P.

acnes

Effect of Citronella Oil on Time Kill Profile,

Leakage and Morphological Changes of
Propionibacterium acnes
Pilanthana Lertsatitthanakorn,
Faculty of Pharmacy, Mahasarakham University, Mahasarakham 44150, Thailand

Suwimol Taweechaisupapong,
Faculty of Dentistry, Khon Kaen University, Khon Kaen 40002, Thailand

Channarong Arunyanart,
Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand

Chantana Aromdee and Watcharee Khunkitti,*

Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand
Abstract
The aim of this study was to investigate the effect of citronella oil (Java type) (Cymbopogon winterianus Jowitt)
on Propionibacterium acnes DMST 14916. Citronella oil compositions were determined by gas chromatography mass
spectrometry (GC/MS). Anti-P. acnes activity of citronella oil and its major components were also investigated. After
exposure with P. acnes at various concentrations of citronella oil (0100 mL/mL), time-kill profiles, pentose leakage
and electron microscopic characteristics were determined. The GC/MS results revealed that the major constituents
of the oil were citronellal, geraniol and citronellol. At all concentrations above Minimum Bactericidal Concentrations
(MBCs; 0.625mL/mL), this essential oil killed P. acnes in a dose-dependent manner. The intracellular materials leakage and electron microscopic characteristics evidenced that the major target sites of antibacterial activity appeared
to be cell wall, cytoplasmic membrane and intracellular materials. Citronella oil exerted satisfactory bacteriostatic
and bactericidal actions against P. acnes.
Key Word Index
Cymbopogon winterianus, Poaceae, citronella oil, essential oil composition, citronellal, citronellol, geraniol,
Propionibacterium acnes.

Introduction
Citronella grass (family: Poaceae) is widely distributed in
the semi-temperature to tropical parts of the world. About
140 species of Cymbopogon are known. Citronella oil is obtained from the steam distillation of the fresh or partly dried
aerial parts of citronella grass. Sri Lanka and Java citronella
oils are the two main commercially available varieties of this
essential oil (1). The difference between both types, are the
main constituents of the oil. In the Sri Lanka type citronella
oil [(C. nardus (L.) Rendle var. nardus], the five main constituents are geraniol (15.023.0%), limonene (7.011.5%),
methyl isoeugenol (7.011.0%), camphene (7.010.0%), and
citronellol (3.08.5%)(2). In the Java type citronella oil (C.
winterianus Jowitt), the five main constituents are citronellal
(3140%), geraniol (2025%), citronellol (8.514%), geranyl
acetate (2.55.5%) and elemol(1.34.0%) (3) .

Apart from insect repellent property (4,5), citronella oil of

also posseses pronounced antibacterial activity against P. acnes
which is a major cause of acne vulgaris (6), anticandidiasis (7,8),
and antifungal property (9). An in vitro study demonstrated that
citronella oil has a very good free radical scavenging activity and
anti-inflammatory action (6). Additionally, dermal sensitization
by a local lymph node assay demonstrated that citronella oil at a
concentration of 50% w/v was classified as a non-sensitizer (10).
The United States of America Food and Drug Administration
(FDA) classify citronella oil as GRAS (Generally Recognized
As Safe). Therefore, citronella oil could be used as potential
anti-acne oil. However, the mechanism of action of citronella
oil on P. acnes has not been reported. In this study, the effect
of citronella oil on P. acnes was investigated by its lethal action and its ability to induce membrane damage as well as its
ability to induce morphological changes after treatment with
Received: February 2008

*Address for correspondence

Revised: August 2008

1041-2905/10/0003-0270$14.00/0 2010 Allured Business Media
270/Journal of Essential Oil Research

Accepted: November 2008

Vol. 22, May/June 2010

Khunkitti et al.

Figure 1. Time-kill curves of citronella oil on P. acnes.

Concentrations of citronella oil (mL/mL): (u) 0; (n) 0.625, (s) 12.50,
( ) 50, () 100

Figure 2. TEMs of P. acnes.

Arrows indicate ruptured cell wall and cytoplasmic membrane;

(a) Untreated (control), (b) Treated with 0.625 mL/mL citronella oil
Magnification 58,300 (bar, 0.5 mm), (c) Treated with 12.5 mL/mL
citronella oil Magnification 87,400 (bar, 0.2 mm), (d) Treated with 50
mL/mL citronella oil Magnification 87,400 (bar, 0.2 mm), (e) Treated
with 100 mL/mL citronella oil. Magnification 87,400 (bar, 0.2 mm).

various concentrations of citronella oil using scanning (SEM)

and transmission (TEM) electron microscopy.

Experimental
Materials: Citronella oil was purchased from Thai China
Flavours & Fragrances Industry Co. (Thailand). Tryptic casein
soy agar was provided by Hispanlab (Spain) and fluid thioglycollate broth was obtained from Difco (USA). All other chemicals
were of reagent grade.
Determination of citronella oil constituents by gas
chromatography -mass spectrometry (GC/MS): Constituents
of citronella grass oil were determined by Gas Chromatograph
Vol. 22, May/June 2010

Mass Spectrometer (GCMS-QP 2010, Shimadzu). Conditions were as follows: Column: Rtx-5 MS (30 m x 0.25 mm x
0.25 mm film thickness), Carrier gas: He, Oven temperature:
80230C (10C/min) fold for 1 min. Injection temperature:
270C; Injection volume: 1mL, Split mode 1:50. Flow rate:
2 mL/min. MS conditions: positive ion detection, Interface
temperature: 250C, Ion source: 0.98 kV, Scan Mode: positive
ion, full scan 35550 m/z. The compounds were identified by
their linear retention indices and comparing their mass spectra
with the NIST mass spectral libraries. Some constituents were
also compared with the synthetic reference compounds (Fluka,
Switzerland). The purity of citronellal was 8090%, geraniol
was 96.0% and citronellol was 9095%.
Bacterial suspensions: Propionibacterium acnes DMST
14916 was obtained from the culture collection of the Department of Medical Sciences, Ministry of Public Health, Thailand.
Two colonies of P. acnes DMST 14916 from overnight cultures
on Tryptic casein soy agar was inoculated into 5 mL of fluid
thioglycollate broth and incubated at 37C for 72 h under
anaerobic conditions to give approximately 1 x 109 CFU/mL.
Determination of bacteriostatic and bactericidal activities
by broth microdilution method: Bacteriostatic and bactericidal
activities of the oil sample towards the bacteria were determined
as described previously (11). Briefly, 50 mL of the oil samples
(50 mL/mL) was two-fold serially diluted with in thioglycolate
broth containing 1% w/v Tween 80 in a microtitre plate. An
equal volume of the bacterial suspension was added in each
well to make a final concentration approximately 10 6 CFU/mL.
The plates were incubated for 48 h, at 37C in an anaerobic
jar. Then bacterial growths were examined and the lowest
concentration of the oils which inhibited the visible growth
of bacteria was recorded as the minimum growth inhibitory
concentration (MIC). The positive growth of P. acnes cultured
in the broth without oils served as a positive control and the
mixture of the broth and oils without microorganism served
as a negative control. Aliquots of the mixture of the oils and
bacteria which showed negative-visible growth after the first
48 h of incubation were inoculated onto the surface of Trypic
casein soy agar. The lowest concentration of the oils giving
negative growth of bacteria was recorded as the minimum
bactericidal concentration (MBC).
Lethal effects: A suspension of P. acnes (0.1 mL) was
added to 0.9 mL of various concentrations (0100 mL/mL) of
citronella oil in fluid thioglycollate broth containing 1% Tween
80 as an oil solubilizing agent to give approximately 10 7 CFU/
mL. Samples (0.1 mL) were transferred to 0.9 mL of normal
saline solution at 0, 1, 3 and 6 h at 37C. Dilution was used to
reduce carry over effect. The samples were then serially diluted
in normal saline solution for viable counting by pour plate
method. The plates were incubated at 37C under anaerobic
conditions for 48 h. Each concentration of citronella oil was
carried out in triplicate. The 1% Tween 80 in fluid thioglycollate
broth treated with the same manner as the sample solutions was
used as control. Results are presented graphically as time-log
survivors curves with bars representing the standard deviation
of an average of log CFU/mL in triplicate plates.
Leakage of intracellular materials: Equal volumes (2.5
mL) of double-washed P. acnes suspension at a cell density of
2 mg dry weight/mL and either citronella oil solution using 1%
Journal of Essential Oil Research/271

P. acnes

Tween 80 in normal saline solution (NSS) or NSS treated with

the same manner was used as blank, were mixed to give the
required final concentrations of the oil (0100 mL/mL). After
incubation at 37C under anaerobic condition for 30 min, the
cells were removed by centrifugation at 2,000 x g for 10 min
and the supernatant fluid was assayed for pentose concentration using colorimetry. Briefly, volumes (3 mL) of freshly
prepared reagent containing 200 mg of orcinol monohydrate
and 5.1 mg of cuprous chloride in 100 mL of concentrated
hydrochloric acid were added to 3 mL of either supernatant
solution (sample) or standard pentose solution (221 mg/mL)
and mixed well. The solution was held in boiling water bath
for 40 min. After cooling, the color complex was measured
by reading the absorbance of pentose, whose represents as
intracellular materials leaked from the bacteria, at 665 nm
using double-beam spectrophotometer (UV-160A, Shimadzu)
(12). The results were expressed as pentose leakage (mg/mg dry
weight). Each experiment was performed in triplicate.
Sample preparation for electron microscopy: Equal
volumes (0.75 mL) of double-washed P. acnes suspension
at a cell density of 2 mg dry weight/mL and citronella oil in
1% Tween 80 in NSS were mixed to give the required final
concentrations of the oil. P. acnes treated with NSS were
used as control. After incubation at 37C under anaerobic
conditions for 30 min, the cells were double-washed with
NSS, centrifuged and the supernatant was removed. Sample
fixation method was modified from previous work (13). Firstly,
samples were fixed with the mixture of 5% glutaraldehyde and
1% osmium tetroxide in 0.2 M phosphate buffer and kept in
refrigerator overnight. The suspension was then washed three
times with 0.1 M phosphate buffers and twice with distilled
water, respectively.
Scanning electron microscopy (SEM): The method of
SEM was modified from Khunkitti et al. (14). The fixed samples
were freezed with liquid nitrogen for 1 min, kept at -40C
for 2 h and dried overnight in freeze-dryer. Finally, samples
were coated with gold before examined by Scanning Electron

Table I. Percentage composition of the identified chemical

constituents of citronella oil
Constituents
LRI
Percentage

limonene
linalool
citronellal
isopulegol
citronellol
geraniol
citral
citronellyl acetate
eugenol
geranyl acetate
b-elemene
a-amorphene
germacrene-D
a-muurolene
g-cadinene
d-cadinene
elemol
endo-1-bourbonanol

-
1100
1153
1164
1227
1255
1273
1352
1364
1383
1400
1487
1494
1510
1526
1534
1560
1590

Mode of
identification

3.3
0.7
34.4
0.3
11.1
23.4
0.6
5.2
0.7
6.3
1.8
0.4
3.6
0.8
0.8
2.9
2.7
0.5

a
a, b
a, b, c
a, b
a, b, c
a, b, c
a, b
a, b
a, b, c
a, b
a, b
a
a, b
a, b
a, b
a
a, b
a

Percentage were calculated on the basis of results obtained on Rtx-5 MS

column (unidentified compounds were not shown); LRI = linear retention index;
Mode of identification: a = mass spectra; b = LRI; c = comparing with authentic
compounds.

Table II. Susceptibility of Propionibacterium acnes to the major

constituents of citronella oil by broth microdilution method
(n=3)
Substance

MIC (mL/mL)

MBC (mL/mL)

Citronella oil
Citronellal*
Citronellol
Geraniol

0.078
3.12-6.25
1.95
0.78

0.625
1.56

* The data is indicated as a range due to the replications showing different values;
- = Not inhibit.

Table III. Pentose leakage and morphological changes of Propionibacterium acnes after 30 min-treated with citronella oil
Group

Concentration
(mL/mL)

Control
Citronella
oil

-
0.156
0.625
1.250
3.125
6.500
12.500
25.000
50.000
60.000
100.000

Pentose
leakage*
SD

Length SD

(mg/mg dry wt)

Morphology (n = 100)
(mm)
Round-end

Tapered-end

width SD

width SD

0.434 0.052
nd
0.391 0.056
nd
nd
nd
0.395 0.045
nd
0.400 0.047
nd
0.399 0.046

0.378 0.043
nd
0.346 0.037
nd
nd
nd
0.345 0.038
nd
0.351 0.041
nd
0.349 0.037

(n = 3)
-
1.610 1.552
2.062 0.832
4.300 0.789
10.538 2.150
20.087 0.437
20.624 2.391
20.794 1.989
25.864 3.821
13.909 3.334
12.665 2.955

0.943 0.209
nd
1.208 0.277
nd
nd
nd
1.238 0.278
nd
1.204 0.251
nd
1.232 0.262

nd = not determined; * P-value < 0.05 by Kruskal Wallis test when comparison between treated groups; P-value < 0.05 by Student t- test when comparison between control
and each treated group.

272/Journal of Essential Oil Research

Vol. 22, May/June 2010

Khunkitti et al.

Microscope (JSM-6460 LV, Jeol). One hundred cells per each

sample were randomly chosen from the SEM monitor. Length,
tapered-end width and round-end width of the chosen cells
were measured directly.
Transmission electron microscopy (TEM): The fixed
samples were double-washed with propylene oxide before
immersed in mixture of low viscosity embedding media: propylene oxide (1:2) overnight. The samples were then immersed
in mixture of low viscosity embedding media:propylene oxide
(2:1) for 3 h before immersed overnight in pure embedding
media. After that they were transferred into flat embedding
mold containing pure embedding media and incubated at
80C for 8 h. The resulting polymerized samples were then
cut into thin section and mounted on carbon-coated copper
grids, stained with 2% uranyl acetate in 50% methanol for 10
min and washed with 50% methanol. Finally, the thin sections
were stained with 0.4% lead citrate for 15 min and washed
with stain water. Ultrastructural examination of the samples
was performed using Transmission Electron Microscope
(JEM-1230, Jeol).
Statistical analysis: SPSS 11.5 for windows was used to
perform statistical analysis. The averages were expressed as
mean SD Student t-test was used to investigate morphological changes of P. acnes between each treated group and the
untreated one. Kruskal-Wallis was used to compare a difference of pentose leakage between various concentrations of
the essential oil treated groups. P values less than 0.05 were
considered statistically significant.

Results and Discussion

The constituents of citronella oil are listed in Table I. The
percent relative content of citronellal, geraniol, and citronellol
were 34.4, 23.4 and 11.1, respectively. The MICs of citronella oil
against P. acnes was less than geraniol, citronellol and citronellal,
respectively. In the range of concentrations tested (0.01225.0
mL/mL), citronellal and citronellol had no bactericidal effect.
However, MBCs of geraniol was 1.56 mL/mL and citronella
oil was 0.625 mL/mL (Table II). The time-kill curves of P.
acnes were, therefore, performed at the concentrations of 1 x
MBC, 20 x MBC, 80 x MBC and 160 x MBC, respectively. The
results revealed that viable count of P. acnes were reduced in
a dose-dependent manner (Figure 1). At high concentrations
of citronella oil (50 and 100 ml/mL), it took about 30 min to
reach 1-log reduction and about 3.5 h to reach 3-log reduction.
The results can be supported by the intercellular leakage and
morphological changes of P. acnes when exposed with the same
concentrations as the lethal curve (Table III and Figure 2). As
demonstrated in Table III, pentose leaked from P. acnes cells
treated with citronella oil for 30 min showed a biphasic pattern
with a significant difference between treated groups (P < 0.05).
As the concentrations increased, pentose leakage was gradually
increased and reached the maximum at the concentration of
50 mL/mL and the leakage was gradually decreased thereafter.
Morphological changes could be seen in Table III and Figure
2. The length, round-end and tapered-end width measured
from SEM micrographs revealed that P. acnes cells treated
with citronella oil at MBC and all above concentrations were
elongated and thinned in comparison with control (untreated
Vol. 22, May/June 2010

group). This aspect represented by the increasing of length but

the decreasing of round-end width including tapered-end width.
There were significant differences between the morphology of
P. acnes in control group and each citronella oil treated group
(P < 0.05).Among treated groups; P. acnes cells treated with
50 mL/mL of citronella oil (maximum pentose leakage concentration) showed the shortest length but adopted the longest
width at both round-end and tapered-end. The ultrastructure
of the cells investigated using TEM revealed that untreated P.
acnes possessed a rigid and complete cell structure (Figure 2a).
Whereas cell wall of the treated groups at all concentrations
was ruptured. The cytoplasmic membrane was also damaged
resulting in amorphous and depleted cytoplasmic content of
treated P. acnes (Figure 2b-c) and at maximum pentose leakage
(50 mL/mL), the cytoplasmic leakage were clearly seen (Figure
2d). At the highest concentration of the oil (100 mL/mL), the
cytoplasm was coagulated and precipitated (Figure 2e).
According to the chemical constituents of citronella oil,
it appeared to be the Java type. Although the major chemical compounds of the oil finding in this study was similar to
previous reports (9,15), their amounts were different. These
are probably affected by many factors such as the cultivation
conditions of the plant, variety and isolation techniques (16).
As seen in Table II, the susceptibility of P. acnes to the major
compounds alone was much less than that of citronella oil. It
implied that the anti-P. acnes activity of citronella oil might
be due to the synergistic effect of the oil components as well
as the amount of each components in the oil. In addition, the
activity of the oil could be related to the oil components and
the composition of the bacterial cell. Even though the major
compounds were considered for antimicrobial activity, it might
be noteworthy to consider that perhaps some minor compounds
may play a role in synergistic activities. As shown in Table III,
citronella oil caused intercellular leakage and morphological
changes. In this study, citronellal, which is a monoterpene
aldehyde, is considered to be a principle active component of
citronella oil. The potential targets of aldehydric antimicrobial
agents are membrane functional proteins causing membrane
permeability changes (17). The anti-P. acnes activity of monoterpene aldehydes found in this essential oil might be due to
electronegative compounds interfering with vital nitrogen
components of proteins at cytoplamic membrane, cytoplasmic
contents and nucleic acids. The similar findings against other
Gram-positive bacteria were also demonstrated (18,19). In
addition, monoterpene alcohols found in citronella oil were
geraniol, citronellol, linalool and isopulegol while sesquiterpene
alcohol was elemol and endo-1-bourbonanol. The total alcoholic
compounds were 38.7% while phenolic compound namely
eugenol was 0.7%. It is generally known that alcohol possess
bactericidal rather than bacteriostatic activity. However, the
activity of alcoholic compounds in the oil were dose dependent
that evidenced by the time-killing profiles. Alcohol compounds
probably act as dehydrating agents at low dose and as protein
denature at higher dose (18). In addition, alcohols and phenols
cause cytoplasmic membrane rupture and cell wall damage on
bacterial cells (2022). This study revealed that cell wall of the
treated groups lost rigid structure and the wall components were
ruptured. Consequently, cytoplasmic membrane was damage
leading to progressive leakage of intracellular materials and
Journal of Essential Oil Research/273

P. acnes

cell lysis (Figure 2b-e). Horne et al (23) found that citronellol

and geraniol appeared to be the substances causing gross cell
wall and cytoplasmic membrane damage and provoking lysis of
Streptococcus pneumoniae. Similarly, the study of de Billerbeck
et al. (24) which was found that citronella oil might cause the
hyphal diameter and the hyphal wall thinning as well as caused
plasma membrane damage and disorganized the mitochondria
structure of Aspergillus niger by causing irregularity of the
plasmalemma and the formation of lomasomes. Additionally,
the monoterpene esters in this essential oil were citronellyl
acetate (5.2%) and geranyl acetate (6.3%). Esters can disrupt
bacterial cytoplasmic membrane and hence causing leakage
of intracellular constituents (20,25). Apart from the major
constituents in the oil, there were sesquiterpene hydrocarbons
e.g. b-elemene, a-amorphene, germacrene-D, a-murolene,
and d-cadinene. Particularly, d-cadinene exhibited antibacterial
activity against P. acnes (26) and Staphylococcus aureus (27).
Moreover, germacrene D was weakly active against Proteus
vulagaris, Bacillus cereus and Staphylococcus epidermidis (19).
In this study, d-limonene was only one monoterpene hydrocarbon found in the oil (3.25%), its antibacterial activities have
been reported (18,28,29). Limonene might probably destroy
the cellular integrity and inhibited respiratory activity of the
bacteria (30). Since the essential oil of citronella possessed
pronounced antibacterial activity against P. acnes. Citronellal, geraniol and citronellol would be the key constituents of
citronella grass oil accounting for acne control efficacy. Due to
the complexity of its chemical constituents, the sites of action
of citronella oil might depend on the combination effect of its
chemical constituents.
Acknowledgments

The authors thank the Co-operative Research Network (CRN)

grant in Pharmacy, Ministry of Education, Thailand and The Khon
Kaen Universitys Graduate Research for supporting this study.
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Vol. 22, May/June 2010