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Journul of EthnophaImacology, 23 (1988) 127- 149 Elsevier Scientific Publishers Ireland Ltd.
127
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, 19821 and 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.
TA
BL
E
1
DIF
FU
SIO
N
ME
TH
OD
S
(Al
Dis
k m
etho
d.
Its
esse
ntia
l fe
atur
e is
the
pla
cing
of
filt
er p
aper
dis
ks w
ith
the
anti
biot
ic
on t
he s
urfa
ce o
f ag
ar i
mm
edia
tely
af
ter
inoc
ulat
ion
with
the
org
anis
m
test
ed.
Und
ilute
d ov
erni
ght
brot
h cu
ltur
es
shou
ld
neve
r be
use
d as
an
inoc
ulum
. R
outi
ne
dire
ct
appl
icat
ion
of d
isks
to
pla
tes
seed
ed
with
cl
inic
al m
ater
ial
is n
ot r
ecom
men
ded
beca
use
of p
robl
ems
with
in
ocul
um
cont
rol
and
mix
ed
cult
ures
. T
his
tech
niqu
e w
as
orig
inal
ly
stan
dard
ized
by
Bau
er e
t al
. (1
9661
and
by E
rics
on
and
Sher
ris
(197
1) a
nd t
hen
chan
ged
in t
he R
epor
t of
the
WH
O (
1977
1.
Gen
eral
met
hods
Sa
mpl
es
Ref
eren
ces
Filt
er p
aper
dis
ks (
Wha
tman
no.
2 o
f 6.
5 m
m i
n di
amet
erl’
satu
rate
d w
ith a
ntib
ioti
c so
luti
on
are
plac
ed o
n th
e su
rfac
e of
a b
lood
aga
r se
eded
w
ith a
clin
ical
spe
cim
en
(tw
o di
sks/
plat
e)
Pen
icill
in
and
othe
r an
tibi
otic
s at
the
ti
me
of p
rim
ary
isol
atio
n A
queo
us
and
MeO
H e
xtra
cts
Bon
di e
t al
. (1
9471
Aja
o et
al.
(198
41
Smal
l st
erile
di
sks
(6 m
m i
n di
amet
er1
are
moi
sten
ed
and
plac
ed o
n Sa
bour
aud’
s m
alto
se
agar
pl
ates
whi
ch h
ad p
revi
ousl
y be
en s
eede
d w
ith
2 m
l of
the
fun
gi i
n br
oth
cult
ure
or 1
ml
of t
he b
roth
cu
ltur
e of
the
org
anis
m (
bact
eria
1
Sim
ilar,
but
the
oil
to b
e te
sted
is
dilu
ted
with
E
tOH
(29
% v
/v1
Ess
enti
al o
ils d
ilute
d w
ith T
wee
n 59
0.5
% (
v/v)
; di
sks
are
dipp
ed i
n so
luti
on o
f 1:
5&l
: 199
and
12
99
The
oil
is m
ixed
with
T
wee
n 80
at
a ra
tio
of 0
.5:
199
and
this
mix
ture
is
dilu
ted
with
aq.
EtO
H 1
0.59
an
d 19
9% (
v/v)
Aga
r su
rfac
e is
see
ded
with
cl
inic
al s
peci
men
; 6
4 of
eac
h es
sent
ial
oil
are
adde
d to
the
dis
k;
6-
12 d
isks
are
pla
ced
on e
ach
plat
e
Ess
enti
al
oils
Ess
enti
al
oils
M
anun
ta e
t al
. (1
9871
Ess
enti
al
oils
G
arg
and
Kas
era
(198
31
Ess
enti
als
oils
Ess
enti
al
oils
Mar
usse
lla
and
Lig
uori
(19
581
Mar
usse
lla
and
Hen
ry
(195
81
Pra
sad
et a
l. (1
9881
Bat
ra a
nd M
ehta
(19
851
Val
net
et a
l. (1
9781
TA
BL
E 1
(c~
t~n
~d
~
Gen
eral
met
hod
s S
ampl
es
Ref
eren
ces
0.2
ml o
f ba
cter
ial
brot
h c
ult
ure
is
adde
d to
th
e su
rfac
e of
th
e ag
ar. D
isk
s ar
e sa
tura
ted
wit
h t
he
oil
and
plac
ed i
n t
he
cen
ter
of t
he
inn
er s
urf
ace
of t
he
Pet
ri d
ish
cov
er.
Wh
en t
he
plat
es a
re i
nve
rted
an
d in
cuba
ted,
th
e di
sks
are
at a
dis
tan
ce o
f ab
out
8 m
m f
rom
th
e su
rfac
e gr
owth
of
the
mic
roor
gan
ism
. Th
e pr
esen
ce o
f a
clea
r xo
ne
on t
he
surf
ace
of t
he
agar
abo
ve t
he
disk
is i
ndi
cati
ve o
f an
tiba
cter
ial a
ctiv
ity.
Ess
enti
al o
il v
apor
s M
aru
xxel
la a
nd
Sic
ure
lla
&M
O1
A s
trai
n o
f B
adlt
ls
sub
tih
is
th
e or
gan
ism
te
sted
. 26.
0 m
l of
Bac
toS
trep
tom
ycin
aga
r ar
e ad
ded
to P
etri
dis
hes
. Wh
ile
the
plat
es a
re s
till
war
m,
4.0
ml
of s
eede
d ag
ar a
re a
dded
an
d di
stri
bute
d ov
er t
he
surf
ace.
A c
once
ntr
atio
n o
f ap
prox
imat
ely
250.
000
spor
es/m
l in
th
e se
eded
aga
r gi
ves
the
desi
red
resu
lts.
Sam
ples
are
dil
ute
d in
ph
osph
ate
buff
er (
pH 7
.91t
0.2
Ml
and
subs
equ
ent
dilu
tion
s w
ith
0.
1 M
bu
ffer
. 0.9
08 m
l sa
mpl
e is
pip
ette
d on
to
filt
er p
aper
dis
ks
(4-6
di
sks/
plat
e1
Mu
elle
r-H
i&on
med
ium
is u
sed.
Sam
ples
are
dis
solv
ed
in w
ater
, MeO
H o
r m
ixtu
res
of b
oth
. In
ocu
lum
sh
ould
be
dilu
ted
at l
east
lo-
fold
or
pref
erab
ly t
o a
den
sity
equ
ival
ent
to t
he
bari
um
su
lph
ate
stan
dard
D
isk
s are
impr
egn
ated
wit
h 0
.602
ml o
f ea
ch so
luti
on
Equ
ilib
rati
on at
4 “
C, b
efor
e in
cuba
tion
Sim
ilar
, bu
t th
e ex
trac
ts a
re s
olu
bili
zed
in
phos
phat
e bu
ffer
. D
isk
s ar
e so
aked
wit
h a
sol
uti
on
of 4
mg
extr
act/
ml
Dis
ks
are
impr
egn
ated
wit
h 0
.1 m
l of
eac
h e
xtra
ct.
Equ
ilib
rati
on a
t 5°
C f
or 4
4 -
55 m
in
Str
epto
myc
in
Alc
ohol
ic e
xtra
cts
Ext
ract
s of
cre
scen
t
pola
rity
E
sen
tial
oil
s
An
tibi
otic
s B
auer
et
al. (
1966
1 E
xtra
cts
of c
resc
ent
WH
O (
1977
1 po
lari
ty
Bar
baga
llo
and
Ch
isar
i (19
871
Fla
von
oids
T
erpe
noi
ds
Ext
ract
s of
cre
scen
t po
lari
ty
Wat
er/M
eOH
ext
ract
s an
d fl
avon
oids
isol
ated
Loo
et
al. (
1945
1 B
oss
et a
l. (1
98O
bl
Sin
gh e
t al
. (19
831
Ros
s et
al.
(198
Oal
Waa
ge a
nd
Hed
in (
1985
1 M
ath
ur
and
Gon
zale
z (1
9821
Em
eru
wa
(198
21
Har
sh a
nd
Nag
(19
841
The
pla
tes
are
allo
wed
to
sta
nd a
t ro
om t
empe
ratu
re
for
30 m
in a
nd t
hen
are
incu
bate
d
Ess
enti
al
oil
is d
ilute
d w
ith
MeO
H a
nd p
aper
dis
ks
are
impr
egna
ted
with
0.0
1 m
l of
the
se
solu
tion
s
Ba&
ti
8ubt
ilis
is
gro
wn
in t
wo
diff
eren
t m
edia
. F
ilter
pap
er d
isks
ar
e di
pped
in
a s
obrt
ion
of t
he c
ompo
und
in E
tOH
/wat
er
(1: l
), dr
ied
for
10
min
te
rem
ove
the
EtO
H a
nd p
lace
d in
the
aga
r pl
ate.
The
Pet
ri d
ishe
s ar
e pr
einc
ubat
ed
at 5
X
for
12 h
to
perm
it t
he m
axim
um
diff
usio
n of
the
dr
ug.
Aft
er
this
, th
e pl
ates
ar
e in
cuba
ted
0.2
ml
of t
he,a
queo
us
susp
ensi
on
of s
pore
s an
d m
ycel
ial
frag
men
ts
are
pipe
ted
on t
he s
urfa
ce o
f ag
ar p
late
s (S
abou
raud
’s g
luco
se
agar
), T
wo
hour
s af
ter
inoc
ulat
ion
the
surf
aces
of
the
pla
tes
are
cove
red
with
pap
er d
isks
(W
hatm
an n
o. 1
of
9 m
m)
and
they
are
soa
ked
for
2 s
in t
he a
queo
us
solu
tion
s of
the
tes
ted
subs
tanc
es
0.1
ml
of t
he o
rgan
ism
cul
ture
is
add
ed t
o a
fres
h un
solid
aga
r m
ediu
m a
nd t
he m
ixtu
re
is p
oure
d on
to
agar
med
ium
in
Pet
ri d
ishe
s.
Dis
ks
of 6
.3 m
m i
n di
amet
er o
n w
hich
0.6
-20
c(g o
f a
test
co
mpo
und
are
appl
ied.
The
ant
imic
robi
al a
ctiv
ity
is
expr
esse
d in
ter
ms
of m
inim
um i
nhib
itio
n co
ncen
trat
ion
bgld
isk)
1 g
of c
rush
ed pl
ant
mat
eria
l is
add
ed t
o 5
ml
of
95%
EtO
H,
Pap
er d
isks
, 7
mm
in
diam
eter
, ar
e al
low
ed t
o so
ak i
n th
e E
tOH
ext
ract
ove
rnig
ht.
Bef
ore
sssa
y th
e di
sks
are
rem
oved
an
d ai
r-dr
ied
in s
teri
le
Pet
ri
dish
es
in t
he d
ark
Alk
aloi
ds
Ess
enti
al
oils
and
th
eir
frac
tion
s
EtO
H e
xtra
cts
and
sesq
uite
rpen
ic
acid
Alk
aloi
ds
Qui
none
s
EtO
H e
xtra
cts
Aya
for
et a
l. (1
982)
Yaa
phe
et a
l. (1
979)
Baa
and
Alv
arez
11
9811
Hej
tman
kova
et
al.
(198
41
Tab
ats
et a
l. (1
982)
Wat
et
al.
(198
01
TA
BL
E 1
(co
ntiw
ed)
(B)
Hol
epla
te
assa
y m
etho
d.
Thi
s m
etho
d de
pend
s up
on t
he d
iffu
sion
of
the
ant
ibio
tic
from
a v
erti
cal
hole
thr
ough
th
e so
lidif
ied
agar
lay
er o
f a
g
Pet
ri
dish
or
plat
e to
suc
h an
ext
ent
that
gr
owth
of
the
ad
ded
mic
roor
gani
sm
is p
reve
nted
en
tire
ly
in a
cir
cula
r ar
ea o
r zo
ne a
roun
d th
e ho
le
cont
aini
ng a
sol
utio
n of
the
ant
ibio
tic.
Gen
eral
met
hods
Sa
mpl
es
Ref
eren
ces
Sam
des
are
diss
olve
d w
ith
dist
illed
w
ater
. ph
osph
ate
buff
ers
of d
iffe
rent
pH
, or
MeO
H.
Mue
ller-
H&
on
or S
abou
raud
aga
r is
pou
red
into
pr
e-st
erili
aed
Pet
rf d
ishe
s.
Aft
er
cong
ealin
g,
the
agar
med
ium
is
hom
ogen
eous
ly
inoc
ulat
ed
with
a
cult
ure
of t
he t
est
orga
uism
. Si
x ho
les
(12
mm
in
diam
eter
) ar
e du
g w
ith a
fla
med
cor
k bo
rer
and
asep
tica
lly
fille
d w
ith 0
.02
ml o
f ea
ch s
olut
ion
to b
e te
sted
. T
he p
late
s ar
e fe
ft a
t 4%
fo
r 1-
2
h an
d th
en a
re i
ncub
ated
Ant
ibio
tics
A
lkal
oids
M
eOH
and
aqu
eous
ex
trac
ts
USP
XIX
(19
751
Vlll
ar e
t al
. (1
988a
l A
desi
na
and
Akl
nwus
i (1
9841
Sim
ilar,
but
the
hol
e is
fill
ed w
ith 0
.2 m
l of
eac
h ex
trac
t.
The
lip
ophi
lic f
ract
ions
ar
e di
ssol
ved
in
PE
G 4
991p
hosp
hate
buf
fer
(4:6
1 pH
7.4
and
phy
siol
o-
gic
buff
er f
or h
ydro
phyl
ie
frac
tion
s
Idem
. P
EG
409
1pho
spha
te b
uffe
r at
a r
atio
of
1: 1
Fiv
e w
ells
of
1 c
m i
n di
amet
er,
0.1
ml
of t
he
frac
tion
s ar
e pl
aced
in
each
wel
l
20 m
l of
Dif
co A
ntib
ioti
c M
ediu
m 2
are
ove
rlai
d w
ith 5
ml
of D
ifco
Ant
ibio
tic
Med
ium
1 p
revi
ousl
y in
ocul
ated
. F
our
wel
ls
of 1
6 m
m i
n di
amet
er
whi
ch
rece
ived
0.3
ml
of t
he s
ubst
ance
s to
be
test
ed
0.2
ml
of e
ach
extr
act
are
care
fully
ad
ded
to t
he
cups
and
allo
wed
to
dif
fuse
at
room
tem
pera
ture
fo
r 2
h
Ext
ract
of
cre
scen
t po
lari
ty
Ext
ract
s of
cre
scen
t po
lari
ty
EtO
H e
xtra
cts
and
thei
r fr
acti
ons
Van
Hoo
f et
al.
(198
01
Van
Hoo
f et
al.
(198
31
MeO
H e
xtra
cts
Lyn
ch-B
rath
w~t
e et
at.
(197
51
EtO
H e
xtra
cts
Xan
thon
es
CH
CI,
and
MeO
H
extr
acts
Lev
en
et a
l. (1
9791
G
undi
dsa
(198
6, 1
9871
Deb
ro a
nd W
ard
(197
91
Sund
aram
et
al.
(198
31
Far
ouk
et a
l. (1
9831
Pla
tes
for
assa
y ar
e un
ifor
mly
pre
pare
d by
see
ding
st
erile
, pa
rtia
lly
cool
ed,
mol
ten
agar
with
di
luti
ons
of t
est
mic
roor
gani
sm
grow
n in
bro
th o
r su
spen
sion
s of
con
idia
. T
he s
eede
d ag
ar m
ediu
m i
s po
ured
int
o st
erile
P
etri
dis
hes.
H
oles
hav
ing
a di
amet
er
of 1
1 m
m
are
fiie
d w
ith 1
00 p
l of
a s
olut
ion
or s
uspe
nsio
n of
an
extr
act,
fr
acti
on o
r pu
re c
ompo
und
Ext
ract
s an
d fl
avon
oids
al
kalo
ids
isol
ated
F
lavo
noid
s
Huf
ford
et
al.
(197
5.19
801
Huf
ford
and
Las
swel
l (1
9781
The
alk
aloi
ds a
re d
isso
lved
in
a b
uffe
r m
ade
up o
f eq
ual
part
s of
1%
ace
tic
acid
and
1%
sod
ium
ace
tate
in
wat
er
(pH
5.5
1
Alk
aloi
ds
Ver
poor
te
et a
l. (1
9781
Sam
ples
are
dis
solv
ed
in c
itra
te-p
hosp
hate
bu
ffer
pH
4.0
E
tOH
ext
ract
s
The
ext
ract
s ar
e di
ssol
ved
in t
he s
ame
extr
acti
ve
solv
ent
The
sol
vent
is
a m
ixtu
re
of a
caci
a gu
m i
n w
ater
4-
5%
The
res
idue
fro
m e
ach
extr
act
is t
reat
ed
with
di
still
ed
wat
er
to m
ake
50,2
5,20
,10
and
5%
solu
tion
s (w
/w).
Thr
ee d
rops
of
each
ext
ract
in
ea
ch w
ell.
Equ
ilibr
atio
n at
roo
m t
empe
ratu
re
Ext
ract
s of
cre
scen
t po
lari
ty
Ext
ract
s of
cre
scen
t po
lari
ty
Aqu
eous
an
d al
coho
lic
extr
acts
Van
Bee
k et
al.
(198
4al
Alm
agbo
ul
et a
l. (1
9851
Ikra
m a
nd I
nam
ul-H
aq (
1980
1
Aja
o et
al.
(198
51
Cl
Cyl
iude
r m
etho
d.
Thi
s m
etho
d is
sim
ilar
to t
he h
ole-
plat
e m
etho
d.
Stai
nles
s st
eel
or p
orce
lain
cy
linde
rs
are
used
for
ass
ay.
Aft
er
incu
bati
on,
the
cylin
ders
ar
e re
mov
ed,
and
the
aver
age
diam
eter
of
eac
h zo
ne o
f gr
owth
in
hibi
tion
is
mea
sure
d an
d re
cord
ed.
Gen
eral
met
hods
Sa
mpl
es
Ref
eren
ces
Six
cylin
ders
are
dis
trib
uted
on
the
Mue
ller-
H
into
n ag
ar.
0.1
ml
of t
he a
ntib
ioti
c on
eac
h cy
linde
r. T
he s
olve
nt
can
be w
ater
, M
eOH
. or
ph
osph
ate
buff
ers
of d
iffe
rent
pH
Ant
ibio
tics
P
olar
ext
ract
s H
ydro
acet
onic
ex
trac
ts
Ext
ract
s of
cre
scen
t po
lari
ty
USP
XIX
(19
751
Lau
rens
et
al.
(198
21
Mou
rey
et a
l. (1
9851
B
arba
gallo
et
al.
(198
21
TA
BL
E
1 (c
onti
nued
)
Gen
eral
met
hods
Sa
mpl
es
Ref
eren
ces
Inoc
ula
are
prep
ared
in
liqui
d A
ntib
ioti
c M
ediu
m
no.
3 K
lifeo
). 0
.2 m
l of
the
ino
culu
m d
ilute
d ar
e se
eded
an
d m
ixed
with
10
ml
of A
ntib
ioti
c M
ediu
m
no.
1 @
life0
1 and
10
ml
of A
ntib
ioti
c M
ediu
m n
o. 1
1 (D
ifco
) and
mai
ntai
ned
at 4
5% i
n a
ther
mos
tati
c ba
th.
Ster
ile
cylin
ders
(b
/pla
te)
are
disp
osed
on
the
aga
r su
rfac
e of
the
coo
led
and
drie
d P
etri
dis
hes.
0.
15 m
l of
ext
ract
ar
e ad
ded
on e
ach
cylin
der.
T
he s
ame
met
hod
is u
sed
for
dete
rmin
ing
the
anti
fung
al a
ctiv
ity
usin
g Sa
bour
aud-
mal
tose
ag
ar.
EtO
H e
xtra
cts
EtO
H e
xtra
cts
and
flav
onoi
ds
isol
ated
Gut
hind
et
al.
(196
1)
PaI
acio
s et
al.
(193
3)
Aro
mat
ic
chem
ical
va
pors
M
aruz
zeB
a et
al.
(196
1)
0.5
mi
of t
he a
rom
atic
che
mic
al a
re p
lace
d in
the
ce
nter
of
the
Pet
ri d
ish
top.
Whe
n th
e di
shes
ar
e in
vert
ed
and
incu
bate
d,
the
surf
ace
of t
he s
ubst
ance
in
the
cup
is
abou
t 5
mm
fro
m t
he a
gar
surf
ace.
V
apor
s of
the
che
mic
al a
re a
llow
ed
to e
man
ate
thro
ugho
ut
the
peri
od o
f in
cuba
tion
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
TA
BL
E 2
DIL
UT
ION
ME
TH
OD
S
(A)
“Tu
be”
aw
ay
or t
urb
idim
etri
c m
eth
od.
It i
s ba
sed
on t
he
hom
ogen
ous
disp
ersi
on o
f a
sam
ple,
dis
solv
ed i
n p
uri
fied
wat
er,
MeO
H,
wat
er/
MeC
H m
ixtu
res,
ph
osph
ate
buff
ers,
in
th
e br
oth
cu
ltu
re r
equ
ired
for
th
e or
gan
ism
ass
ayed
. A
fter
in
cuba
tin
g 3-
4 h
. tr
ansm
itta
nce
or
abso
rb
ante
is r
ead
in a
su
itab
le s
pect
roph
otom
eter
fit
ted
wit
h a
530
nm
fil
ter
WS
P X
IX,
1975
; PE
, 19
711.
Gen
eral
met
hod
s S
ampl
es
Ref
eren
ces
0.25
g o
f ex
trac
t/l0
ml
of s
teri
le w
ater
are
se
rial
ly d
ilu
ted
to g
ive
the
desi
red
con
cen
trat
ion
s.
1 m
l of
a se
rial
ly d
ilu
ted
plan
t ext
ract
is
adde
d.
Th
e tu
bes
are
incu
bate
d an
d m
easu
red
spec
trop
hot
o-
met
rica
lly
Alc
ohol
ic e
xtra
cts
Tw
aij
et a
l. (1
986)
Ess
enti
al o
il i
s di
lute
d w
ith
Tw
een
20
at a
rat
io
of 1
0%
Ess
enti
al o
ihl’w
een
20
at a
rat
io o
f 1:
8 (w
/w).
T
he
solu
tion
is s
eria
lly
dilu
ted
twic
e in
ste
rile
br
oth
Ess
enti
al o
ihl’w
een
80
at a
rat
io 6
:4
Tw
o-fo
ld d
ilu
tion
in b
roth
. Th
e co
nce
ntr
atio
n o
f pu
re c
ompo
un
ds in
th
e in
itia
l tu
be i
s 50
pg/
ml
10 m
g of
eac
h a
lhal
oid
are
susp
ende
d in
10
ml
of
wat
er. 5
ml
of e
ach
dil
uti
on a
re d
ilu
ted
to 2
5 m
l w
ith
cu
ltu
re m
ediu
m
Ess
enti
al o
ils
Fou
rnie
r et
al.
(19’
781
Ess
enti
al o
ils
You
sef
and
Taw
il(1
980)
Ess
enti
al o
ils
Fla
von
oids
Alk
aloi
ds
Ch
alch
at e
t al
. (1
9871
Hu
ffor
d an
d L
assw
ell
(197
81
Lu
mon
adio
et
al. (
1986
1
tB) A
gar
dlbt
tion
met
hod.
T
he s
ampl
e is
dis
solv
ed
or s
uspe
nded
in
an
appr
opri
ate
solv
ent
and
mix
ed w
ith a
gar
med
ium
. T
he r
esul
ts
obta
ined
w
ith t
his
met
hod
are
equi
vaIe
nt
to t
hose
obt
aine
d w
ith d
iffu
sion
and
diiu
tion
met
hods
.
Gen
eral
met
hods
Sa
mpl
es
Ref
eren
ces
10 m
g ex
trac
t/92
ml
solv
ent
(wat
er,
MeO
H,
acet
one
or o
ther
sol
vent
w
ithou
t an
timic
robi
al
activ
ity
at
a f
ina
l co
ncen
trat
ion
of 2
%).
Bef
ore
cong
ealin
g,
10 m
l of
Mue
Ber
-Hin
~n
agar
is
adde
d as
eptic
ally
to
eac
h of
the
plat
es a
nd t
hey
are
swir
led
care
iirlly
un
til t
he a
gar
begi
ns t
o se
t. T
he a
ctiv
e ex
trac
ts
are
re-a
ssay
ed
at a
con
cent
ratio
n of
100
ccg
hnl.
Thi
s m
etho
d ca
n be
em
ploy
ed i
n th
e M
IC d
eter
min
atio
n.
Sim
ilar
to t
he M
isch
er m
etho
d ab
ove,
but
app
lied
to
esse
ntia
l oi
Is. T
wee
n 8O
less
entia
l oi
l at
a r
atio
15
. T
he m
ixtu
re
is d
ilute
d w
ith t
he a
gar
untiI
diI
utio
ns o
f l/l
00
to l
/800
.
Ext
ract
s ar
e ad
ded
to t
he c
ultu
re
med
ium
at
a
conc
entr
atio
n of
100
mg
of d
ried
pla
nt/m
l m
ediu
m.
If t
he a
ssay
is
posi
tive,
dos
es o
f 10
.5,4
.2
and
1 m
g/m
I mus
t be
ass
ayed
.
Milt
Sab
oura
ud
sgar
mix
ed w
ith t
he e
xtra
cts
or
com
poun
ds,
shak
en,
and
20 m
l of
thi
s m
ediu
m
are
put
in t
he p
late
s
Phos
phat
e bu
ffer
tpH
7.4
) to
mak
e a
solu
tion
at a
ra
tio1
: 1 (w
/w).
1 m
l of e
ach
extr
act
is a
dded
to
each
ste
rile
pl
ate
with
aga
r m
ediu
m o
r bl
ood
agar
bef
ore
cong
eahn
g
MeO
H e
xtra
cts
AIk
aIoi
ds,
flav
onoi
ds
Alk
aloi
ds
CH
Cl,
and
MeO
H
Sequ
iterp
enes
Ess
entia
l oi
ls
Eth
eric
, E
tOH
and
wat
er
extr
acts
Wat
er e
xtra
cts
and
isol
ated
an
thra
quin
ones
Aqu
eous
ext
ract
s
Mits
cher
et
al.
(197
2)
Mits
cher
et
al.
(197
51,
(198
0)
Al-
Sham
ma
et a
l. (1
9811
(1
982)
R
ios
et a
l. (1
987)
C
aBad
a et
al.
(198
01
Vi&
r et
al.
(198
8b)
Bia
rd e
t al
. (1
980)
Fuze
hier
et
al.
(198
2)
El-
Said
(19
71)
TA
BL
E 3
BIO
AU
TO
GR
AP
HIC
ME
TH
OD
S
(A)
Co
nta
ct
bio
au
tog
rap
hy
. It
is
base
d on
th
e di
ffu
sion
of
sepa
rate
d co
mpo
un
ds b
y T
LC
or
PC
fro
m s
hee
ts o
r cb
rom
atop
laqu
es.
Th
ese
are
plac
ed o
n t
he
surf
ace
of l
arge
nu
trie
nt
agar
pla
tes
inoc
ula
ted
wit
h m
icro
orga
nis
ms
that
are
sen
siti
ve t
o th
e an
tibi
otic
s be
ing
anal
yzed
. A
fter
15
-36
min
, th
e sh
eets
or
chro
mat
opla
ques
are
rem
oved
. In
bot
h i
nst
ance
s, a
nti
biot
ics
diff
use
in
to t
he
agar
lay
er a
nd
inh
ibit
th
e gr
owth
of
the
test
m
icro
orga
nis
ms.
Th
e pl
ates
are
th
en i
ncu
bate
d at
an
app
ropr
iate
tem
pera
ture
un
til
a th
in f
ilm
of
the
grow
ing
mic
roor
gan
ism
s is
vis
ible
on
th
e su
rfac
e. Z
ones
of
inh
ibit
ion
are
th
en c
lear
ly v
isib
le.
Inh
ibit
ion
zon
es c
an b
e m
ade
mor
e co
nsp
icu
ous
and
visi
ble
earl
ier
by u
sin
g de
hyd
roge
nas
e-
acti
vity
in
dica
tors
.
Gen
eral
met
hod
s S
ampl
e R
efer
ence
s
Pet
ri d
ish
es w
ith
a c
ult
ure
med
ium
are
see
ded
wit
h a
cel
l or
spo
re s
usp
ensi
on in
th
e ap
prop
riat
e li
quid
med
ium
an
d th
e T
LC
-pla
tes
are
plac
ed o
n t
he
agar
su
rfac
e. T
he
TL
C-p
late
s ar
e re
mov
ed,
and
Pet
ri d
ish
es a
re i
ncu
bate
d. I
nh
ibit
ion
zon
es o
f th
e se
para
ted
anti
biot
ics
are
obse
rved
An
tibi
otic
s
CH
Cl,
and
MeO
H e
xtra
cts
Alk
aloi
ds
Bic
kel
et
al. i
n
Bet
ina
(197
31
Rao
et
al. (
1982
1 A
l-S
ham
ma
et a
l. (1
982)
Th
e P
etri
dis
hes
wit
h t
he
chro
mat
opla
ques
are
fr
ozen
at
4%.
Th
e pl
ate
is r
emov
ed f
rom
th
e ag
ar a
fter
24
h a
nd
then
th
e P
etri
dis
h is
in
cuba
ted.
Bu
ffer
ed W
hat
man
pap
er n
o. 1
is p
lace
d on
th
e ch
rom
atop
laqu
e an
d pr
esse
d. A
fter
15
min
it is
w
ith
draw
n a
nd
plac
ed o
ver
the
agar
-in
ocu
late
d su
rfac
e. T
hen
th
e pl
ate
is i
ncu
bate
d. T
he
inh
ibit
ion
zon
es c
orre
spon
d to
th
e ac
tive
su
bsta
nce
s se
para
ted
by c
hro
mat
ogra
phy
Ext
ract
s an
d se
squ
i-
terp
ene
com
pou
nds
E
xtra
cts
of c
resc
ent
pola
rity
A
lkal
oids
An
tibi
otic
s an
d re
late
d su
bsta
nce
s
McC
alli
on e
t al
. 119
82)
Zah
ir S
hah
et
al. (
1986
1
Ver
poor
te e
t al
. (19
82,1
983)
V
an B
eek
et
al. (
1984
bJ98
5)
Wal
lhii
use
r (19
69)
Alu
foli
en (M
erck
) is
use
d. T
he
chro
mat
ogra
m is
cu
t in
str
ips
(2.5
-5
mm
1 an
d pl
aced
on
th
e ag
ar s
urf
ace
prev
iou
sly
inoc
ula
ted
wit
h fu
ngi
spo
res.
Th
ere
is n
o gr
owth
of
myc
eliu
m o
n t
he
stri
ps w
ith
fu
ngi
cida
l su
bsta
nce
s
Fu
ngi
cide
s C
HC
$ ex
trac
ts
Wol
ters
(19
691
Wol
ters
an
d E
iler
t (1
981)
(Bl
Dir
ect
bioa
uto
grap
by.
A m
icro
orga
nis
m s
usp
ensi
on i
n l
iqu
id m
ediu
m i
s sp
raye
d on
a d
evel
oped
ch
rom
atop
laqu
e af
ter
rem
ovin
g th
e so
l-
ven
ts.
It i
s th
en in
cuba
ted.
Wit
h t
his
met
hod
, th
ere
is n
o di
ffu
sion
an
d th
e pr
oble
ms
of t
he
con
tact
met
hod
are
eli
min
ated
.
Gen
eral
met
hod
s S
ampl
e R
efer
ence
s
Aft
er l
ocat
ing
UV
-abs
orpt
ion
spo
ts,
the
chro
mat
ogra
ms
are
spra
yed
wit
h a
con
idia
l su
spen
sion
of
the
test
fu
ngu
s in
a b
roth
med
ium
. Aft
er s
pray
ing,
th
e th
in-
laye
r pl
ates
are
in
cuba
ted.
In
hib
itio
n z
ones
in
dica
te
pres
ence
of
the
orig
inal
fu
ngi
toxi
c pr
odu
ct
Fu
ngi
toxi
c pr
odu
cts
Hom
ans
and
Fu
chs
(197
01
TL
C l
s sp
raye
d w
ith
a s
usp
ensi
on o
f th
e ge
rm i
n T
SB
. In
hib
itio
n z
ones
are
in
ten
sifi
ed b
y th
e u
se o
f te
trax
ollu
m s
alts
(de
hyd
roge
nas
e-ac
tivi
ty i
ndi
cato
r re
agen
t). T
he
resu
lts
show
th
at t
he
met
hod
can
be
appl
ied
succ
essf
ull
y to
com
pou
nds
in
a c
rude
ext
ract
fr
om p
lan
t ti
ssu
e
Aqu
eou
s an
d M
eOH
ex
trac
ts
Lu
nd
and
Lyo
n (
1975
1
Tes
t or
gan
ism
is a
ppli
ed t
o th
e T
LC
pla
te a
s a
spra
y su
spen
sion
of
the
con
idio
spor
es.
Th
e te
sts
are
don
e w
ith
an
d w
ith
out
addi
tion
of
B_g
luco
sida
se
(0.5
mg/
mll
to
the
con
idia
l su
spen
sion
spr
ay
5-6
ml
of s
pray
su
spen
sion
(mic
roor
gan
ism
s in
TS
B
cult
ure
med
ium
) are
spr
ayed
on
ch
rom
atop
laqu
e 20
x
20 cm
. In
hib
itio
n z
ones
are
vis
ual
ized
w
ith
tet
raso
liu
m s
alts
Fu
ngl
toxi
c pr
odu
cts
are
mea
sure
d qu
alit
ativ
ely
and
quan
tita
tive
ly u
sin
g a
bioa
uto
grap
hic
tec
hn
iqu
e. T
he
thin
-lay
er p
late
s ar
e sp
raye
d w
ith
a m
ixtu
re o
f ag
ar
and
mic
roor
gan
ism
spo
res.
Th
e di
amat
er o
f th
e zo
nes
of
in
hib
itio
n is
rel
ated
to
the
amou
nt
of f
un
gito
xic
chem
ical
in th
e sp
ot
Fu
ngi
toxi
c pr
odu
cts
Com
pou
nds
from
hig
her
pl
ants
Fu
ngi
toxi
c pr
odu
cts
Van
der
Slu
is a
nd
Lab
adie
(1
9811
V
an d
er N
at e
l al
. (1
9821
Ham
burg
uer
an
d C
orde
ll
(198
71
Pet
erso
n a
nd
Edg
ingt
on
(198
91
TA
BL
E 3
(co
ntin
ued)
(C) I
mm
ersi
on
bioa
utog
naph
y.
In t
his
met
hod
the
chro
mat
opla
que
mus
t be
inc
lude
d in
the
med
ium
. T
he d
evel
oped
pl
aque
is
cove
red
with
fu
sed
agar
. T
he s
olid
ifie
d ag
ar i
s in
ocul
ated
w
ith
the
mic
roor
gani
sms
and
the
plat
es
are
left
at
4O
C in
the
fre
ezer
or
at
room
tem
pera
ture
. A
fter
4
h,
the
plat
es a
re i
nocu
late
d an
d in
cuba
ted.
Gen
eral
met
hods
Sa
mpl
e R
efer
ence
s
An
agar
med
ium
, co
ntai
ning
TT
C a
s a
dehy
drog
enas
e in
dica
tor
is s
eede
d w
ith t
he t
est
mic
roor
gani
sm
and
is p
oure
d ov
er t
he d
evel
oped
chr
omat
opla
que
Ant
ibio
tics
N
icol
aus
et a
l. in
B
etin
a (1
9731
The
aga
r m
ediu
m i
s sp
raye
d on
the
dri
ed T
LC
-pla
ques
. A
noth
er a
gar
med
ium
, co
oled
to
48%
and
see
ded
with
a
test
mic
roor
gani
sm,
is p
oure
d di
rect
ly
over
the
su
rfac
e of
the
pre
pare
d pl
ate.
Inh
ibit
ion
zone
s ar
e id
enti
fied
af
ter
incu
bati
on b
y vi
ewin
g th
e op
aque
pl
ates
dir
ectl
y
Ant
ibio
tics
The
aga
r m
ediu
m l
s sp
raye
d on
the
dri
ed T
LC
-pla
tes.
A
fter
sol
idif
icat
ion,
th
e pl
ates
ar
e se
eded
w
ith
the
mic
roor
gani
sm
and
the
grow
th
or i
nhib
itio
n ba
nds
can
be v
iew
ed
usin
g a
tetr
azol
lum
sa
lt
Ant
ibio
tics
an
d re
late
d su
bsta
nces
Bic
kel
et a
l. in
B
etin
a (1
9731
Wal
lhau
ser
(196
91
The
dev
elop
ed
and
drie
d ch
rom
atog
am
is p
ut i
n a
ster
ile
Pet
ri p
late
. T
he p
late
is
cove
red
with
ag
ar a
nd c
oole
d at
roo
m t
empe
ratu
re
(16
min
i an
d fr
ozen
1 h
at
0 O
C fo
r di
ffus
ion.
The
TT
C is
inc
lude
d in
the
cul
ture
med
ium
The
TL
C p
late
s ar
e fl
oode
d w
ith t
he a
gar
cont
aini
ng
spor
es o
f th
e m
icro
orga
nism
. A
fter
a
pred
iffu
sion
pe
riod
of
24 h
at
4% t
he p
late
s ar
e in
cuba
ted
and
the
inhi
biti
on
zone
s ar
e re
cord
ed
Ant
ibio
tics
an
d de
riva
tive
s
Alk
aloi
ds
Ran
dera
th
(197
41
Ver
poor
te
et a
l. (1
9781
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. (19721 and WHO (19771 to 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. (19871 have 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., 19871 used 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
References
Adeoye, A.D., Oguntimein. B.O. and Clark, A.M. (1988) 3Dimethylallylindone: an antibacterial and antifungal metabolite from Monodora tenuifolia. Journal of Natural Products 49, 534- 537.
Adesina, SK. and Akinwusi, D.D. (1984) Biological effects of Co&u&u portoricensis and Tagen- aria breviflom extracts. Fitotempk 55, 339 - 342.
Ajao, A.O., Shonukan, 0. and Femi-Onadeko, B. (1985) Antibacterial effect of aqueous and alco- holic extracts of Spondiaa monbin and Alchornia cordifolia Fitotempia 56,337-339.
Allegrini, J., Simeon de Buochberg. M.. Maillois, H. and Boillot. A. (1973) Emulsions d’huiles essentielles fabrication et applications en microbiologic. Travuux de la Soci&k ok Pharmacie de Montpellier 33,73-86.
Almagboul, A.Z., Farouk, A., Bashir. A.K., Salih. A. and Karim. A. (1985) Antimicrobial activity of certain Sudanese plants used in folkloric medicine. Screening for antibacterial activity (II). Fitotempk 56,103- 199.
Al-Shamma, A., Drake, S., Flynn, D.L., Mistcher, L.A., Park, Y.H., Rao. G.S.R., Simpson, A., Swayze, J.K.. Veysogiu, T. and Wu, T-S. (1981) Antimicrobial agents from Peganum hannalu seeds. Journal of Natuml Products 44,745-747.
Al-Shamma. A., Drake, S.. Guagliardi, L.E., Mitscher, L.A. and Swayze, J.K. (1982) Antimi- crobial alkaloids from Boehmeti cylindrica Journal of Chromatogaphy 21,485-487.
Ayafor, J.F.. Sondengam, B.L. and Ngadjui, B.T. (1982) Veprisinium salt, a novel antibacterial quaternary alkaloid from Vepria lorisii Planta Medica 44,139- 142.
Barbagallo, C., Maugeri, T.L., Meli. R., Pavone. P. and Scoglio, M.E. (1982). Attivitd antibatterica della radice di Astmgulus siculus Biv. Fitotempk 52.87-90.
Barbagallo, C. and Chisari, G. (1987) Antimicrobial activity of three Hypetim species. Fitotem- pia 58,175- 177.
Baron, E.J. and Bruckner, D.A. (1984) Comparison of susceptibilities of anaerobic bacteria deter- mined by agar dilution and by a microbroth method. Reviews of Znfectiow Diseases 1984 Suppl. 1,249-253. (Chemical Abstmcts 101 (1984) 29123~).
Batra, A. and Mehta, B.K. (1985) Chromatographic analysis and antibacterial activity of the seed oil of Asgyreia speciosa Fitotempia 56,357 - 359.
Bauer. A.W., Kirby, W.M.M., Sherris, J.C. and Turck. M. (1988) Antibiotic susceptibility testing by a standardized single disk method. The American Journal of Clinical Pathology 45, 493- 496.
Begit, W.J. and Kline, R.M. (1972) The use of tetrazoiium salt in bioautographic procedures. Journal of Chmmatogmphy 64,182- 183.
Betina, V. (1973) Bioautography in paper and thin-layer chromatography and its scope in the anti- biotic field. Journal of Chromatography 78, 41- 51.
Biard, J.F., Verbist, J.F., Le Boterff, J.. Ragas. G. and Lecocq. M. (1980) Alques fixees de la tote Atlantique franqaise contenant des substances antibacteriknnes et antifungiques. Planta Med- ica Suppl., 138-- 151.
Bondi, A., Spaulding, E.H. and Smith, D.E. (1947) Rapid determination of susceptibility to penieil- lin and the other antibiotics. Amekan Journal of the Medical Sciences 231,221- 225.
Calaada. J., Ciceio. J.F.. Echandi. G. (1989) Antimicrobial activity of the heliangolide chromalaen- ide and related sesquiterpene la&ones. Phytochemia try 19,987 - 988.
Chnlchat, J-C., Garry, R-PI. Michet. A. and Bastide, P. (1987) Correlation composition chimique/ activite antimicrobienne. I. Activite de l’huile essentielle de pin sylvestre a chemotypes differents vis-a-vis d’Escherichia coli Plantes Me’dicinales et Phytothhmpie 21,26-35.
Clark, A.M., El-Feraly. F.S. and Li, W.-S. (1981) Antimicrobial activity of phenolic constituents of Magnolia gmndifim Li. Journal of Pharmaceutical Sciences 70,951- 952.
Clark, A.M., Huddleston, D.L., Ma, C.-Y., and Ho, C.-H. (1984) In vitro antimicrobial activity of benwquinolinediones. Pharmaceutical Research, 289-271.
146
Debra, L.H. and Ward, H.B. (19791 Antibacterial activity of freshwater green algae. P&&a Med- ica 36,375- 378.
El-Feraly, F.S., Cheatham, S.F. and Breedlove, R.L. (1983) Antimicrobial neolignans of Sassafras ramiaiense roots. Journal of Natural Products 46,493-498.
El-Said, F., Fadulu, S.O., Kuye, J.O. and Sofowora, E.A. (1971) Native cures in Nigeria. III: The antimicrobial properties of the buffered extracts of chewing sticks. Lloydia 34,172- 174.
Emeruwa, A.C. (19821 Antibacterial substance from Carica papaya fruit extract. Lloyd& 45, 123 - 127.
Ericsson, H.M. and Sherris, J.C. (1971) International collaborative study on antibiotic sensitivity testing. Acta Pathologica et Microbiolgica Scandinavica Sect. B suppl. 217, 3-88.
Farouk, A., Bashir, A.K. and Salih, K.M. (19831 Antimicrobial activity of certain Sudanese plants used in folkloric medicine. Screening for antimicrobial activity I. Fitotempiu 54, 3 - 7.
Fournier, G., Paris, M.R., Fourniat, M.C. and Quero, A.M. (19781 Activite bacteristatique d’huiles essentielles de Cannabis sativa L. Ann&s Phannaceutiques Fmnqaise 36,603-606.
Fuzellier, M.C., Mortier, F., Girard, Th. and Payen, S. (19811 Etude des propriktes antibiotiques de quelques anthraquinones a l’aide de microplaques de chromatographie. Ann&s Pharma- ceutiquesFmnGaises39,313-318.
Fuzellier, M.C., Mortier, F. and Lectdard, P. (19821 Activite antimicrobienne de Cassia a&a L. Ann&es Phannaceutiques Fmncaises 40,357 - 363.
Gabrielyan, S.A., Bogdanova, L.F., Vorob’eva, L.S. and Givental, N.I. (1985) Comparative estima- tion of agar dilution and agar diffusion methods in determination of Pseudomonas aeruginosa sensitivity to certain antibiotics. Antibiotiki Medisinische Biotekhnologii 30. 511- 516. (Chemical Abstmcts 101 (1984120123~1.
Garg. S.C. and Kasera, H.L. (19831 In vitro antibacterial activity of the essential oil of Sphaeran- thus indicus L. Fito terapia 54,37 - 39.
Gundidza, M. (19861 Screening of extracts from Zimbawean higher plants. II: Antifungal activi- ties. Fitoterapiu 57, ill- 116.
Gundidza, M. (1987) Antimicrobial activities of Helinus integr#olius. Fitotempia 58,180- 183. Gutkind, G.O., Martino, V., Grana, N., Coussio, J. and De Torres, R. (19811 Screening of South-
American plants for biological activities. 1: Antibacterial and antifungal activity. Fitoterapia 52,213-218.
Hamburguer, M.O. and Cordell, G.A. (19871 A direct bioautographic TLC assay for compounds possessing antibacterial activity. Journal of Natuml Products 50, 19 - 22.
Harsh, M.L. and Nag, T.N. (19841 Antimicrobial principles from in vitro tissue culture of Peganum harmala. Journul of Natural Products 47,365-367.
Hejtmbnkova, N., Walterova, A., Preininger, V. and SimOnek, V. (19841 Antifungal activity of quaternary benzo (cl phenantridine alkaloids from Chelidonium majus. Fitoterapiu 55, 291- 294.
Homans, A.L. and Fuchs, A. (19701. Direct bioautography on thin-layer chromatograms as a method for detecting fungitoxic substances. Journal of Chromatogmphy 51,325-327.
Hufford, C.D., Funderburk, M.J., Morgan, J.M. and Robertson, L.W. (19751 Two antimicrobial alkaloids from heart wood of Liriodendron tulipifem L. Journol of Pharmaceutical Sciences 64,789 - 792.
Hufford, C.D. and Lassell, W. (19781 Antimicrobial activity of constituents of Uvati chamae. LZoydiu 41.156-160.
Hufford, C.D., Sharma, A.S. and Oguntimein, B.O. (1980) Antibacterial and antifungal activity of liriodenine and related oxoaporphine alkaloids. Journal of Pharmaceutical Sciences 69, 1180 - 1183.
Ikram, M. and Inamul-Haq (19801 Screening of medicinal plants for antimicrobial activity. Part I. Fitotempia 51,231- 235.
Janssen, M., Scheffer, J.J.C. and Svendsen, A.B. (19871 Antimicrobial activity of essential oils: a 1976-1986 literature review. Aspects of the test methods. Planta Medic% 395-398.
Laurens, A., Mboup, S., Giond-Barber, P., Sylla, 0. and David-Prince, M. (19821 Etude de l’action antibacterienne des extraits d’Anucardium occidentale L. Ann&s Pharmaceutiques Fran- Faises 40,143 - 146
147
Leven, M., Van den Berghe, D.A., Mertens, F., Vlietinck, A. and Lammens, E. (1979) Screening of higher plants for biological activities. I: Antimicrobial activity. Plunta Medica 36, 311- 321.
Loo, Y.H., Shell, P.S., Thornberry, E.J., Sylvester, J.C., Ehrlich, J., McGuire, J.M. and Savage, GM. (1945) Assay of streptomycin by the paperdisk plate method. Joumul of Bacteriology 50,701-769.
Lumonadio, L., Vanhaelen, M. and Devleeschouwer, M.J. (1986) Antibacterial activity of three indole alkaloids extracted from Hannoa klaineana roots. FitoteroM 57, 291- 293.
Lund, B.M. and Lyon, G.D. (19751 Detection of inhibitors of ErwinM carotovom and E. herbicolu on thin-layer chromatograms. Journul of Chmmatogmphy 110,193- 196.
Lynch-Brathwaite, B.A., Duncan, E.J. and Seaforth, C.E. (1975) A survey of ferns of Trinidad for antibacterial activity. Planta Medica 27, 173- 178.
Manunta, A., More& I. and Picci, V. (1987) L’huile essentielle du Buppbumm fruticosum L. Plantes M~dicinales et Phytothe’rapie 21,20-25.
Maruzzella, J.C., Chiaramonte, J.S. and Garofalo, M.M. (1961) Effects of vapors of aromatic chem- icals on fungi. Journul of Phunnaceutical Sciences 50,665-668.
Maruzzella, J.C. and Henry, P.A. (1958) The antimicrobial activity of perfume oils. Journal of the American Phunnaceutical Association 47,471-475.
Maruzzella, J.C. and Liguori, L. (1958) The in vitro antifungal activity of essential oils. Joum~l of the American Pharmaceutical Ausocicrtion 47,251- 253.
Maruzzella, J.C. and Sicurella, M.A. (1960) Antibacterial activity of essential oil vapors. Journal of the American Pharmaceutical Association 49,692 - 694
Mathur, S.B. and Gonzalez, L. (1982) Identification of terpenoids from the leaves of Piptocarpha peredoxa and their biological activities. Journal of Natural Products 45, 495-496.
McCallion, R.F., Cole, A.L.J., Walker, J.R.L., Blunt, J.W. and Munro, M.H.G. (1982) Antibiotic substances from New Zealand plants. II: Polygodial, an anti-candida agent from Pseudowin- tern cokwata. Planta Medica 44,134- 138.
Miski, M., Vlubelen, A., Johansson, C. and Mabry, T.J. (1983) Antibacterial activity studies of flavonoids from Salvia palaestinu Journal of Natural Products 46, 874 - 875.
Mitscber, L.A., Leu. R.P., Bathala, M.S., Wu, W-N., Beal, J.L. and White, R. (1972) Antimicrobial agentsfrom higherplants.I:Introduction,rationaleandmethodology.lloydirr35,157- 166.
Mitscher, L.A., Bathala, M.S., Clark, G.W. and Beal, J.L. (1975) Antimicrobial agents from higher plants. The antimicrobially inactive components of Ptelea trifoliata L. Lloydia 38, 117- 123.
Mitscher, L.A., Han-Park, Y. and Clark, D. (1980) Antimicrobial agents from higher plants. Antimicrobial isoflavonoids and related substances from Glycyrrhiza glabra L. var. typiea Journal of Natural Products 43,259- 269.
Mitscher, L.A., Drake, S., Gollapudi, S.R. and Okwute, K. (1987). A modern look at folkloric use of anti-infective agents. Journul of NaturalProducts 50,1025- 1040.
Mourey, M., Mortier, F. and Mourey, A. (1985) Activitb antimicrobienne d’extraits de feuilles de Ginkgo biloba L. Plantes Mkdtinules et Phyto thkmpie 19,270 - 276.
Naqvi, S.A.H., Khan, M.S.Y. and Vohora, S.B. (1976) Antibacterial, antifungal and anthelmintic studies on Ochrocarpus longa~olius. Planta Medica 29,98- 100.
Nadir, D.M.T., Abdval-Baqi, J., Al-Sarraj, S.M. and Hussein, W.A. (19861 The effect of different methods of extraction on the antimicrobial activity of medicinal plants. Fitolerapia 57, 359- 363.
Palacios, P., Gutkind, G., Rondina, R.V.D., De Torres, R. and Coussio, J.D. (1983) Genus &c-
charis. II: Antimicrobial activity of B. c&pa and B. notosergila Plunta Medica 49, 128- 132. Pellecuer, S., Allegrini, J. and Simeon de Buochberg, M. (1976) Huiles essentielles bactericides et
for&ides. Revue de l’lnstitut Pasteur de Lyon 9, 135- 159.
Peterson, C.A. and Edgington, L.U. (1969) Quantitative estimation of the fungicide benomyl using a bioautograph technique. Journal of Agricultural and Food Chemistry 17.898-899.
Pharmacopke Europkenne (1971) Titrage microbiologique des antibiotiques. Titrage turbidimkt- rique. Pharrnacopge Europe’enne, Vol. 2 Maisonneuve, France, p. 51.
Prasad, G., Abhay Kumar. Sin& A.K., Bhattacharya, A.K., Singh, K. and Sharma, V.D. (1986) Antimicrobial activity of essential oils of some O&mum species and clove oil. FitoteTaph 57, 429-431.
148
Banderath, K. (1974) Cromatogm$k de capafiua Urmo, BiIbao. p. 207. Rae, K.V. and Alvarez, FM. (1981) Antibiotic principles of Eupatorium capillifolium. Journal of
Natuml Products 44,252 - 256. Rae, G.8.K Gerhart, M.A., Lee III, R.T., Mitscher, L.A. and Drake, S. (1982) Antimicrobial
agents from higher plants. Dragon’s blood resin. Journal of Natural Products 45,646-647. Rios, J.L., R-40, M.C. and ViIlar, A. (1987) Antimicrobial activity of selected plants employed in
the Spanish Mediterranean area. Journal of Ethnopharmacobgy 21,X39- 152. Riot, J.L., Sime611. S., JimBnez. F.J., Zafra-Polo, M.C. and ViBar. A. (1986) Reagents for screen-
ing medicinal plants by thin-layer chromatography. A review. Fitotempia 57,158- 162. Ross, S.A., El-Keltawy, N.E. and Megalla, S.E. WSOa) Antimicrobial activity of some Egyptian
aromatic plants. Fitotempia 51, 201- 206. Ross, S.A., Megalla, S.E., Bishay, D.W. and Awad, A.H. (198Ob) Studies for determining anti-
biotic substances in some Egyptian plants. Part I. Screening for antimicrobisl activity. Fib tempia 51,303-308.
Singh, K.V., Bhatt, S.K. and Sthapak, J.K. (1988) Antimicrobial and anthebninic properties of the seeds of Aegk marmelos. Fitotempia 54,261- 264.
Sundaram, B.M., Gopalakrishnan, C., Subramanian, S., Shankaranayanan, D. and Kameswaran, L. (1988) Antimicrobial activities of Gartinfe mangortana Plunta Medica 48,59-60.
Tabata, M., Tsukada, M. and Fukui, H. (1982) Antimicrobial activity of quinone derivatives from Echium lycopsis callus cultures. Planta Medica 44,234 - 236.
Twaij, H.A.A., Saiih. F.M., Nadir, M.T. and Aaix, N.K. (1986) Antimicrobial and some pharmacol- ogical activity of Centaurea phyllocephala and C. beherr Fitotempia 57,41- 45.
United States Pharmacopeial Convention (1975) Biological tests and assays. Antibiotic-microbial assays. In: United States Pharmwopeia XIX, Rockville Mack Pubbshing Co., MO p. 595.
Valnet, J., Duraffourd, Cl., Duraffourd, J. and Laprax, Cl. (1978) L’ Aromatogramme noveaux result&s et essai d’interpretation sur 268 cas cIiniques. P&antes MkdieiMZes et Phytothdmpie 12, 43- 52.
Van Beek, T.A., Deelder, A.M., Verpoorte, R. and Svendsen, B.A. (1984a) Antimicrobila, anti- amoebic and antiviral screening of some Tabernaemoutana species. Pianta Medica 50, 181- 188.
Van Beek, T.A., KuijIaars. F.L.C., Thomaasen, P.H.A.M., Verpoorte, R. and Svendsen, A.B. (1984b) Antimicrobialiy active alkaloids from Tabemaemontana pachysiphon Phytochemistry 23,1771- 1778.
Van Beck, T.A., Verpoorte, R. and Svendsen, A.B. (1985) AntimicrobiaIIy active alkaloids from Tabemaemontaua chippii Journal of Natural products 48,400-423.
Van der Nat, J.M., Van der Sluis, W.G. and Labadie, R.P. (1982) Gentiogenai, a new antimi- crobial iridoid derived from gentiopicrin (gentiopicroside). Phrntu Medica 45,161.
Van der Sluis, W.G. and Labadie, R.P. (1981) Fungitoxic activity of the seeoiridoid glucoside gen- tiopicrin (gentiopicroside). Planta Medica 42,189- 140.
Van Hoof, L., Vanden Berghe, D.A. and Vhetinck, A.J. (1980) Screening of poplar trees for anti- bacterial and antiviral activity. B&log& Pluntarum 22, 265- 273.
Van Hoof, L., Vanden Berghe, D.A., Petit, E. and Vhetinck, A.J. (1988) Antimicrobial and anti- viral screening of Bryophyta Fitotempia 54,223-229.
Verpoorte, R., Kode, E.W., Van Doorne, H. and Svendsen, A.B. (1978) Antimicrobial effect of the alkaloids from Strychnos afzelii Gilg. Pfanto Medica 33,287 - 242.
Verpoorte, R., Kos-Kuyck, E., Tjin A Tsoi, A., Ruigrok, C.L.M., De Jong, G. and Svendsen, A.B. (1988) Medicinai plants of Surinam. III: Antimicrobiaily active aikaloids from Aspidqwnna excelsum. Planta Medica 48,283 - 289.
Verpoorte, R.. Ruigrok, C.L.M. and Svendsen, A.B. (1982) Medicinal plants of Surinam. II: Antim- icrobial active alkaloids from Aspidospewna marcgravum. Planta Medica 46,149- 152.
Viilar, A., Mares, M. and Rios, J.L. (1988) Proprietis antimicrobiennes des feuiiles d’tlnwna cherimotia Mill. Plantee Mkdkinales et Phytothgmpie 17,233 - 265.
Viilar, A.. Mares, M., Rios, J.L., Canton, E. and Gobernado, M. (1987) Antimicrobial activity of benayiisoquinoline alkaloids. Pharmazie 42, H.4,248-250.
149
ViBar, A., Hios. J.L. and Mares, hf. (lQ64) Annona cherimolicr alhsloids: isolation, identification and antimicrobial activity. Farmoceutioch Ti@chrift war Relgie 61,299.
Viilar, A., Bias. J.L., Becio, MC., Cortes, D. and Cave, A. (lQ66aI Antimicrobial activity of hen- zylisoquinoiine akaioids. IL Relation between chemical composition and antimicrobial activ- ity.P&ntoMedfco52.556-557.
VilIar, A., Bias. J.L., Recio, MC. and %&a-Polo. M.C. flQ66b3. Antimicrobial activity of essential oils from Sideritis species. Phu7mke 41, H.4. 298-299.
Villar, A., Bias, J.L.. Zafra-Polo, M.C., Mares, M. and Becio, MC. (1985) Activite antimicrobienne des extraits d%sp&es de Sideritir. Plantee Mkdicinalee et Phytothkmpie 19,248-254.
Wasge, S.K. and Hedin. PA. (1985) Quercetin 3-Ggalactosyl-U+6) glucoside. a compound from narrow leafvetd with antimicrobiaI activity. Phytochemistry 24,242 - 245.
Wsllhiiusser, K.H. (1969) Exeeution of the microbiological test. In: E. Stahl (Ed.), Thin-layer Chromatography. A Labomtory Handbook, Springer-Verlag, Berlin, p. 570.
Wat, C.K., Johns, T. and Towers, G.H.N. flQ60) Phototoxic and antibiotic activities of plants of the Asteraceae used in folh medicine. Journal of Ethnopharmacology 2,279--290.
Wolters. B. (1969) Zur verwendung vorbeschicbteter foiien dei der diinnschichtchromato graph&hen untersuchung pfianzhcher fungistatica. Plantu Medica 17,42- 50.
Walters, B. and Eilert, U. (1981) Antimicrobial substances in callus cultures of Ruta gmveolens. Plantu Medica 42,166 - 174.
World Health Organization (1977) Report of the W.H.O. Expert Committee on Antibiotics. Inform n. 26. Technical Report series n. 610, anexe 5 WHO, Geneva, Switzerland 106- 137.
Yasphe. J.. SegaI, It., Berver, A. and Erdreich-NaftaIi. G. (1979) Antibacterial activity of Artemi- sia herba-alba Journal of Pharmaceutical Sciences, 68,924 - 925.
Yousef, R.T. and Tawil, G.G. (1966) Antimicrobial activity of volatile oils. Phurma& 35, H.11, 4 -11.
Zahir Shah, Verpoorte, It. and Svendsen. A.B. (1988) Antimicrobial investigation of Strychnos camptoneuru Fitotempia 57,4X+427
