(tea tree) oil
, C.F. Carson
and T.V. Riley
Discipline of Microbiology, School of Biomedical and Chemical Sciences, The University of Western Australia, Crawley, WA, Australia,
Division of Microbiology and Infectious Diseases, Western Australian Centre for Pathology and Medical Research, Queen
2003/159: received 26 February 2003, revised and accepted 13 June 2003
A B S T R A C T
K . A . H A M M E R , C . F . C A R S O N A N D T . V . R I L E Y . 2003.
Aims: To investigate the in vitro antifungal activity of the components of Melaleuca alternifolia (tea tree) oil.
Methods and Results: Activity was investigated by broth microdilution and macrodilution, and time kill methods.
Components showing the most activity, with minimum inhibitory concentrations and minimum fungicidal
£0Æ25%, were terpinen-4-ol, a-terpineol, linalool, a-pinene and b-pinene, followed by 1,8-cineole.
The remaining components showed slightly less activity and had values ranging from 0Æ5 to 2%, with the exception
of b-myrcene which showed no detectable activity. Susceptibility data generated for several of the least water-
soluble components were two or more dilutions lower by macrodilution, compared with microdilution.
Conclusions: All tea tree oil components, except b-myrcene, had antifungal activity. The lack of activity reported
for some components by microdilution may be due to these components becoming absorbed into the polystyrene of
the microtitre tray. This indicates that plastics are unsuitable as assay vessels for tests with these or similar
Signiﬁcance and Impact of the Study: This study has identiﬁed that most components of tea tree oil have
activity against a range of fungi. However, the measurement of antifungal activity may be signiﬁcantly inﬂuenced by
the test method.
Keywords: Candida albicans, 1,8-cineole, monoterpenes, tea tree oil, terpinen-4-ol.
I N T R O D U C T I O N
The essential oil of Melaleuca alternifolia, also known as tea
tree oil, is commonly used in Australia as a topical
therapeutic agent. The medicinal uses of tea tree oil relate
primarily to the anti-inﬂammatory (Brand et al. 2002; Koh
et al. 2002) and antimicrobial (Carson et al. 2002; Hammer
et al. 2002) properties of the oil. Use as a topical antimi-
crobial agent is supported by a growing body of clinical data
indicating that tea tree oil is effective in the treatment of
infections or conditions such as herpes labialis (Carson et al.
2001), acne (Bassett et al. 1990), tinea (Tong et al. 1992;
Satchell et al. 2002a), onychomycosis (Buck et al. 1994),
dandruff (Satchell et al. 2002b) and oral candidiasis (Vaz-
quez and Zawawi 2002), and in the clearance of methicillin-
resistant Staphylococcus aureus carriage (Caelli et al. 2000).
In addition, several recent publications have characterized
the in vitro activity and mechanisms of action of tea tree oil
against bacteria (Cox et al. 2000; Mann et al. 2000; Carson
et al. 2002) and, to a lesser extent, fungi (Hammer et al.
2000, 2002). However, little is known about the in vitro
activity of tea tree oil components against fungi.
The components of tea tree oil, of which there are ca 100,
are mostly monoterpenes, sesquiterpenes and their related
alcohols (Brophy et al. 1989). The chemical composition of
tea tree oil is well characterized and the International
Standard ISO 4730 for oil of Melaleuca, terpinen-4-ol type
(tea tree oil) contains a chromatographic proﬁle that
Correspondence to: Katherine A. Hammer, Discipline of Microbiology (M502),
School of Biomedical and Chemical Sciences, The University of Western Australia,
35 Stirling Hwy, Crawley, WA 6009, Australia (e-mail: khammer@
ª 2003 The Society for Applied Microbiology
Journal of Applied Microbiology 2003, 95, 853–860
values for 14 components (International Organisation for
Standardisation 1996). The three major components, terp-
inen-4-ol, c-terpinene and a-terpinene, comprise ca 70% of
whole oil while q-cymene, terpinolene, a-terpineol and a-
pinene account for ca 15% of the oil (Brophy et al. 1989).
Given the lack of data relating to the antifungal activity of
the components of tea tree oil, the purpose of this study was
to examine the in vitro susceptibility of some medically
important fungi to tea tree oil components, using several
different investigative tools.
M A T E R I A L S A N D M E T H O D S
A total of 14 fungal isolates were obtained from the
Discipline of Microbiology at The University of Western
Australia and the Division of Microbiology and Infectious
Diseases at The Western Australian Centre for Pathology
and Medical Research. They included reference and
clinical isolates and were Candida albicans ATCC 10231,
C. albicans ATCC 90028, C. parapsilosis ATCC 90018,
Saccharomyces cerevisiae ATCC 10716, Trichosporon sp.,
Rhodotorula rubra, Epidermophyton ﬂoccosum, Microsporum
canis, Trichophyton mentagrophytes var. interdigitale, T. men-
tagrophytes var. mentagrophytes, Aspergillus niger, A. ﬂavus,
A. fumigatus and Penicillium sp. Yeast isolates were grown
and maintained on Sabouraud dextrose agar (SDA) stored
°C, and ﬁlamentous fungi were grown and main-
Tea tree oil and components
Tea tree oil (batch 971) was kindly supplied by Australian
Plantations Pty Ltd (Wyrallah, NSW, Australia) and its
composition complied with the International Standard ISO
4730 (International Organisation for Standardisation 1996).
The following tea tree oil components, listed together with
their percentage composition in batch 971, were investigated
for antifungal activity: (+)-terpinen-4-ol (41Æ5%) (Fluka
Chemie AG, Buchs, Switzerland); c-terpinene (21Æ2%)
(Aldrich Chemical Company Inc., Milwaukee, WI, USA);
a-terpinene (10Æ2%) (Sigma Chemical Co., St Louis, MO,
USA); terpinolene (3Æ5%) (Fluka); a-terpineol (2Æ9%)
(Aldrich); a-pinene (2Æ5%) (Aldrich); 1,8-cineole (2Æ1%)
(Sigma); q-cymene (1Æ5%) (Aldrich); (+)-aromadendrene
(1Æ0%) (Fluka); (+)-limonene (0Æ9%) (Sigma); b-myrcene
(Sigma); (+)-b-pinene (Fluka); (±)-linalool (Sigma) and
))-(a)-phellandrene (Fluka). The concentrations of
971 of tea tree oil were not determined. All components
‡97% purity, except for terpinolene, b-myrcene and
Broth microdilution assay
Yeast inocula were prepared by growing isolates on SDA for
24–48 h at 35
°C and then suspending growth in ca 2 ml of
sterile distilled water (SDW). The density of this suspension
was adjusted to 1 McFarland, and then serially diluted in
SDW to correspond to a ﬁnal inoculum concentration of
. Inocula for the remaining fungi
were prepared as described previously (Hammer et al.
2002). Final inocula concentrations for all organisms were
conﬁrmed by viable counts.
Broth microdilution assays were performed according to
National Committee for Clinical Laboratory Standards
(NCCLS) methods (NCCLS 1997, 1998) with minor
modiﬁcations. Brieﬂy, doubling dilutions of components,
with ﬁnal concentrations ranging from 8 to 0Æ002% (v/v)
were prepared in 96-well microtitre trays (Becton Dickinson
Labware, Franklin Lakes, NJ, USA) in RPMI Medium 1640
(Gibco BRL, Grand Island, NY, USA). Tween 80 (Sigma)
was included at a ﬁnal concentration of 0Æ001% (v/v) to
enhance the solubility of each component or tea tree oil. The
activity of terpinen-4-ol, terpinolene, 1,8-cineole, c-terpin-
ene, a-terpinene, q-cymene, a-terpineol and b-myrcene
against C. albicans ATCC 10231 was also determined with a
ﬁnal concentration of 0Æ1% Tween 80 to ascertain whether
an increased concentration of surfactant signiﬁcantly inﬂu-
enced results. For yeasts, minimum inhibitory concentra-
tions (MICs) and minimum fungicidal concentrations
(MFCs) were determined by subculturing 10 ll from each
well of the microtitre tray, spot inoculating onto SDA and
incubating aerobically at 35
°C. MICs were determined as
the lowest concentration of agent resulting in the mainten-
ance or reduction of the inoculum and MFCs were
determined as the lowest concentration of agent resulting
in no growth. For the remaining fungi, MICs were
determined visually as described by the NCCLS (1998)
and MFCs were determined by subculture as described
previously (Hammer et al. 2002). All isolates were tested on
at least two separate occasions and were re-tested if resultant
MIC or MFC values differed. Modal values were then
Broth macrodilution assay
The activity of tea tree oil and components against
C. albicans ATCC 10231 was also determined by the broth
macrodilution method. Doubling dilutions of tea tree oil or
component, with ﬁnal concentrations ranging from 2 to
0Æ016% (v/v) were prepared in RPMI Medium 1640 in
0Æ5 ml volumes in glass McCartney bottles, with a ﬁnal
ª 2003 The Society for Applied Microbiology, Journal of Applied Microbiology, 95, 853–860, doi:10.1046/j.1365-2672.2003.02059.x
ﬁnal concentration of 0Æ001% (v/v) Tween 80 showed that
results for terpinolene and c-terpinene were not reprodu-
cible whereas results with 0Æ1% Tween 80 were. Inocula
were prepared as described for the microdilution assay and
volumes of 0Æ5 ml were added to each dilution of oil or
component. Dilutions were incubated at 35
statically or with shaking. After 24 h incubation, MICs
and MFCs were determined as described above. For a-
terpineol, terpinen-4-ol, a-terpinene and q-cymene, MICs
and MFCs were also determined after 48 h by incubating
dilutions for a further 24 h.
Time kill assay
Inocula for the time kill assays were prepared by inoculating
one to two colonies of C. albicans ATCC 10231 into ca 8 ml
of Sabouraud dextrose broth (SDB) and incubating for 18 h
centrifugation for 3 min at 1300 g, washed twice in SDW,
and ﬁnally resuspended in phosphate-buffered saline (PBS)
pH 7Æ4 to 6
. This was halved
upon inoculation, resulting in a starting inoculum concen-
tration of ca 5
. Treatments containing
component or whole oil at one or more ﬁnal concentrations
ranging from 1 to 0Æ125% (v/v) were prepared in 1 ml
volumes of PBS with a ﬁnal concentration of 0Æ001%
Tween 80, which was similar to controls. At 1-min
intervals, 1 ml of inocula was added to each treatment
and mixed for 20 s. Treatments were incubated with
shaking at 35
°C and samples were taken at 0Æ5 and
30 min, and at 1, 2, 3, 4 and 6 h. Viable counts were
performed by serially diluting each sample 10-fold in SDW
and spreading 100 ll volumes from the appropriate dilu-
tions onto SDA in duplicate. Alternatively, duplicate pour
plates were prepared by aliquoting 1 ml of the appropriate
dilution into the centre of an empty 90 mm plastic Petri
dish and adding ca 19 ml of molten, cooled SDA. Petri
dishes were swirled during and after the addition of agar to
ensure even mixing. After incubation at 35
°C, plates with
30–300 colonies were counted and viable counts deter-
mined. The limit of detection, calculated from 30 colonies
in the 10
dilution, was 3
plates or 3
for pour plates. Assays were
repeated at least twice and mean and standard error values
were calculated from viable count data.
Viable count data from time kill assays were compared
using a Student’s t-test (two-tailed, two-sample assuming
unequal variance). P-values of <0Æ05 were considered
R E S U L T S
Broth microdilution assay
Data obtained by broth microdilution are shown in
Table 1. In addition, MICs of a-pinene were 0Æ008% for
A. niger and 0Æ016% for A. ﬂavus, A. fumigatus and
Penicillium sp. MFCs of a-pinene were 0Æ03% for Penicil-
lium sp. and 0Æ016% for A. niger, A. ﬂavus and A. fumig-
atus. For the dermatophytes, MICs of a-pinene were
<0Æ004% and further testing of these organisms, and yeasts
other than C. albicans ATCC 10231, was not pursued.
Components with the lowest MICs and MFCs were
terpinen-4-ol and a-terpineol, followed by 1,8-cineole. In
contrast, a-terpinene, c-terpinene and q-cymene showed
little activity. Comparison of the different fungal groups
showed that the dermatophytes were most susceptible to
components, with the lowest MICs and MFCs for each
component often occurring within this group. The group
with the highest MICs was the yeasts and the highest
MFCs, disregarding values of >8, were observed among
the nondermatophyte ﬁlamentous fungi. MICs and MFCs
obtained with the increased Tween concentration (0Æ1%)
were either equivalent or one concentration lower for
terpinen-4-ol, 1,8-cineole and tea tree oil, compared with
values obtained with 0Æ001% Tween. For q-cymene,
c-terpinene and b-myrcene all values remained unchanged
at >8% whereas values for a-terpinene were 8% with
increased Tween. For terpinolene, values were consider-
ably lower with 0Æ1% Tween 80, with an MIC of 1Æ0% and
MFC of 2Æ0%, compared with values of >8% obtained
with 0Æ001% Tween.
By the macrodilution method, all components except
b-myrcene showed activity at
£2Æ0% (Table 2). Comparison
of values obtained at 24 and 48 h showed that MICs and
MFCs did not change for terpinen-4-ol and a-terpineol
(data not shown).
a-terpinene and q-cymene were one or more concentrations
higher at 48 h compared with 24 h, for assays conducted
standing and with shaking. Similarly, comparison of results
obtained with standing or shaking did not differ for
terpinen-4-ol, a-terpineol, terpinolene and tea tree oil at
24 h. However, for c-terpinene and a-terpinene, MICs were
1Æ0% when obtained standing, compared with 0Æ5%
obtained with shaking. Susceptibility data obtained by
micro- and macrodilution methods were equivalent or
differed by only one dilution for terpinen-4-ol, a-terpineol,
a-pinene, aromadendrene, a-phellandrene and tea tree oil.
In contrast, values were two or more concentrations lower
by macrodilution for terpinolene, 1,8-cineole, c-terpinene,
a-terpinene, q-cymene, limonene and linalool.
C O M P O N E N T S O F T E A T R E E O I L
Results of time kill assays are shown in Fig. 1. Data for 1%
c-terpinene, a-terpinene and q-cymene were very similar
and were indistinguishable by statistical analyses. The
results for c-terpinene only are shown as a representative
(Fig. 1e). Treatments causing decreases in viability of >3
log CFU ml
within 30 min were 0Æ25% terpinen-4-ol, 1%
viable organisms were recovered from either 1,8-cineole
treatment. Treatments causing similar decreases in viability
but over a longer time period were 0Æ25% a-terpineol, 1%
a-terpinene, 1% c-terpinene, 1% q-cymene, and 0Æ25 and
0Æ5% tea tree oil. Treatments having only moderate or
negligible effects on C. albicans viability were 0Æ12%
a-terpineol, terpinen-4-ol and tea tree oil, 0Æ25% 1,8-cineole
and 1% terpinolene.
D I S C U S S I O N
Quantiﬁcation of the antimicrobial activity of particular
essential oil components appears to be heavily inﬂuenced by
the test method, as evidenced by the large differences
between the MICs and MFCs obtained by the broth micro-
and macrodilution methods. While technical factors such as
Table 2 In vitro activity of tea tree oil and components (% v/v)
against Candida albicans ATCC 10231 obtained by the broth micro-
dilution and macrodilution methods
Tea tree oil/
MIC, minimum inhibitory concentration; MFC, minimum fungicidal
*Final concentration of 0Æ001% Tween 80, data obtained at 48 h.
Final concentration of 0Æ1% Tween 80, data obtained at 24 h.
0 1 2 3 4 5 6
Log CFU ml
Fig. 1 Time kill curves of (a) tea tree oil, (b)
terpinen-4-ol, (c) a-terpineol, (d) 1,8-cineole,
(e) c-terpinene and (f) terpinolene against
Candida albicans ATCC 10231. Cells were
treated with 0% (j), 0Æ12% (
), 0Æ25% (m),
oil (v/v). Mean ±
tration of Tween 80 may have contributed to the contra-
dictory results, differences may also relate speciﬁcally to the
physical, molecular and chemical characteristics of tea tree
oil components. Most terpenes are of only limited solu-
bility in aqueous media. For example, the solubilities of
c-terpinene, a-terpinene, q-cymene, terpinolene and limon-
ene have been reported as being between 1Æ0 and 8Æ2 ppm
(Grifﬁn et al. 1999). A major consideration for antimicrobial
activity assays is, therefore, how to achieve and maintain
adequate solubilization of the compound and physical
contact with the test organism. Surfactants have often been
incorporated into these assays to address this issue (Janssen
et al. 1987). In the current study, increasing the Tween 80
concentration from 0Æ001 to 0Æ1% in the microdilution assay
did not result in signiﬁcantly lower MICs or MFCs, with
the exception of terpinolene. This suggests that the lack of
activity observed for these components in the microdilution
assay was not solely due to inadequate solubilization. The
prospect that the tea tree oil components may be dissolving
into or becoming otherwise irreversibly associated with the
polystyrene of the microtitre tray is supported by data in the
literature. Tea tree oil is known to interact with certain
plastic types and has been shown to both deform and
migrate through polymers such as low-density polyethylene
(Rowe 1999), although these effects are poorly documented.
The sorption of these less water-soluble components into
polystyrene has also been used as a technique for removing
them from solutions of tea tree oil (Brand et al. 2001).
Removal of these compounds by their association with the
polystyrene results in less of the component being in
solution and available to interact with the test organism, and
may explain why MICs and MFCs were considerably lower
when performed in glass bottles. Interestingly, MICs and
MFCs for terpinen-4-ol, a-terpineol and tea tree oil did not
differ signiﬁcantly between methods, suggesting that the use
of microtitre trays, although not ideal, may still be
acceptable for these components and oil. Component
solubility must be considered when designing assays for
evaluating the antimicrobial activity of essential oil compo-
nents such as terpenes. Methods such as disc or well
diffusion are particularly unsuitable given that the size of the
zone of inhibition is dependent on the diffusion of mostly
water-insoluble compounds through an aqueous agar
medium (Janssen et al. 1987; Carson and Riley 1995).
Methodological considerations aside, most of the compo-
nents tested showed antifungal activity, and by grouping
those components similar in chemical composition and
structure, generalizations about their antifungal activity can
1,8-cineole and linalool had relatively good antifungal
activity with MICs and MFCs for some components that
were slightly lower those of tea tree oil. In addition,
terpinen-4-ol, a-terpineol and 1,8-cineole showed relatively
rapid killing effects against C. albicans in time kill assays.
Previously published data for both fungi and bacteria are in
agreement with these results (Moleyar and Narasimham
1986; Carson and Riley 1995; Grifﬁn et al. 1999; Adegoke
et al. 2000; Cox et al. 2001; Inouye et al. 2001). Although
linalool differs slightly from the other monoterpene alcohols
because it is acyclic, this component still showed signiﬁcant
antifungal activity. This suggests that the presence of the
alcohol moiety is a greater determinant of antifungal activity
than whether the component has a cyclic or acyclic
structure. The antimicrobial activity of terpenes, including
the monoterpene alcohols, has been attributed to their
interactions with cellular membranes (Sikkema et al. 1995).
At relatively low concentrations, these interactions may
result in changes such as inhibition of respiration (Uribe
et al. 1985) and alteration in permeability (Uribe et al. 1985;
Cox et al. 2000) and at higher concentrations effects such as
total loss of homeostasis, gross membrane damage and death
may occur (Carson et al. 2002). The monoterpene alcohols
are thought to be particularly antimicrobially active because
of their relatively high water solubility and the presence of
the alcohol moiety (Grifﬁn et al. 1999; Dorman and Deans
pinolene, q-cymene, limonene and a-phellandrene showed
MICs and MFCs by macrodilution that were one or two
concentrations higher than those for tea tree oil. Similarly,
time kill assays showed these components to have antifun-
gal activity, although the rate of killing for c-terpinene,
a-terpinene, terpinolene and q-cymene could not be regar-
ded as rapid. Relatively slow kill rates have been reported
previously for these compounds (Cox et al. 2001). Earlier
reports of antimicrobial activity for these components are
confounded by methodological issues and a lack of
meaningful data for the ﬁlamentous fungi. As a result,
activity ranging from negligible (Carson and Riley 1995;
Dorman and Deans 2000) through to moderate or good
(Moleyar and Narasimham 1986; Himejima et al. 1992;
Grifﬁn et al. 1999; Adegoke et al. 2000; Cox et al. 2001)
has been reported for these compounds. While not as active
as the monoterpene alcohols, these compounds are likely to
be active by similar mechanisms but activity may be limited
by their low solubilities in both aqueous media and
microbial membranes. Aromadendrene, the only sesquiter-
pene tested, showed activity similar to the monocyclic
terpenes, although a previous report found little antimi-
crobial activity, albeit by well diffusion (Dorman and
Myrcene, an acyclic monoterpene, showed no antifungal
activity, which is consistent with the little amount of
published data available for this component (Dorman and
K . A . H A M M E R ET AL.
for this acyclic monoterpene suggests that the cyclic
structure of the cyclic monoterpenes may be contributing
signiﬁcantly to their activity. However, generalizations from
this study are limited since only one nonalcohol acyclic
terpene was tested.
The bridged bicyclic terpenes a- and b-pinene showed
considerable antifungal activity, with b-pinene showing the
most. In general, these data are in agreement with previous
studies (Himejima et al. 1992; Adegoke et al. 2000),
although some studies have demonstrated little activity for
these components (Raman et al. 1995; Consentino et al.
1999; Mourey and Canillac 2002). The present study
showed that b-pinene was more active against C. albicans
than a-pinene, whereas previously, activities have been
shown as equivalent for C. utilis (Himejima et al. 1992) or
a-pinene was shown as the more active against C. albicans
(Grifﬁn et al. 1999). There is no clear consensus yet as to
which pinene isomer is more antimicrobially active and the
differing activities of the enantiomers of both compounds
ought not to be discounted (Lis-Balchin et al. 1999). The
solubility of terpenes is hypothesized as correlating with
their antimicrobial activity (Sikkema et al. 1995). However,
the pinenes are examples of compounds with very low water
solubilities (Grifﬁn et al. 1999) but relatively high antimi-
crobial activities. The reasons for this are so far unknown,
but may relate to properties of these components other than
With the use of appropriate methods, this study has
identiﬁed that most of the components of tea tree oil have
activity against C. albicans and other fungi. This contradicts
several previous reports (Raman et al. 1995; Consentino
et al. 1999; Cox et al. 2001) and challenges beliefs about the
various attributes and properties of tea tree oil components,
such as that tea tree oil contains a single ÔactiveÕ component
terpinen-4-ol while many of the other components lack
activity (Mann et al. 2000).
The importance of investigating the activity of tea tree oil
components lies in gaining an understanding of the activity
of each component, and of how each component contributes
to the activity of the whole oil. Although some of the
components tested in this study are present at only very low
levels in whole oil, each may contribute to total activity and
attempts to eliminate components considered inactive may,
therefore, be counter-productive. In addition, several
aspects of the properties of tea tree oil components such
as synergistic action between two or more components or
other beneﬁcial pharmacological or medicinal properties
have not yet been explored fully.
In conclusion, this study showed that a- and b-pinene and
the monoterpene alcohols had the lowest MICs and MFCs
against fungi, followed by the monocyclic terpenes. Data
from this study suggests that the use of polystyrene
microtitre trays in microdilution assays may result in an
underestimation of the antimicrobial activity of some
essential oil components and should, therefore, be avoided.
Appropriate methods for determining the susceptibility of
microorganisms to essential oil components require further
investigation, as does the activity of these components
A C K N O W L E D G E M E N T S
The assistance of the Discipline of Microbiology at The
University of Western Australia and the Division of
Microbiology and Infectious Diseases at The Western
Australian Centre for Pathology and Medical Research in
obtaining isolates is appreciated. This work was supported
by grants UWA-57A and 58A from the Rural Industries
Research and Development Corporation, Australia, and
Australian Bodycare Pty Ltd (Vissenbjerg, Denmark).
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