Journal of Microbiological Methods 40 (2000) 199206

Journal
of
Microbiological
Methods
www.elsevier.com / locate / jmicmeth

Chromogenic plate assay distinguishing bacteriolytic from

bacteriostatic activity of an antibiotic agent
Gonzalo Mardones, Alejandro Venegas*

, Pontificia Universidad Catolica

, Departamento de Genetica
Molecular y Microbiologa
de Chile,
Laboratorio de Bioqumica
Casilla 114 -D, Santiago, Chile
Received 26 April 1999; received in revised form 20 December 1999; accepted 5 January 2000

Abstract
A solid agar plate assay was devised to discriminate bacteriolytic from bacteriostatic activity for a given antibacterial
agent. The assay uses a bacterial culture harboring b-galactosidase enzyme as reporter of cellular lysis. When a drop of
bacteriolytic compound is placed on the agar, b-galactosidase is released from the bacteria to the external solid medium
where it hydrolyzes X-Gal substrate analogue, developing a blue halo at the edge of the inhibition growth zone. The assay
was successfully evaluated against several antibiotics with well-known mechanism of action. It was found that bacteriostatic
compounds consistently did not display blue halo at the inhibition zone. 2000 Elsevier Science B.V. All rights reserved.
Keywords: Antibiotics; Apidaecin; Bacteriolytic assay; X-Gal plates

1. Introduction
The knowledge about the mechanism of action of
a new antimicrobial agent is basic to understanding
the events occurring during bacterial growth inhibition. This issue is very important for the development of any antibacterial compound for therapeutic
use.
Recently, several efforts have focused on studying
the mechanisms of a number of new antibacterial
peptides. Surface active peptides which bind and
alter amphipatic surfaces, including membranes and
receptors, have been extensively studied (DeGrado et
al., 1981; Kaiser and Kezdy, 1983, 1984; Kaiser,
1988). Some of these antibacterial agents act in a
*Corresponding author. Tel.: 1 56-2-686-2661; fax: 1 56-2-2222810.
E-mail address: avenegas@genes.bio.puc.cl (A. Venegas)

similar way to hormones by binding to specific

cellular receptors which require specific peptide
conformation. In contrast, the mechanisms of action
of other antibacterial agents is less dependent on
such stringent structural requirement. Among these
compounds, cytolytic cationic peptides with a wide
spectrum of action have been isolated from mammalian macrophages the so called defensins
(Ganz et al., 1990), from insects, melittin (Habermann, 1972), cecropins (Steiner et al., 1981) and
sarcotoxins (Okada and Natori, 1985) and from
amphibians, magainin (Zasloff, 1987). The target
for these surface-active peptides seems to be the lipid
bilayer of the cellular membrane. It has been reported that their activity is exclusively due to their
unique structural features, which allow them to bind
to the corresponding cells, modulating the membrane
voltage and affecting membrane permeability (Westerhoff et al., 1989; Ganz et al., 1990). Participation

0167-7012 / 00 / $ see front matter 2000 Elsevier Science B.V. All rights reserved.
PII: S0167-7012( 00 )00125-1

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G. Mardones, A. Venegas / Journal of Microbiological Methods 40 (2000) 199 206

of voltage-dependent ionic channels have also been

proposed to explain lytic activity (Christensen et al.,
1988; Cruciani et al., 1988; Duclohier et al., 1989;
Kagan et al., 1990).
The entire structure of the bactericidal compound
under study seems to be the most important feature,
as found for some peptides, since their all-D-enantiomers also have biological properties similar to those
of the corresponding native L-enantiomers (Bessalle
et al., 1990). This assumption, however, is not valid
for the receptor-oriented-type of compound (Flouret
and du Vigneaud, 1965; Morley et al., 1965; Stewart
and Woolley, 1965; Casteels and Tempst, 1994). At
present, a variety of natural and synthetic products is
under study, searching for new antibiotic compounds.
We propose here a simple, inexpensive assay to
screen compounds with bacteriolytic activity.

2. A new chromogenic plate test assay for

evaluation of bacteriolytic compounds
There are several approaches to establish the
bacteriolytic or bacteriostatic nature of an antibacterial compound. For instance, bacterial lysis may be
followed by permeability assays for the inner and
outer membranes in liquid media (Lehrer et al.,
1988), using electrophysiological techniques (Saberwal and Nagaraj, 1994), or by studying the enantiomer biological activities (Bessalle et al., 1990;
Casteels and Tempst, 1994). However, some of these
techniques are expensive and time-consuming.
In this report we present a simple strategy based
on the use of b-galactosidase as an appropriate
marker of cellular lysis. The antibiotic to be tested is
laid as a small drop (16 ml) on a plate with soft
agar containing an Escherichia coli growing lawn
which expresses b-galactosidase activity. If lysis
occurs, then the enzyme activity is released outside
the bacterium and detected on the plate. When the
enzyme reaches the agar medium, it hydrolyzes the
5-bromo-4-chloro-3-indolyl-b-D-galactoside (X-Gal),
a chromogenic compound included in the agar and
extensively used in alpha-complementation assays
(Sambrook et al., 1989). After overnight incubation,
X-Gal forms a blue circle staining the edge of the
inhibition zone produced by the antibiotic applica-

tion. Only compounds causing cellular lysis produce

a blue-colored edge at the inhibition zone.
Some of the advantages of this method are low
cost, simplicity and the possibility to deal with
several samples at once in a single Petri dish,
providing a comparative direct observation of the
results on a particular bacterial strain.

3. Procedure

3.1. Strains
Escherichia coli strain BL21 (DE3) (F 2 ompT r B2
m ) obtained from Novagen Inc. was used to
standardize the assay. Also, E. coli strains BL21
(Novagen Inc.), C600 lacY 2 (Clowes and Hayes,
1968), and UH302 (Cole et al., 1982), as well as
Erwinia carotovora spp. carotovora Ecc193 (kindly
provided by Dr Chatterjee) and Citrobacter freundii
and Shigella flexneri (provided by Dr Guido Mora)
were utilized.
2
B

3.2. Antibiotics
Ampicillin, carbenicillin, cephaloridine, cephalosporin C, cephradine, chloramphenicol, erythromycin, gentamicin, kanamycin, kasugamycin, moxalactam, nalidixic acid, spectinomycin, streptomycin,
sulfadiazine, tetracycline and trimethoprim were
from Sigma (St. Louis, MO). Apidaecin Ib (Casteels
et al., 1989), cecropin B (Gazit et al., 1994) and
cecropin P1 (Christensen et al., 1988) were chemically synthesized by Bios Chile I.G.S.A. (Santiago,
Chile). Cephamezine and ceftizoxime were from
Instituto Beta (Santiago, Chile). The stock solutions
of the antimicrobial compounds were prepared in
ethanol or distilled water depending on their solubility properties.

3.3. Reagents
X-Gal was from Promega (Madison, WI).
Isopropyl-b-D-thiogalactopyranoside (IPTG) was
from Sigma. Bacto-agar, bacto-tryptone were purchased from Difco (Detroit, MI). NaCl was from

Merck (Darmstadt,
Germany). Yeast extract powder
was from HiMedia (Bombay, India).

G. Mardones, A. Venegas / Journal of Microbiological Methods 40 (2000) 199 206

3.4. Minimal inhibitory concentration ( MIC)

determinations
This was done in liquid cultures following the
procedure described by Braude (1981) with few
modifications. Ten microliters of an overnight culture of E. coli BL21(DE3) cells were diluted into 1
ml Luria broth and then, aliquots of 100 ml were
transferred to eight sterile tubes. The first tube
contains the highest antimicrobial concentration to be
tested. To this tube additional 100 ml of bacterial
cells were added and after mixing, 100 ml were
withdrawn and transferred to the next tube. The
two-fold serial dilution was repeated for the other
tubes. The tubes were incubated 12 h at 378C and the
bacterial growth was measured at 600 nm. The MIC
value (a-b) expresses the highest antimicrobial concentration at which cells were able to grow (a) and
the lowest concentration at which no growth was
detected (b).

3.5. Bacteriolytic plate assay

Escherichia coli strain BL21(DE3) which contains
a chromosomal IPTG-inducible b-galactosidase
gene, was used for most of the assays. Other lacZ 1
strains tested were E. coli UH302, E. coli C600 (a
lacY 2 derivative), Citrobacter freundii, Erwinia
carotovora sp. carotovora Ecc93 and Shigella flexneri. First, an inoculum with this strain was grown
overnight in 2 ml LB media (10 g / l tryptone, 5 g / l
NaCl, 5 g / l yeast extract powder), at 378C with
shaking. Then, a soft agar-incubation mix containing
10 ml of 0.8% agar previously melted at 458C with
50 ml of the bacterial cell inoculum, 10 ml of 1 mM
IPTG, and 50 ml of 50 mg / ml X-Gal was vortexmixed and carefully overlaid on LB plates containing
20 ml of 1.5% agar prepared the day before. Once
the soft agar was solidified and dried (23 h), single
1-, 1.5-, 3-, or 6-ml drops (depending upon the
antibiotic tested), containing the appropriate concentration of the antibiotic, were deposited on the
soft agar layer using fine disposable tips. Then the
plates were incubated at 378C for 916 h. After
incubation, the inhibition zones were visually inspected by color formation along the edge of spots
and the plates were photographed. Plates can be

201

stored at 48C for several weeks without loss of the

blue color.

4. Results and discussion

The method presented here allowed us to distinguish a bacteriolytic from a bacteriostatic modeof-action of antimicrobial compounds. To test the
new assay, a selected group of antibiotics was
analyzed (Table 1). All the antibiotics assayed gave
the expected pattern, a blue edge at the inhibition
zone for bacteriolytic agents, and no color for
bacteriostatic compounds. The only exception to the
pattern was apidaecin which behaved as a bacteriolytic agent, in contrast to the proposed non lytic
mode-of-action (Casteels and Tempst, 1994).
Fig. 1 presents the assay plate for some of the
antibiotics listed in Table 1, including two bacteriostatic agents (chloramphenicol and tetracycline) and
five bacteriolytic compounds (nalidixic acid, ampicillin, cecropin B, cecropin P1 and apidaecin Ib).
Notice the sharp blue halos around the bacteriolytic
compounds.
We used tetracycline and ampicillin as bacteriostatic and bacteriolytic agents, respectively, to determine appropriate conditions for the assay. Different strains in the bacterial lawn, incubation time and
suitable amount of X-Gal for color detection and
sensitivity were also used to determine conditions.
Results shown in Table 2 validated the assay for
different Gram negative lac 1 strains. E. coli HB101
strain was included as a lac 2 control. In order to find
the most appropriate X-Gal concentration, plates
containing soft agar with 62.5, 125, 250 and 375
mg / ml were assayed (not shown). At the highest
X-Gal concentration only the plate background was
increased with rather modest improvement of color
intensity at the inhibition zone. Under the standard
X-Gal concentration described for the assay (250
mg / ml), no blue color appeared at the inhibition
zone when the bacteriostatic compound was tested.
In addition, plate incubation was also tested at 28
and 428C, keeping other assay conditions as described in Section 3.5, with no significant improvement of the assay.
To evaluate the sensitivity of the method (the
smallest inhibition zone at which the blue color

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G. Mardones, A. Venegas / Journal of Microbiological Methods 40 (2000) 199 206

Table 1
Pattern of halo at inhibition zone for various antibacterial agents in the chromogenic plate assay a
Antibacterial
agent

Amount added
(mg)

Ampicillin
Apidaecin Ib
Carbenicillin
Cecropin B
Cecropin P1
Cephamezine
Ceftizoxime
Cephaloridine
Cephalosporin C
Cephradine
Chloramphenicol
Erythromycin
Gentamicin
Kanamycin
Kasugamycin
Moxalactam
Nalidixic acid
Spectinomycin
Streptomycin
Sulfadiazine
Tetracycline
Trimethoprim

6
15
20
6
6
30
30
20
30
20
37.5
100
10
20
20
10
37.5
20
20
100
9.5
2

Type of halo b

Mechanism
(Reference)

Observed

Expected

1
1
1
1
1
1
1
1
1
1
2
1/2
1
1
2
1
1
2
1
2
2
2

1
2
1
1
1
1
1
1
1
1
2
1
1
1
2
1
1
2
1
2
2
2

Tomasz, 1979
Casteels and Tempst, 1994
Maki et al., 1978
Gazit et al., 1994
Christensen et al., 1988
Mandell and Sande, 1991
Ogawa et al., 1981
Rolinson, 1980
Flynn, 1972
Neiss, 1973
Pratt and Fekety, 1986
and Trieu-Cuot, 1988
Brisson-Noel
Rosselot et al., 1964
Bryan, 1989
Bakker, 1992
Labia, 1982
Hooper et al., 1987
Schoutens et al., 1972
Bryan, 1989
Woods, 1962
Chopra and Howe, 1978
Ferone et al., 1969

a
All antibacterial compounds were tested as described in Section 3.5, using E. coli BL21(DE3) strain, except for erythromycin which was
assayed in E. coli UH302.
b
Halo indicated as ( 1 ) blue color, (2) colorless, and ( 1 / 2 ) faint blue.

could be detected in the halo), lower ampicillin

concentrations were tested. Results showed that the
inhibition zone should be at least 3 mm or larger in
diameter for the blue halo to be noticed (spot of 0.6
mg ampicillin in Fig. 2A). In the case of a bacteriostatic agent, no color was detected over a wide range
of tetracycline (0.154.5 mg in Fig. 2B). In addition,
it should be mentioned that the appropriate amount
of ampicillin to be used in the testing plate is close to
that of the MIC value determined in liquid media
(referable to 1 ml of cultured cells) in such a way
that the halo can be easily distinguished. For instance, 1 ml containing 1.3 mg ampicillin (MIC value
determined as 1.3 mg / ml for BL21(DE3) strain)
showed an inhibition blue halo of 5 mm in diameter
(Fig. 2A).
Since IPTG is a strong inducer of b-galactosidase
activity, the effect of this compound was evaluated.
It was found that 1 mM IPTG in the soft agar
containing BL21(DE3) cells was just enough to

optimize the blue color at the inhibition zone compared to a plate without IPTG (not shown). This
effect may vary due to the particular E. coli strain
used. Moreover, when BL21(DE3) cells were used,
concentrations higher than 1 mM IPTG increased the
blue background of the plate rather than the blue
color of the halo. Concentrations higher than 50 mM
IPTG stained blue the entire plate, precluding any
discrimination between bacteriostatic and bacteriolytic agents (not shown).
The reproducibility of this method was tested at
least four times for several antibiotics with well
characterized mode-of-action giving the results summarized in Table 1.
An interesting point to be mentioned is that among
22 tested antibiotics (Table 1) only apidaecin Ib
behaved differently with respect to its assigned
mode-of-action (Casteels and Tempst, 1994).
Apidaecin Ib is a unique antibacterial peptide found
in immune honeybee lymph. It consists of 18 amino

G. Mardones, A. Venegas / Journal of Microbiological Methods 40 (2000) 199 206

203

Fig. 1. Plate assay showing bacteriolytic or bacteriostatic activity

for some antibiotics. The assay was done as described in Section
3.5. A, ampicillin, 1.5 ml of a stock solution 20 mg / ml; T,
tetracycline, 1.5 ml of 6.3 mg / ml; C, chloramphenicol, 1.5 ml of
25 mg / ml; API, apidaecin Ib, 1.5 ml of 4 mg / ml; CB, cecropin B,
6 ml of 1 mg / ml; CP, cecropin P1, 6 ml of 1 mg / ml; N, nalidixic
acid, 1.5 ml of 25 mg / ml. Ten milliliters of 0.7% soft agar
containing 50 ml of 50 mg / ml X-Gal and 50 ml of saturated
culture of E. coli BL21(DE3) were added on the top of a
Luria-agar plate and incubated at 378C for 16 h.

acids including six proline residues, and is very

stable at high temperature and at low pH (Casteels et
al., 1989). Casteels and Tempst (1994) have proposed that apidaecin functions as a bacteriostatic
agent, specifically toward Gram negative bacteria,
through a non-pore forming mechanism. In contrast, our results suggest a lytic mechanism. However, there are few differences in the assays that may
explain the divergence in the results. First, our plate
assay was evaluated after 1012 h of incubation with
the peptide, while Casteels and Tempst measured
ONPG hydrolysis spectrophotometrically after 25
min. Second, our assay was done in solid medium,
the other was done in solution. Third, we used E.
coli BL21(DE3), a lacZ 1 derivative, and the other
authors, E. coli ML-35p. These strains may differ in
membrane permeability. Independently determined,
apidaecin MIC values in liquid cultures were 0.63
mg / ml for BL21(DE3) (our data) and 0.050.1 mg /

Fig. 2. Sensitivity of the assay. The assay was done as described

in Section 3.5, but inhibition zones were formed adding a 1-ml
drop on the lawn. (A) Drops containing 0.3 (center), 0.6, 1.3, 2.5,
5, 10 and 20 mg of ampicillin. (B) Drops containing 0.08 (center),
0.15, 0.3, 0.6, 1.2, 2.3 and 4.5 mg of tetracycline.

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G. Mardones, A. Venegas / Journal of Microbiological Methods 40 (2000) 199 206

Table 2
Chromogenic plate assay done with different lacZ 1 bacterial strains to distinguish a bacteriolytic agent from a bacteriostatic compound a
Strain

Citrobacter freundii
Escherichia coli B
E. coli BL21
E. coli BL21(DE3)
E. coli C600
E. coli HB101 (lacZ 2 control strain)
E. coli K-12
E. coli UH302
Erwinia carotovora spp. carotovora Ecc193
Shigella flexneri

Halo b
Ampicillin
(10 mg / drop)

Tetracycline
(2.3 mg / drop)

1
1
1
1
1
2
1
1
1
1

2
2
2
2
2
2
2
2
2
2

a
One microliter was laid on the bacterial lawn using ampicillin as a bacteriolytic agent and tetracycline as a bacteriostatic antibiotic and
the assay conditions were done as described in Section 3.5, except for the Erwinia strain which was grown at 288C.
b
Halo indicated as ( 1 ) blue color, and (2) colorless.

ml for ML-35p (Casteels and Tempst, 1994). In

addition to the differences mentioned above, we have
expressed a synthetic apidaecin Ib gene in
BL21(DE3) cells (unpublished results) and 2 h after
IPTG induction of the apidaecin gene we detected
b-galactosidase activity in the supernatant fraction.
This result indicates that bacterial lysis is induced by
cytoplasmic expression of apidaecin and lysis can be
detected as early as 2 h after IPTG induction. We
also found that expression of apidaecin drastically
affected bacterial growth in a similar way as reported
by other authors (Taguchi et al., 1994), during
expression of apidaecin fused to the inhibitor of
Streptomyces subtilisin.
A different case is erythromycin which, in addition to its bacteriolytic effect on Gram-positive
bacteria, has shown a bacteriostatic effect at very
low concentration such as 0.001 mg / ml (Brisson and Trieu-Cuot, 1988). This result cannot be
Noel
evaluated in our assay conditions because it is
beyond the sensitivity of the method, but the method
allowed us to detect the bacteriolytic effect described
for erythromycin, suggesting that, in some cases, the
actual effect on bacterial cells depends on the
concentration of the agent used.
The formation of a blue halo when a bacteriolytic
compound is being tested could be explained by
X-Gal hydrolysis occurring at the edge of the
inhibition zone. This could be due to two factors: (1)
the radial diffusion of bacteriolytic agent that gener-

ates a concentration gradient at which an equilibrium

between growing and lysed cells is reached, and (2)
certain number of lysed cells release a sufficient
amount of b-galactosidase enzyme able to hydrolyze
a visible quantity of X-Gal substrate. Bacterial cells
located close to the center of the inhibition zone did
not have the chance to grow nor to accumulate the
enzyme, because of the diffusion of the antimicrobial
compound in a radial way, starting from the application point on the agar plate.
The light blue background observed at concentrations of X-Gal higher than 125 mg / ml in the plate,
may be due to a slight and slow X-Gal diffusion into
the dividing cells, providing a soft blue background
rather than the dark blue circle in the inhibition zone.
Another explanation could be that the X-Gal may
enter the bacterial cells using the lactose permease
system. Regarding this point, we tried E. coli C-600
which is a lacY 2 mutant. However, no improvement
to reduce the blue background was observed. We
favour the explanation that the background may be
related to the intrinsic X-Gal permeability for a
defined strain. For instance, we did not observed a
lawn with blue background when Shigella flexneri
was used in our standard assay conditions, even at
X-Gal concentrations higher than 125 mg / ml.
We conclude that the method described here
allows discrimination between a bacteriolytic or
bacteriostatic mechanism-of-action of antimicrobial
molecules. The assay is simple, economical, and

G. Mardones, A. Venegas / Journal of Microbiological Methods 40 (2000) 199 206

reliable. It requires only a minimal amount of the

compound to be tested and facilitates the analysis of
an extensive number of different compounds at the
same time. We believe that this method should
facilitate investigations of the mechanism of action
of new antibiotics.

Acknowledgements
This research was supported by grants from Fondo
de Chile (FONNacional de Ciencia y Tecnologa
DECYT [1940713 and [1971010). We gratefully
acknowledge Dr Jorge Delgado and Steve Nguyen
for critical reading of the manuscript.

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