of the

sample in water. They are used to determine, principally, the minimum
concentration (MIC) values of an extract, essential oil or pure substance. They
can be used in the preliminary screening of antimicrobial activity.
TABLE 1
DIFFUSION METHODS
(Al Disk method. Its essential feature is the placing of filter paper disks with the antibiotic on the surface of agar immediately after inoculation
with the organism tested. Undiluted overnight broth cultures should never be used as an inoculum. Routine direct application of disks to plates
seeded with clinical material is not recommended because of problems with inoculum control and mixed cultures. This technique was originally
standardized by Bauer et al. (19661and by Ericson and Sherris (1971) and then changed in the Report of the WHO (19771.

General methods Samples References

Filter paper disks (Whatman no. 2 of 6.5 mm in Penicillin and other Bondi et al. (19471
diameterl’saturated with antibiotic solution antibiotics at the
are placed on the surface of a blood agar time of primary
seeded with a clinical specimen (two disks/plate) isolation
Aqueous and Ajao et al. (19841
MeOH extracts
Small sterile disks (6 mm in diameter1 are Essential oils Marussella and Liguori (19581
moistened and placed on Sabouraud’s maltose agar Marussella and Henry (19581
plates which had previously been seeded with 2 ml Prasad et al. (19881
of the fungi in broth culture or 1 ml of the broth
culture of the organism (bacteria1
Similar, but the oil to be tested is diluted with Essential oils Manunta et al. (19871
EtOH (29% v/v1
Essential oils diluted with Tween 59 0.5% (v/v); Essential oils Garg and Kasera (19831
disks are dipped in solution of 1:5&l : 199 and
1299
The oil is mixed with Tween 80 at a ratio of 0.5: 199 Essentials oils Batra and Mehta (19851
and this mixture is diluted with aq. EtOH 10.59 and
199% (v/v)
Agar surface is seeded with clinical specimen; 6 4 Essential oils Valnet et al. (19781
of each essential oil are added to the disk;
6- 12 disks are placed on each plate
TABLE 1 (c~t~n~d~

General methods Samples References

0.2 ml of bacterial broth culture is added to Essential oil vapors Maruxxella and
the surface of the agar. Disks are saturated Sicurella &MO1
with the oil and placed in the center of the
inner surface of the Petri dish cover. When the
plates are inverted and incubated, the disks
are at a distance of about 8 mm from the surface
growth of the microorganism. The presence of a
clear xone on the surface of the agar above the
disk is indicative of antibacterial activity.
A strain of Badltls subtih is the organism Streptomycin Loo et al. (19451
tested. 26.0 ml of BactoStreptomycin agar are added Alcoholic extracts Boss et al. (198Obl
to Petri dishes. While the plates are still warm, Extracts of crescent Singh et al. (19831
4.0 ml of seeded agar are added and distributed polarity
over the surface. A concentration of approximately Esential oils Ross et al. (198Oal
250.000 spores/ml in the seeded agar gives the
desired results. Samples are diluted in phosphate
buffer (pH 7.91t0.2 Ml and subsequent dilutions with
0.1 M buffer. 0.908 ml sample is pipetted onto
filter paper disks (4-6 disks/plate1
Mueller-Hi&on medium is used. Samples are dissolved Antibiotics Bauer et al. (19661
in water, MeOH or mixtures of both. Inoculum Extracts of crescent WHO (19771
should be diluted at least lo-fold or preferably to polarity Barbagallo and Chisari (19871
a density equivalent to the barium sulphate standard
Disks are impregnated with 0.602 ml of each solution Flavonoids Waage and Hedin (19851
Equilibrationat 4 “C, before incubation Terpenoids Mathur and Gonzalez (19821
Similar, but the extracts are solubilized in Extracts of crescent Emeruwa (19821
phosphate buffer. Disks are soaked with a solution polarity
of 4 mg extract/ml
Disks are impregnated with 0.1 ml of each extract. Water/MeOH extracts Harsh and Nag (19841
Equilibration at 5°C for 44 - 55 min and flavonoids isolated
The plates are allowed to stand at room temperature Alkaloids Ayafor et al. (1982)
for 30 min and then are incubated
Essential oil is diluted with MeOH and paper disks Essential oils and Yaaphe et al. (1979)
are impregnated with 0.01 ml of these solutions their fractions
Ba&ti 8ubtilis is grown in two different EtOH extracts and Baa and Alvarez 119811
media. Filter paper disks are dipped in a sobrtion sesquiterpenic acid
of the compound in EtOH/water (1: l), dried for 10
min te remove the EtOH and placed in the agar
plate. The Petri dishes are preincubated at 5X
for 12 h to permit the maximum diffusion of the
drug. After this, the plates are incubated
0.2 ml of the,aqueous suspension of spores and Alkaloids Hejtmankova et al.
mycelial fragments are pipeted on the surface of (19841
agar plates (Sabouraud’s glucose agar), Two hours
after inoculation the surfaces of the plates are
covered with paper disks (Whatman no. 1 of 9 mm)
and they are soaked for 2 s in the aqueous
solutions of the tested substances
0.1 ml of the organism culture is added to a fresh Quinones Tabats et al. (1982)
unsolid agar medium and the mixture is poured onto
agar medium in Petri dishes. Disks of 6.3 mm in
diameter on which 0.6-20 c(g of a test compound
are applied. The antimicrobial activity is
expressed in terms of minimum inhibition
concentration bgldisk)
1 g of crushedplant material is added to 5 ml of EtOH extracts Wat et al. (19801
95% EtOH, Paper disks, 7 mm in diameter, are
allowed to soak in the EtOH extract overnight.
Before sssay the disks are removed and air-dried
in sterile Petri dishes in the dark
TABLE 1 (contiwed)
g
(B) Holeplate assay method. This method depends upon the diffusion of the antibiotic from a vertical hole through the solidified agar layer of a
Petri dish or plate to such an extent that growth of the added microorganism is prevented entirely in a circular area or zone around the hole
containing a solution of the antibiotic.

General methods Samples References

Samdes are dissolved with distilled water. Antibiotics USP XIX (19751
phosphate buffers of different pH, or MeOH. Alkaloids Vlllar et al. (1988al
Mueller-H&on or Sabouraud agar is poured into MeOH and aqueous Adesina and Aklnwusi (19841
pre-steriliaed Petrf dishes. After congealing, the extracts
agar medium is homogeneously inoculated with a
culture of the test orgauism. Six holes (12 mm in
diameter) are dug with a flamed cork borer and
aseptically filled with 0.02 ml of each solution
to be tested. The plates are feft at 4% for 1- 2 h
and then are incubated
Similar, but the hole is filled with 0.2 ml of each Extract of crescent Leven et al. (19791
extract. The lipophilic fractions are dissolved in polarity Gundidsa (1986, 19871
PEG 4991phosphate buffer (4:61 pH 7.4 and physiolo-
gic buffer for hydrophylie fractions
Idem. PEG 4091phosphate buffer at a ratio of 1: 1 Extracts of crescent Van Hoof et al. (19801
polarity
EtOH extracts and Van Hoof et al. (19831
their fractions
Five wells of 1 cm in diameter, 0.1 ml of the MeOH extracts Lynch-Brathw~te et at. (19751
fractions are placed in each well
20 ml of Difco Antibiotic Medium 2 are overlaid EtOH extracts Debro and Ward (19791
with 5 ml of Difco Antibiotic Medium 1 previously Xanthones Sundaram et al. (19831
inoculated. Four wells of 16 mm in diameter which
received 0.3 ml of the substances to be tested
0.2 ml of each extract are carefully added to the CHCI, and MeOH Farouk et al. (19831
cups and allowed to diffuse at room temperature extracts
for 2 h
Plates for assay are uniformly prepared by seeding Extracts and flavonoids Hufford et al. (1975.19801
sterile, partially cooled, molten agar with dilutions alkaloids isolated
of test microorganism grown in broth or suspensions Flavonoids Hufford and Lasswell (19781
of conidia. The seeded agar medium is poured into
sterile Petri dishes. Holes having a diameter of 11 mm
are fiied with 100 pl of a solution or suspension
of an extract, fraction or pure compound
The alkaloids are dissolved in a buffer made up of Alkaloids Verpoorte et al. (19781
equal parts of 1% acetic acid and 1% sodium acetate
in water (pH 5.51
Samples are dissolved in citrate-phosphate buffer EtOH extracts Van Beek et al. (1984al
pH 4.0
The extracts are dissolved in the same extractive Extracts of crescent Almagboul et al. (19851
solvent polarity
The solvent is a mixture of acacia gum in water Extracts of crescent Ikram and Inamul-Haq (19801
4-5% polarity
The residue from each extract is treated with Aqueous and alcoholic Ajao et al. (19851
distilled water to make 50,25,20,10 and 5% extracts
solutions (w/w). Three drops of each extract in
each well. Equilibration at room temperature

Cl Cyliuder method. This method is similar to the hole-plate method. Stainless steel or porcelain cylinders are used for assay. After incubation,
the cylinders are removed, and the average diameter of each zone of growth inhibition is measured and recorded.

General methods Samples References

Six cylinders are distributed on the Mueller- Antibiotics
Hinton agar. 0.1 ml of the antibiotic on each Polar extracts
cylinder. The solvent can be water, MeOH. or Hydroacetonic extracts
phosphate buffers of different pH Extracts of crescent
polarity
USP XIX (19751
Laurens et al. (19821
Mourey et al. (19851
Barbagallo et al. (19821
TABLE 1 (continued)

General methods Samples References

Inocula are prepared in liquid Antibiotic Medium EtOH extracts Guthind et al. (1961)
no. 3 Klifeo). 0.2 ml of the inoculum diluted are EtOH extracts and PaIacios et al. (1933)
seeded and mixed with 10 ml of Antibiotic Medium flavonoids isolated
no. 1 @life01and 10 ml of Antibiotic Medium no. 11
(Difco) and maintained at 45% in a
thermostatic bath. Sterile cylinders (b/plate)
are disposed on the agar surface of the cooled
and dried Petri dishes. 0.15 ml of extract are
added on each cylinder. The same method is used
for determining the antifungal activity using
Sabouraud-maltose agar.
0.5 mi of the aromatic chemical are placed in the Aromatic chemical MaruzzeBa et al. (1961)
center of the Petri dish top. When the dishes are vapors
inverted and incubated, the surface of the substance
in the cup is about 5 mm from the agar surface.
Vapors of the chemical are allowed to emanate
throughout the period of incubation
 135

The physico-chemical properties of the dispersions used are important for

observing the activity, and surface-active substances such as the different
polysorbatum (Tween 20 or Tween 80) can be used.
In the liquid dilution method, turbidity is taken as an indication of bacterial
density. When no growth takes place, the medium remains clear; when the
sample is inactive against the germ tested and there is growth, it appears tur-
bid. The grade of inhibition is related to the turbidity of the medium and meas-
ured by spectrophotometry.
With the agar dilution method, a fixed amount of an antibiotic-containing
mixture is mixed with nutrient agar and allowed to set. The advantages of this
method are its simplicity and speed and the possibility of using it in the
antimicrobial study of water-soluble or insoluble samples such essential oils.
Six microorganisms can be seeded in a Petri dish and there is antimicrobial
activity when the germs do not grow.
Table 2 summarizes both methods and the kind of samples that can be
assayed.

3. Bioautographic methods

According to Betina (19731, bioautography is the most important detection

method for new or unidentified antimicrobial compounds. It is based on the
biological (antibacterial, antiprotozoal, antitumoral, etc.) effects of the
substances under study. In comparison with paper chromatography (PC), thin-
layer chromatography (TLC) has greater resolving power and is the more rapid
of the two techniques. The typical bioautography procedure is based on the so-
called agar-diffusion technique, whereby the antibacterial compound is
transferred from the chromatographic layer to an inoculated agar plate.
Inhibition zones are visualized by dehydrogenase-activity-detecting reagents
(Begit and Kline, 19721. The initial procedure had several disadvantages which
have been corrected by introducing certain modifications, all of which are
described in a review by Betina 11973). Bioautography can be classified into
three general variants as described in Table 3.

Discussion and conclusions

Diffusion methods
The diffusion methods are those most often employed in research in spite of
certain difficulties, but they are models with a low credibility for samples that
are difficult to diffuse in the media because there is no relation between diffu-
sion power and antimicrobial activity. Pellecuer et al. (19761 showed the
different results that can be obtained for two different samples (phenol and
essential oil from Thymus) against Escherichia co& Similar activity was
observed when they used diffusion (disks) with inhibition halos of 42 and 41 mm,
respectively; but when assayed by a dilution method, the essential oil was more
active (l/30001 than the phenol (l/10001. When the authors compared the activity
TABLE 2

DILUTION METHODS

(A) “Tube” away or turbidimetric method. It is based on the homogenous dispersion of a sample, dissolved in purified water, MeOH, water/
MeCH mixtures, phosphate buffers, in the broth culture required for the organism assayed. After incubating 3-4 h. transmittance or absorb
ante is read in a suitable spectrophotometer fitted with a 530 nm filter WSP XIX, 1975; PE, 19711.

General methods Samples References

0.25 g of extract/l0 ml of sterile water are Alcoholic extracts Twaij et al. (1986)
serially diluted to give the desired concentrations.
1 ml of a serially diluted plant extract is added.
The tubes are incubated and measured spectrophoto-
metrically
Essential oil is diluted with Tween 20 at a ratio Essential oils Fournier et al. (19’781
of 10%
Essential oihl’ween 20 at a ratio of 1: 8 (w/w). Essential oils Yousef and Tawil(1980)
The solution is serially diluted twice in sterile
broth
Essential oihl’ween 80 at a ratio 6:4 Essential oils Chalchat et al. (19871
Two-fold dilution in broth. The concentration of Flavonoids Hufford and Lasswell (19781
pure compounds in the initial tube is 50 pg/ml
10 mg of each alhaloid are suspended in 10 ml of Alkaloids Lumonadio et al. (19861
water. 5 ml of each dilution are diluted to 25 ml
with culture medium
tB) Agar dlbttion method. The sample is dissolved or suspended in an appropriate solvent and mixed with agar medium. The results obtained
with this method are equivaIent to those obtained with diffusion and diiution methods.

General methods Samples References

10 mg extract/92 ml solvent (water, MeOH, acetone MeOH extracts Mitscher et al. (1972)
or other solvent without antimicrobial activity at AIkaIoids, flavonoids Mitscher et al. (19751,
a final concentration of 2%). Before congealing, (1980)
10 ml of MueBer-Hin~n agar is added aseptically Alkaloids Al-Shamma et al. (19811
to each of the plates and they are swirled careiirlly (1982)
until the agar begins to set. The active extracts CHCl, and MeOH Rios et al. (1987)
are re-assayed at a concentration of 100 ccghnl. Sequiterpenes CaBada et al. (19801
This method can be employed in the MIC determination.
Similar to the Mischer method above, but applied to Essential oils Vi&r et al. (1988b)
essential oiIs. Tween 8Olessential oil at a ratio
15. The mixture is diluted with the agar
untiI diIutions of l/l00 to l/800.
Extracts are added to the culture medium at a Etheric, EtOH and water Biard et al. (1980)
concentration of 100 mg of dried plant/ml medium. extracts
If the assay is positive, doses of 10.5,4.2 and 1
mg/mI must be assayed.
Milt Sabouraud sgar mixed with the extracts or Water extracts and Fuzehier et al. (1982)
compounds, shaken, and 20 ml of this medium are isolated anthraquinones
put in the plates
Phosphate buffer tpH 7.4) to make a solution at a Aqueous extracts El-Said (1971)
ratio1 : 1 (w/w).1 ml of each extract is added to
each sterile plate with agar medium or blood
agar before congeahng
TABLE 3
BIOAUTOGRAPHIC METHODS
(A) Contact bioautography. It is based on the diffusion of separated compounds by TLC or PC from sheets or cbromatoplaques. These are
placed on the surface of large nutrient agar plates inoculated with microorganisms that are sensitive to the antibiotics being analyzed. After 15
-36 min, the sheets or chromatoplaques are removed. In both instances, antibiotics diffuse into the agar layer and inhibit the growth of the test
microorganisms. The plates are then incubated at an appropriate temperature until a thin film of the growing microorganisms is visible on the
surface. Zones of inhibition are then clearly visible. Inhibition zones can be made more conspicuous and visible earlier by using dehydrogenase-
activity indicators.

General methods Sample References

Petri dishes with a culture medium are seeded Antibiotics Bickel et al. in
with a cell or spore suspension in the appropriate Betina (19731
liquid medium and the TLC-plates are placed on the CHCl, and MeOH extracts Rao et al. (19821
agar surface. The TLC-plates are removed, and Alkaloids Al-Shamma et al. (1982)
Petri dishes are incubated. Inhibition zones of
the separated antibiotics are observed
The Petri dishes with the chromatoplaques are Extracts and sesqui- McCallion et al. 11982)
frozen at 4%. The plate is removed from the terpene compounds
agar after 24 h and then the Petri dish is Extracts of crescent Zahir Shah et al. (19861
incubated. polarity
Alkaloids Verpoorte et al. (1982,1983)
Van Beek et al. (1984bJ985)

Buffered Whatman paper no. 1 is placed on the Antibiotics and related Wallhiiuser (1969)
chromatoplaqueand pressed. After 15 min it is substances
withdrawn and placed over the agar-inoculated
surface. Then the plate is incubated. The
inhibition zones correspond to the active
substances separated by chromatography
Alufolien (Merck) is used. The chromatogram is cut Fungicides Wolters (19691
in strips (2.5-5 mm1and placed on the agar surface CHC$ extracts Wolters and Eilert (1981)
previously inoculated with fungi spores. There is no
growth of mycelium on the strips with fungicidal
substances
(Bl Direct bioautograpby. A microorganism suspension in liquid medium is sprayed on a developed chromatoplaque after removing the sol-
vents. It is then incubated. With this method, there is no diffusion and the problems of the contact method are eliminated.

General methods Sample References

After locating UV-absorption spots, the chromatograms Fungitoxic products Homans and Fuchs (19701
are sprayed with a conidial suspension of the test
fungus in a broth medium. After spraying, the thin-
layer plates are incubated. Inhibition zones indicate
presence of the original fungitoxic product
TLC ls sprayed with a suspension of the germ in TSB. Aqueous and MeOH Lund and Lyon (19751
Inhibition zones are intensified by the use of extracts
tetraxollum salts (dehydrogenase-activity indicator
reagent). The results show that the method can be
applied successfully to compounds in a crude extract
from plant tissue
Test organism is applied to the TLC plate as a spray Fungitoxic products Van der Sluis and Labadie
suspension of the conidiospores. The tests are (19811
done with and without addition of B_glucosidase Van der Nat el al.
(0.5 mg/mll to the conidial suspension spray (19821
5-6 ml of spray suspension (microorganisms in TSB Compounds from higher Hamburguer and Cordell
culture medium) are sprayed on chromatoplaque plants (19871
20 x 20 cm. Inhibition zones are visualized
with tetrasolium salts

Fungltoxic products are measured qualitatively and Fungitoxic products Peterson and Edgington
quantitatively using a bioautographic technique. The (19891
thin-layer plates are sprayed with a mixture of agar
and microorganism spores. The diamater of the zones
of inhibition is related to the amount of fungitoxic
chemical in the spot
TABLE 3 (continued)

(C) Immersion bioautognaphy. In this method the chromatoplaque must be included in the medium. The developed plaque is covered with fused
agar. The solidified agar is inoculated with the microorganisms and the plates are left at 4OC in the freezer or at room temperature. After 4 h,
the plates are inoculated and incubated.

General methods Sample References

An agar medium, containing TTC as a dehydrogenase Antibiotics Nicolaus et al. in

indicator is seeded with the test microorganism Betina (19731
and is poured over the developed chromatoplaque

The agar medium is sprayed on the dried TLC-plaques. Antibiotics Bickel et al. in
Another agar medium, cooled to 48% and seeded with Betina (19731
a test microorganism, is poured directly over the
surface of the prepared plate. Inhibition zones are
identified after incubation by viewing the opaque
plates directly
The agar medium ls sprayed on the dried TLC-plates. Antibiotics and Wallhauser (19691
After solidification, the plates are seeded with related substances
the microorganism and the growth or inhibition bands
can be viewed using a tetrazollum salt
The developed and dried chromatogam is put in a Antibiotics and Randerath (19741
sterile Petri plate. The plate is covered with derivatives
agar and cooled at room temperature (16 mini and
frozen 1 h at 0 OCfor diffusion. The TTC is included
in the culture medium
The TLC plates are flooded with the agar containing Alkaloids Verpoorte et al.
spores of the microorganism. After a prediffusion (19781
period of 24 h at 4% the plates are incubated and
the inhibition zones are recorded
141
142

of the essential oil against two different microorganisms Wcherichia coli and
Staphylococcus aureus), they obtained contradictory results: the essential oil
was more active against S. aweus by the disk diffusion method but E. coli was
more sensitive when they used the liquid dilution method. Yousef and Tawil
09801 obtained contradictory results in the study of 22 essential oils when they
used hole-plate diffusion or the liquid dilution method.
These methods (disk, hole-plate or cylinder) are not acceptable when the
samples are not highly soluble in water as is the case with essential oils or non-
polar extracts. On the other hand, some water-soluble compounds may have a
higher diffusion power and lower antimicrobial activity.
In most studies (Naqvi et al., 1976; Leven et al., 1979; Emeruwa 1982; Singh
et al., 19831,inhibition zones are compared with those obtained for antibiotics.
This is useful in establishing the sensitivity of the test organism, but a
comparison of the antimicrobial potency of the samples and antibiotics cannot
be drawn from this (Janssen et al., 19871.Some researchers relate MIC values
with inhibition diameters (Ayafor et al., 1982) but there is no relationship
between the two. A WHO committee of experts recommends the use of the dilu-
tion method for MIC determination of pure samples, as such antibiotics, alka-
loids, etc. They propose the application of regression lines that relate inhibition
halos and MIC (Ericsson and Sherris, 19711. However, this method is not
acceptable when the samples are essential oils or complex mixtures from
higher plants.
The advantages of these methods is the small size of the sample used in the
screening and the possibility of testing five or six compounds against a single
microorganism. However, these methods should not be employed when the
sample is lipophilic or to determine the MIC of a sample. In some cases the
diffusion techniques can be used for antimicrobial screening but they can never
be used as a definitive method. The diffusion methods are well-suited for
preliminary screening of pure substances (alkaloids, flavanoids, terpenoids,
etc.). The optimum conditions have been established by Bauer et al. (19661,
Mitscher et al. (19721and WHO (19771to be Mueller-Hinton agar and standard
microorganisms (ATCC or similar). Isolated pathogenic microorganisms should
never be used. Results can be expressed by + (growth) or - (inhibition) and
then compared with dilution methods.

Dilution methods
This includes dilution in a liquid medium and in a solid medium. Both
methods are based on the homogeneous dispersion of the sample in a
microorganism-selective culture medium. These methods are the best when it
is necessary to assay water-soluble or lipophilic samples and to determine the
MIC of compounds (Clark et al., 1981, 1984; Miski et al., 1983; El-Feraly et al.,
1983; Adeoye et al., 19861.
Dilution in liquid medium is the most complicated but also the most precise
technique. This method is recommended for the determination of MIC of a pure
sample and it is the only method for determination of minimum bactericidal
 143

concentration (MBC). MBC is determined by subculture of the tube with

inhibition in a agar plate or liquid medium. When the germ does not grow, the
sample is a microbicide.
Gundiza (19871 has assayed the extracts of crescent polarity of Helinus
integrifolius using the hole-plate diffusion and the tube-assay dilution methods.
All the fractions exhibited activity against Candida albicans when the liquid
dilution method was used. With the hole-plate diffusion method, none of the
fractions exhibited activity against C. albicans. This is due to the fact that plant
extracts frequently have low diffusion properties, while in the test tube dilu-
tion method, the samples are in direct contact with the test microorganism. If
the extract has low solubility in water, at least the suspended particles will still
be in contact with the test organism. To avoid sedimentation of the extract in
the liquid culture medium, shaking during incubation is necessary. Another
advantage to this method is that the antimicrobial activity of plant extracts can
be determined by incorporating the sample emulsified with a surface-agent, such
as Tween 20 or Tween 80 (Allegrini et al., 1973; Yousef and Tawil, 1980; Villar
et al., 1986bl at an ideal percentage of lo%, although this ratio may vary
depending on the sample properties. The stability of the emulsion must remain
constant during the entire assay.
The solid dilution method is comparable to the liquid medium dilution. This
method is quick and time-saving, and the MIC of a product against six
microorganisms can be determined at one time (Mitscher et al., 19721.Baron
and Bruckner (19841 have compared the susceptibility of anaerobic bacteria
using the agar dilution and a microbroth dilution method. The MICs of several
widely-used antibiotics (chloramphenicol, penicilin, clindamycin, etc.) were
determined using 110 anaerobic bacteria. The MICs determined by the two
methods were in general similar.
Dilution and diffusion methods have also been compared. Gabrielyan et al.
0985) studied the sensitivity of eight antibiotics against 200 strains of
Pseudomonas aeruginosa isolated from hospitalized patients, and they obtained
similar results with the two methods.
Using the dilution agar method, Mitscher et al. (19871have screened more
than 1000 extracts from higher plants and found 26% of these to be active. Of
the various testing procedures, we believe that this is the most convenient one
for a small laboratory because it is very difficult to prepare sterile plant
extracts without the use of autoclaving or other extreme conditions. In this
technique, it is not necessary that the samples be sterile because aerobic
organisms do not develop well under the solidified agar. The occasional
contaminating culture which develops on the surface of the agar is no problem
because it can be easily recognized. The Mitscher method establishes the
quantity of sample necessary, which cannot be over 1 mg of sample in 1 ml of
culture medium. The active samples are then re-assayed at a concentration of
0.1 mglml, so that in the case of extracts with only small amounts of antibiotic
substances these samples will be inactive and eliminated. On the other hand,
most of the clinically used antibiotics are active at a concentration of 10 pg/ml.
144

Therefore, if a pure substance is not active at 100 pg/ml, it probably will not be
clinically useful. Plant extracts that are active at 100 rglml have a good potency
level and, depending upon the general chemical nature of the component
responsible for activity, the subsequent purification technique can be decided
upon.
Some researchers (Allegrini et al., 1973; Villar et al., 19871used a multipoint
inoculation system. With this method, about 20-25 microorganisms can be
inoculated in a standardized plate.
The agardilution method is applicable to polar or non-polar samples. When
the sample is lipophilic, e.g. an essential oil, the inclusion can be made as an
emulsion. In this case, the emulsion will usually remain stable until its inclusion
in the medium but then may break down during the assay (different with dilu-
tion liquid medium). In our work with essential oils and non-polar extracts, we
have tested the stability and inocuous nature of a variety of emulsification
agents and our best results were obtained with Tween 20 and Tween 80. This
conclusion is similar to the findings of other authors (Allegrini et al., 1973; Pelle-
cuer et al., 1976; Yousef and Tawil 19801.This method, therefore, seems to be
best when the sample to be tested is a complex extract.

Bioautography
In the study of biologically active compounds from natural sources, it is
evident that rapid and efficient detection of such compounds is a critically
important aspect of the discovery process. Because of the complexity of plant
extracts, relatively few studies have dealt with the isolation of antibiotics from
higher plants. Bioautography is a method that makes it possible to localize
antimicrobial activity on a chromatogram. Contact bioautography is the type
most often employed but it presents certain difficulties and requires the use of
suitable microbiological equipment. The problem of the differential diffusion of
compounds from the chromatogram to the agar plate is simplified by direct
bioautographic detection (Homans and Fuchs, 1970; Lund and Lyon, 19751,but
this method also requires more complex microbiological equipment. Immersion
bioautography is also based on the diffusion of separated compounds and for a
small laboratory is the most appropriate method because it is not affected by
possible contamination.
In both immersion and direct bioautography, inhibition zones are observed
directly on the TLC plate. Comparison with a chromatogram developed under
identical conditions and visualized with an appropriate chromogenic reagent
may provide extremely useful information about the chemical nature of the
active principles. In the method described by Rios et al., (19871 double
chromatography is not necessary, because with the use of Alufolien (Merck)
specific strips of the developed chromatograph (in band) can be revealed with
different reagents (Rios et al., 19861.The fused agar should be added at 50°C
because the SiO, layer can fall down if the temperature is higher.
 145

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