Tea tree essential oil

(Melaleuca alternifolia) on
endospore germination in
Bacillus cereus

Uses of the Oil

Published evidence of antibacterial,
antifungal, antiprotozan, antiviral, and antiinflammatory properties
Also used to treat athlete s foot, head lice,
acne, and other skin irritations
Oil readily available for everyday use
without a prescription
Found in shampoos, skin treatments, etc.

The major components of TTO

Component
terpinen-4-ol
-terpinene
-terpinene
1, 8-cineole
terpinolene
-terpineol
p-cymene
-pinene
aromadendrene
virdiflorene
-cadinene
limonene
-phellandrene
globulol
myrcene
-thujene
-pinene
sabinene
-phellandrene
viridiflorol

Mean a

Min a

Max a

37.93
20.20
9.56
3.87
3.45
3.01
2.80
2.46
1.68
1.68
1.49
1.01
0.94
0.86
0.86
0.83
0.66
0.45
0.44
0.33

28.6
9.5
4.6
0.5
1.6
1.5
0.4
0.8
0.1
0.3
0.1
0.4
0.4
0.1
0.1
0.1
0.1
0.0
0.1
0.1

57.9
28.3
12.8
17.7
5.4
7.6
12.4
3.6
6.6
6.1
7.5
2.7
1.9
3.0
1.8
2.1
1.6
3.2
1.9
1.4

ISO 4730
range in % b
30
10-28
5-13
15
1.5-5
1.5-8
0.5-12
1-6
Trace-7
N/A
Trace-8
0.5-4
N/A
Trace-3
N/A
N/A
N/A
Trace-3.5
N/A
Trace-1.5

Previously Found Active Components

terpinen-4-ol thought to be most active
ingredient (Carson and Riley 1995)
terpinen-4-ol and -terpineol cause majority of
the antibacterial and antifungal action (Carson et
al., 2006)
-pinene, linalool, and limonene also shown to
have antibacterial properties (Raman et al.,
1995)
1,8-cineole thought to play role in allowing active
components into cell

The present study

TTO has many antimicrobial
abilities
Can it prevent endospore
germination?
If so, what component of the oil
can do this?

Endospores
Hardy,
encapsulated
pieces of DNA
Able to survive
through harsh
conditions
Bacillus spp. able
to form them

Bacillus spp.
Using B. cereus as
model for B. anthracis
2001 bioterrorism
attacks using anthrax
spores on mailed
envelopes
22 mail workers
infected and 5 died
from exposure
Most infections from
anthrax are
cutaneous

Methods

B. cereus bacteria placed

in LB on shaker for 8
days
Heat treatment
Spread on LB plate

Methods
B. cereus bacteria
placed in LB on
shaker for 8 days
Heat treatment
Spread on LB plate
3-4 3M discs were
placed on each plate

Methods
B. cereus bacteria placed
in LB on shaker for 8
days
Heat treatment
Spread on LB plate
4 3M discs were placed
on each plate
Added small amount of
TTO or components:
terpinen-4-ol, -terpinene,
-terpinene, 1,8-cineole,
-pinene, p -cymene,
-terpineol, or limonene

Methods
B. cereus bacteria placed in
LB on shaker for 8 days
Heat treatment
Spread on LB plate
4 3M discs were placed on
each plate
Added small amount of TTO
or components: terpinen-4ol, -terpinene, -terpinene,
1,8-cineole, -pinene, p cymene,
-terpineol, or limonene
Incubated for 24 hours at
32 C
Measured zone of inhibition

Methods

B. cereus bacteria placed in

LB on shaker for 8 days
Heat treatment
Spread on LB plate
4 3M discs were placed on
each plate
Added small amount of TTO
or components: terpinen-4ol, -terpinene, -terpinene,
1,8-cineole, -pinene, p cymene,
-terpineol, or limonene
Incubated for 24 hours at
32 C

Measured zone of
inhibition
Dose effects of active
components
Synergistic effects
between active + active
and active + inactive
ANOVA and Tukey
Kramer Post Hoc
performed
Oil and components
checked for purity on
GC/MS

Results
TTO inhibited
endospore
germination
terpinen-4-ol,
-terpinene, and
-terpineol
components active
None significantly
more active than the
others or TTO

Synergisms
2.5

Radial Cleared Zone (mm)

Two active
components:
terpinen-4-ol and
-terpineol
Combination
significantly more
effective than either
component
F = 40.17, df = 2,
p < 0.0001

2
1.5
1
0.5
0
-0.5
terpinen-4-ol -terpineol

both

Synergisms
8

Radial Cleared Zone (mm)

7
6
5
4
3
2
1
0
-1
1,8-cineole

-terpinene

both

Active and inactive:

-terpinene and
1,8-cineole
F = 26.24, df = 2,
p < 0.0001

Radial Cleared Zone (mm)

5.5

-terpinene and
p-cymene
F = 10.50, df = 2,
p = 0.0014

4.5
3.5
2.5
1.5
0.5

-0.5
p-cymene

-terpinene

both

Synergisms
Active and inactive:
-terpineol and
1,8-cineole
F = 56.43, df = 2,
p < 0.0001

Radial Cleared Zone (mm)

2.5
2
1.5
1
0.5
0
-0.5
1,8-cineole

-terpineol

both

-terpineol and
-terpinene
and F = 19.86, df = 2,
p < 0.0001

Radial Cleared Zone (mm)

2.5
2
1.5
1
0.5
0
-0.5
-terpinene

-terpineol

both

GC/MS
The ten most
abundant components
of the commercial
sample of TTO.
The relative
percentages in the oil
as observed by GC/
MS.
The normal range for
-terpinene is 5-13%.

Component

% Peak Area

Retention (min)

terpinen-4-ol

34.00%

12.836

-terpinene

27.14%

10.257

-terpinene

16.23%

9.292

-pinene

5.76%

7.351

3.77%

10.917

o-cymene

3.41%

9.469

1,8-cineole

3.12%

9.636

limonene

2.38%

9.578

-terpineol

2.22%

13.092

-thujene

1.96%

7.184

terpineole
ne

GC/MS
Composition of commercially purchased

components that were active
or part of a
significant synergism
Component

Purity

Contaminant

1,8-cineole

100.00%

p-cymene

99.63%
0.37%

cymene

95.24%
4.24%

o-cymene

94.18%
4.41%

cyclooctan, 1-(diethylboryl)

89.96%
10.04%

-terpineol

76.46%
12.92%
5.99%
2.63%

o-cymene
1,8-cineole
1,3-heptadiene

-terpinene
terpinen-4-ol
-terpineol
-terpinene

Discussion
Terpinen-4-ol is not the only active component,
-terpineol and -terpinene are just as active
Terpenes are shown to cause a loss of membrane
integrity and disrupt proton motive force (Sikkema et
al. 1995; Cox et al. 1998)

terpinen-4-ol

OH

-terpineol

OH

-terpinene

 These components are not active on their own

but contribute to the overall
activity of the oil

In bacteria, 1,8-cineole has been shown to
disrupt the cell membrane to allow active
components in (Carson et al. 2006)
-terpinene

1,8-cineole

OH

p-cymene