Letters in Applied Microbiology 1998, 26, 194198

Effects of tea tree oil on Escherichia coli

J.E. Gustafson1,3, Y.C. Liew1,3, S. Chew2, J. Markham4, H.C. Bell5, S.G. Wyllie4 and
J.R. Warmington1,3
1

Microbiology Group and 2Histology Group, School of Biomedical Sciences, Curtin University of Technology, Perth,
Western Australia, 3Genetica Biotechnologies, Bentley, Western Australia, 4School of Science and Centre
for Biostructural and Biomolecular Research, University of Western Sydney, Hawkesbury, New South Wales, and
5
Australian Tea Tree Oil Research Institute, Southern Cross University, Lismore, New South Wales, Australia
1607/97: received 14 July 1997 and accepted 22 October 1997
J .E . G U ST AF S ON , Y . C. LI E W, S. C HE W, J . M AR K HA M, H .C . B E LL , S . G. WY L LI E A N D J .R .

Tea tree oil (TTO) stimulates autolysis in exponential and stationary

phase cells of Escherichia coli. Electron micrographs of cells grown in the presence of
TTO showed the loss of electron dense material, coagulation of cell cytoplasm and formation
of extracellular blebs. Stationary phase cells demonstrated less TTO-stimulated
autolysis and also had greater tolerance to TTO-induced cell death, compared to exponentially
grown cells. It was also revealed that a subpopulation of stationary phase cells
demonstrated increased tolerance to TTO-bactericidal effects.

W AR MI N GT ON . 1998.

INTRODUCTION

The essential oil of the tea tree, Melaleuca alternifolia, is a

topical antimicrobial which has been in use since early in this
century (Carson and Riley 1993) by Europeans. Tea tree oil
has the ability to kill a wide range of medically important
micro-organisms (Carson and Riley 1993, 1994; Shapiro et al.
1994; Carson et al. 1995; Belaiche et al. 1996; Hammer et al.
1996) and contains over 100 components, of which a-pinene,
terpinen-4-ol, linalool and a-terpineol are lipophilic monoterpenes and the major active antimicrobial components of
TTO (Carson and Riley 1995; Kim et al. 1995; Raman et al.
1995). Despite its widespread acceptance as a natural antimicrobial, nothing is known about the effects TTO has on
micro-organisms. Studies have been undertaken to determine
the effects of TTO on Escherichia coli and provide insights
into the bactericidal action of TTO.

Riley 1993) and 16% TTO (Main Camp TTO, Ballina,

NSW, Australia; batch no. 6081). Serial dilutions of this
mixture were made until a TTO concentration of 00625%
was achieved. Dilution tubes contained a final volume of 1
ml and were stored at 4 C until use. Escherichia coli strain
AG100, a K-12 derivative (George and Levy 1983), was
grown at 37 C in Iso-Sensitest (Oxoid) broth for 1618 h
with shaking (200 rev min1) and diluted to an optical density
of 01 (05 McFarland standard) at A625 nm with Iso-Sensitest
broth containing 05% Tween-80. The serial dilutions of
TTO were then inoculated with 1ml of the diluted culture
and incubated at 37 C for 1620 h with constant shaking.
Iso-Sensitest agar plates were then inoculated with samples
from the dilutions and incubated at 37 C to determine colony
forming units (cfu). The minimum bactericidal concentration
(MBC) was defined as the concentration of TTO that killed
the entire inoculum and was equivalent to the minimum
inhibitory concentration (MIC).

MATERIALS AND METHODS

Minimum bactericidal and inhibitory concentration
determination

Iso-Sensitest (Oxoid, Basingstoke, UK) broth was prepared

containing 05% Tween-80 (BDH, Poole, UK) (Carson and
Correspondence to: J.E. Gustafson, Microbiology Group, School of
Biomedical Sciences, Curtin University of Technology, Perth, Western
Australia 6001 (e-mail: tgustafs@alpha2.curtin.edu.au).

Whole cell autolysis assay

A 2% inoculum from the 1620 h AG100 culture was used

to initiate growth in two flasks containing 100 ml of IsoSensitest broth with 05% Tween-80 (BDH). One culture
was incubated at 37 C with shaking until an optical density
of 10 was reached at A600 nm (exponential phase cells). The
other culture was incubated with shaking at 37 C for 48 h
(stationary phase cells). Both cultures were harvested by cen 1998 The Society for Applied Microbiology

T EA TR E E O IL E FF EC T S O N E . CO LI 195

trifugation (3000 g, 4 C) and washed with sterile cold (4 C)

distilled water before being resuspended in 10 ml of 50 mmol
l1 Tris-Cl buffer (pH 70) containing 05% Tween-80 and
split into 5 ml aliquots in two separate sterile tubes. To one
tube, TTO was added to a final concentration equal to 1
the MBC for AG100 (025%). These tubes were incubated
at 37 C with shaking and optical density was measured at
intervals.

Time kill assays

A 2% inoculum from a 1620 h overnight AG100 culture

was used to inoculate two, 10 ml Iso-Sensitest broth cultures.
One culture was allowed to grow to exponential phase while
the other was grown till the cells reached stationary phase as
described above. Cells were then harvested and washed with
cold sterile distilled water, reharvested and resuspended in
10 ml of 50 mmol l1 Tris-Cl (pH 70) containing 05%
Tween-80. Cell suspensions were then divided into two equal
5 ml portions (control and test) and the appropriate dilutions
were made to determine total cell number in each tube. Tea
tree oil was added to one of the cell suspensions to a final
concentration equalling the MBC for AG100 (025%) or 2
the MBC. Both tubes were incubated at 37 C with shaking
(200 rev min1) and at intervals, dilutions were made for
both tubes and plated onto Luria broth (Gibco BRL, Paisley,
UK) (LB) agar plates. All plates were then incubated at
37 C for 1618 h, cfu were counted and time kill plots were
constructed.

RESULTS
Stimulation of whole cell autolysis by TTO and the
MBC-MIC for AG100

The TTO MBC and MIC for AG100 was 025%. After 4 h,
the percentage initial absorbance (%A) of exponential phase
AG100 cells dropped by 63% in the presence of the MBC
for TTO, while that of the control dropped by only 139%
(Fig. 1). In similar experiments using cells grown in LB
media, 05 the MBC also stimulated autolysis of exponentially grown AG100 cells but not as well as the TTO
MBC, and greater than 2 the TTO MBC (data not shown).
After 4 h, the % A of stationary phase AG100 cells dropped
by 23% in the presence of the MBC for TTO, while that of
the control dropped by only 10% (Fig. 1). Little difference
in whole cell autolytic rates was found between unstimulated
stationary and exponential phase cells.
Time kill assays

A representative of three independent experiments which all

showed similar results is shown in Fig. 2. Fifteen minutes
after the resuspension of cells in buffer containing the MBC
for TTO, the number of exponentially grown AG100 cells
fell by 746 log units (Fig. 2). By 30 min, there were no viable
cells present in a cell suspension which initially had a total
cell number of 43 108 cfu ml1. With stationary phase

Preparation of samples for electron microscopy

Three AG100 cultures were allowed to grow to exponential

phase in LB media at which time 05 or 1 the MBC of
TTO was added to one culture; growth of the two cultures
was allowed to continue for an additional hour. Following
the additional growth time, 15 ml aliquots of these cultures
were transferred to a microfuge tube and the cells were harvested by centrifugation in a microfuge at full speed for 5 min.
The cell pellet was immediately resuspended in 15 ml of
25% glutaraldehyde in 005 mol l1 cacodylate buffer
(pH 72) and fixed at room temperature for 2 h. The cells were
reharvested as described above and resuspended in 05 ml of
25% glutaraldehyde in 005 mol l1 cacodylate buffer until
fixation. The cells were then washed in two changes of cacodylate buffer and stained in tannic acid solution overnight
(Bal and Ramaswamy 1982). The stained cells were embedded
in 125% agar followed by processing in graded alcohols,
epoxy propane and Epon 812 and curing overnight at 70 C.
Ultra-thin sections were cut and stained with uranyl acetate
followed by lead citrate.

Fig. 1 Stimulation of autolysis of exponential and stationary

phase AG100 cells without any additions and in the presence of the
MBC of TTO. Each data point represents the mean of three
experiments. The error bars represent the standard deviation for
each data point. (), Exponential phase AG100; (),
exponential phase AG100+TTO; (), stationary phase
AG100; (), stationary phase AG100+TTO

1998 The Society for Applied Microbiology, Letters in Applied Microbiology 26, 194198

196 J .E . G U ST AF S ON ET A L .

One hour after the addition of the TTO MBC, there are
no viable cells in a previously exponentially growing LB
culture. Some cells, even though dead, still appear to have
intact membranes and cell walls after growth in the presence
of TTO (data not shown).

DISCUSSION

Fig. 2 Representative time kill plot of exponential and stationary

phase AG100 cells without any additions and in the presence

of the MBC of TTO. (), Exponential phase cells; (),
exponential phase cells +TTO; (), stationary phase cells;
(), stationary phase cells +TTO

cells, the number of viable cells dropped dramatically after

15 min (353 log units); then cell death continued at a much
slower rate for the next 105 min, after which the final number
of viable cells was found to equal 10 105 cfu ml1. After
45 min, 2 the TTO MBC was able to kill all the stationary
phase cells.
With non-growing exponential phase cells, after 30 min,
there is a drop in viable cell number from 43 108 cfu ml1
to 0 in cells treated with the TTO MBC, and only a 10%
drop in percentage A in TTO-stimulated autolysis assays
(Figs 2 and 3). In similar assays using stationary phase cells,
the TTO MBC caused a drop in viable cell number from
11 109 to 50 105 cfu ml1 and a drop in percentage A
of only 3% after 30 min (Figs 2 and 3).
Electron micrographs of exponentially growing cells
treated with TTO

Figure 3(a) is a representative electron micrograph of AG100

cells in exponential phase. Figure 3(b) is a representative
picture of AG100 cells in exponential phase treated with
05 the TTO MBC for 1 h while growing in LB media.
Following growth with TTO, there is a loss of cellular electron dense material, coagulation of cytoplasmic constituents
(indicated by asterisks in Fig. 3b) and the formation of extracellular electron dense blebs (indicated by arrows in Fig. 3b)
on the surface of treated cells. None of these aberrations was
observed in untreated cells.

The effects of TTO on growing and non-growing E. coli is

reported. It is clear from the data that both exponential and
stationary phase cells appear to die much more rapidly than
they autolyse following the addition of TTO. One hour after
the addition of the TTO MBC, there are no viable cells in
a previously exponentially growing LB culture. However,
electron microscopy reveals that some of these dead cells still
retain a cell wall structure similar to untreated cells. This
information suggests that autolysis is a secondary event that
occurs following TTO induced cell death.
The loss of electron dense material from the cells treated
with TTO indicates the loss of cell constituents and breakdown of the cell wall. The coagulated material observed in
some cells grown in the presence of TTO could represent
denatured membranes and proteins. Similar to the findings
with TTO, coagulation of cellular constituents and loss of
electron dense material has been reported in bacteria treated
with the membrane active disinfectants chlorhexidine and
quaternary ammonium compounds (Gardner and Peel 1991).
The electron dense blebs on the outside surface of E.
coli cells treated with TTO may represent collections of
coagulated membrane and cytoplasmic constituents which
have pushed through holes produced in the cell wall by
the action of TTO. Chlorhexidine treatment also causes the
formation of electron dense blebs on the cell surface of E. coli
(Davies et al. 1968). Recently, it has been reported that the
treatment of E. coli with a neutral wood oil extracted from
Chamaecyparis obtusa and a component of the oil, yoshixol,
cause a granulation of the cytoplasm and the formation of
blebs at the cell surface. These effects are believed to be
caused by the ability of these substances to disrupt membrane
structure (Koyama et al. 1997).
Tea tree oil can kill a wide variety of micro-organisms
(Gram-negative and -positive bacteria, as well as yeasts) (Carson and Riley 1993, 1994, 1995; Carson et al. 1995; Raman
et al. 1995) as can membrane active disinfectants (Gardner
and Peel 1991). Membrane active disinfectants denature proteins and disrupt membrane structure, leading to cytoplasmic
leakage, cell lysis and cell death or visa versa (Gardner and
Peel 1991). Several studies have shown that some components
of TTO, such as linalool, a-terpineol and 1,8 cineole, have
the ability to disrupt or penetrate lipid structures (Williams
and Barry 1991; Kararli et al. 1995; Takahashi et al. 1996).
With this evidence and our data it is possible to suggest that

1998 The Society for Applied Microbiology, Letters in Applied Microbiology 26, 194198

T EA TR E E O IL E FF EC T S O N E . CO LI 197

Fig. 3 Electron micrographs (7100 )

of AG100 cells grown in LB (a)
without any additions and (b) 1 h
following the addition of 05 the
MBC of TTO

TTO kills micro-organisms in a fashion similar to membrane

active disinfectants.
Stationary phase cells of AG100 demonstrate a biphasic
death phase when incubated in the presence of TTO. In the
first 15 min, cells die precipitously (112 10935 105 cfu
or loss of 9997% of viable cells) and then die at a much
slower rate for the last 105 min, during which time, only an
additional 100 000 cells die. This evidence reveals a small

sub-population of stationary phase cells (003% of total population) that exhibit tolerance to TTO-induced cell death at
1 the TTO MBC. However, at 2 the TTO MBC, all
stationary phase cells were killed. This result shows that the
TTO-tolerant sub-population is not resistant to the action of
TTO. Stationary phase cells also showed a greater tolerance
to TTO-stimulated autolysis compared to exponential phase
cells.

1998 The Society for Applied Microbiology, Letters in Applied Microbiology 26, 194198

198 J .E . G U ST AF S ON ET A L .

A number of genes, such as surA, surB, vtm and katF,

are known to be expressed during stationary phase and are
important for the survival of E. coli during times of starvation
(for review see Kolter 1992). It is also known that most classes
of antibiotics, with the exception of the flouroquinolones, do
not exhibit bactericidal action against non-growing Gramnegative bacteria, including E. coli (Eng et al. 1991). It is
possible that the expression of stationary phase genes, or the
non-growing conditions of stationary phase, contribute to the
TTO tolerance observed in stationary phase E. coli cells.
These studies indicate that a population of E. coli cells in
stationary phase is physiologically different from an exponentially growing population of cells. Therefore, the induction of gene products expressed during stationary phase and
the subsequent physiology of the E. coli cell which follows,
protects a small sub-population of cells against a concentration of TTO equal to the MBC.
More research is required to determine the exact TTOinduced killing event in bacteria and it is also important to
determine whether TTO has similar effects on other microoganisms.
ACKNOWLEDGEMENTS

The authors thank Raelene Lim for preparing samples for

electron microscopy. This work is sponsored wholly by Australian Tea Tree Oil Research Institute (ATTORI).
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1998 The Society for Applied Microbiology, Letters in Applied Microbiology 26, 194198