Journal of Applied Microbiology 1998, 84, 538544

A new method for determining the minimum inhibitory

concentration of essential oils
C.M. Mann and J.L. Markham
Centre for Biostructural and Biomolecular Research, University of Western Sydney, Hawkesbury, Richmond, N.S.W.,
Australia
6244/05/97: received 23 May 1997 and accepted 31 July 1997

A new microdilution method has been developed for

determining the minimum inhibitory concentration (MIC) of oil-based compounds. The
redox dye resazurin was used to determine the MIC of a sample of the essential oil of
Melaleuca alternifolia (tea tree) for a range of Gram-positive and -negative bacteria. Use of
015% (w/v) agar as a stabilizer overcame the problem of adequate contact between the
oil and the test bacteria and obviated the need to employ a chemical emulsifier. A rapid
version of the assay was also developed for use as a screening method. A comparison of
visual and photometric reading of the microtitre plates showed that results could
be assessed without instrumentation; moreover, if the rapid assay format was used,
rigorous asepsis was not necessary. Accuracy of the resazurin method was
confirmed by plate counting from microwells and MIC values were compared with
results obtained using an agar dilution assay. The MIC results obtained by the resazurin
method were slightly lower than those obtained by agar dilution.
C .M . M A NN AN D J. L. M AR KH A M. 1998.

INTRODUCTION

The essential oil of Melaleuca alternifolia has had a long

history of use as a topical antiseptic and is currently enjoying
resurgent popularity. Its activity against a range of bacteria
and fungi has been the subject of many studies (Penfold and
Grant 1925; Walsh and Longstaff 1987; Southwell et al. 1993;
Carson et al. 1995b; Hammer et al. 1996). Oil composition is
variable but comprises about 50% oxygenated monoterpenes
and 50% terpene hydrocarbons, with the principal active
component being terpinen-4-ol (Southwell et al. 1993).
Ninety-seven components have now been reported (Brophy
et al. 1989). Currently there is considerable interest in characterizing the bioactivity of the oil and relating it to chemical
composition.
The antimicrobial effectiveness of a compound is often
described in terms of its minimum inhibitory concentration
(MIC), the lowest concentration of the compound capable of
inhibiting the growth of the challenging organism. Diffusion
assays, in which the agent is applied to a well or paper disc
in the centre of an agar plate seeded with the test microorganism, are unsuited to essential oil testing because the oil
Correspondence to: J.L. Markham, Centre for Biostructural and
Biomolecular Research, University of Western Sydney, Hawkesbury,
Bourke St, Richmond, 2753, Australia.

components are partitioned through the agar according to

their affinity with water (Southwell et al. 1993). Broth and
agar dilution methods are widely used to determine the MIC
of tea tree and other essential oils (Beylier 1979; Janssen et al.
1986; Villar et al. 1986; Southwell et al. 1993; Chand et al.
Dellar et al. 1994; Carson et al. 1995b), but the ability to
compare data from different studies is limited due to differences in test methodologies and in the criteria selected for
the determination of the end-point. For example, Carson
et al. (1995b) describe the MIC as the lowest concentration
which resulted in maintenance or reduction of inoculum
viability over a 24 h contact time. Remmal et al. (1993) apply
a similar definition but extend the incubation time to 2448 h
after the appearance of growth in the control. The rapid
microtitre method reported by Chand et al. (1994) employs a
4 h contact time and does not distinguish between MIC and
MBC. The definition of Carson et al. (1995b) is the most
precise and was chosen for this study.
In the testing of essential oils, broth dilution micro
methods often employ an indicator to determine cell viability,
as the turbidity of oilwater emulsions interferes with the
determination of end-points. Chand et al. (1994) used the
fluorogenic substrate fluorescein diacetate (FDA) and Carson
et al. (1995a, 1995b) used triphenyl tetrazolium chloride
(TTC). Lundgren (1981) had previously noted the problem
1998 The Society for Applied Microbiology

D ET ER M IN IN G MI C O F ES SE N TI AL O IL S 539

of autofluorescence when using FDA for bacteriological

applications and the same difficulty was experienced in our
laboratory. TTC reduction, whilst a useful indicator of bacterial growth, did not correlate exactly with either MIC or
MBC (Carson et al. 1995a). Resazurin, a redox indicator used
in the dairy industry to assess the microbiological quality of
raw milk, was trialed as a visual indicator of MIC in this
assay.
When testing non-water-soluble antimicrobials such as
essential oils, it is necessary to incorporate an emulsifier or
solvent into the test medium to ensure contact between the
test organism and the agent for the duration of the experiment. The agents most commonly used are Tween 80 (polysorbate 80) and Tween 20 (polyoxyethylene (2) sorbitan
mono-laurate) (Beylier 1979; Walsh and Longstaff 1987; Patkar et al. 1993; Chand et al. 1994; Carson et al. 1995b), ethanol
(Biondi et al. 1993; Deans et al. 1994) and DMSO (Scortichini
and Rossi 1991). Such agents, however, may cause changes
in the physicochemical properties of the test system, resulting
in either enhancement or reduction of the antimicrobial
activity. Lipophilic molecules, including the components of
tea tree oil, may become solubilized within the micelles formed by non-ionic surfactants, such as Tween 20 and Tween
80, and are thus partitioned out of the aqueous phase of the
suspension (Schmolka 1973). Kazmi and Mitchell (1978) have
shown that antimicrobials solubilized within the micelles do
not contribute to the antimicrobial activity as they do not
come into direct contact with the micro-organisms. This
effect is more marked at higher concentrations of surfactant
(Van Doorne 1990), and Altman (1991) has reported a
reduction in the bioactivity of tea tree oil in the presence of
Tween. The suitability of ethanol has also been questioned
as it has been reported to have a marked potentiating effect
on the activity of some antimicrobial agents at concentrations
of 5% (Van Doorne 1990). The incorporation of low concentrations of bacteriological agar as a stabilizer of the oil
water mixture has been suggested as an alternative which
overcomes these disadvantages (Remmal et al. 1993).
The objective of this study was to develop a broth microdilution assay suitable for the assessment of antimicrobial
activity of essential oils using a chemically and microbially
inert stabilizer, and an indicator capable of reliably predicting
MIC either visually or instrumentally.
MATERIALS AND METHODS
Reagents

The essential oil of M. alternifolia (tea tree) was supplied by

Wollongbar Agricultural Institute (Wollongbar, NSW, Australia). Terpinen-4-ol and 1,8-cineole contents were 401%
and 46%, respectively. A solution of resazurin sodium salt
(Sigma R-2127) was prepared to 001% (w/v) in sterile dis-

tilled water. Emulsifiers tested were Tween 20 and Tween

80 (Ajax Chemicals), 2% ethanol and DMSO (AR grade,
Ajax) and 015% bacteriological agar (Amyl, RM250) which
was dissolved, sterilized by autoclaving and cooled to room
temperature before use.
Test organisms

The bacteria used in this study were Escherichia coli NCTC

8196, Staphylococcus aureus NCTC 4163, methicillin-resistant Staph. aureus (clinical isolate), Group A Streptococcus
JM12 (clinical isolate), Group B Streptococcus (clinical isolate)
and Proteus vulgaris NCTC 4635.
Culture methods

The test organisms were stored on Nutrient Agar (NA)

slopes, prepared from Nutrient Broth base (Oxoid CM1)
and bacteriological agar (Amyl RM250) at 25 C, except
for streptococcal strains, which were stored on Brain Heart
Infusion Agar (BHIA), prepared from BHI broth (Oxoid
CM225) and bacteriological agar (Amyl RM250).
All micro-organisms used in MIC assays were twice-passaged 1618 h cultures grown in either Nutrient Broth (NB)
(Oxoid CM1) or Brain Heart Infusion Broth (BHIB) (Oxoid
CM225). Optical densities of all inocula were measured at
420 nm using a Pharmacia LKB Ultrospec III spectrophotometer. Cell densities were estimated from standard
curves and confirmed by the pour plate method on Plate
Count Agar (Amyl, AM144).
Assay media

For the resazurin MIC method, the inocula were diluted to

the appropriate cell densities in NB containing 015% (w/v)
agar for all test cultures except the streptococci, for which
BHIB with 015% (w/v) agar was used. Prior to inoculation,
test media were melted by steaming and tempered to 37 C,
at which temperature they remained liquid. Iso-Sensitest
Agar (ISA) (Oxoid CM471) containing 025% (v/v) Tween
20 was used for the agar dilution assay.
Inoculum densities

The cell concentration necessary to cause reduction of resazurin within 2 h was determined for each of the test organisms. Serial 10-fold dilutions of each culture were prepared
in prewarmed NB. Aliquots (17 ml) were dispensed into
tubes containing 02 ml sloppy (015%, w/v) agar, and
01 ml resazurin solution. The tubes were incubated for 2 h at
37 C, after which time aliquots from adjacent blue (oxidized),
mauve and pink (reduced) dilution tubes were tested by the
plate count method.

1998 The Society for Applied Microbiology, Journal of Applied Microbiology 84, 538544

540 C .M . M A NN AN D J. L. M AR KH A M

Resazurin MIC assay

Serial twofold dilutions (0005125%, v/v) of the test oil

were prepared by vortexing the oil in room-temperature
sloppy agar. The resazurin assay medium described above
was then inoculated with the test organism to yield a final
cell density 1 log cycle lower than the cell density required
to reduce resazurin (usually 5 log106 log10 cfu ml1). The
inoculum density was confirmed by plate count. A sterile 96well microtitre tray with lid was set up with each of the test
bacteria (n  8) as follows: column 19, 170 ml inoculum plus
20 ml of an oil dilution; column 10, 170 ml inoculum plus 20 ml
oil diluent (positive control); column 11 and 12, 170 ml sterile
resazurin assay medium plus 20 ml oil diluent (negative control and blank, respectively).
Well contents were thoroughly mixed using the micropipettor. Two trays were prepared for each organism and
incubated at 37 C for either 35 h or 18 h. After incubation,
10 ml of resazurin solution was added to all except column 12,
to which 10 ml of distilled water was added. After a second
incubation of 2 h at 37 C, three methods were used to determine the MIC values. First, wells were assessed visually
for colour change, with the highest dilution remaining blue
indicating the MIC. Absorbance was then read at 570 nm
using a Biorad Model 3550 plate reader blanked against wells
containing only assay medium, oil diluent and distilled water.
The MIC was indicated by a rise in absorbance at 570 nm.
Immediately afterwards, plate counts were carried out on
samples from the microwells, to determine whether bacterial
numbers correlated with either indicator of MIC.

teria was not sustained (data not shown). To overcome this

problem, a number of emulsifying agents and solvents were
compared. Mixtures of tea tree oil and the various emulsifying
and solubilizing agents were prepared in nutrient broth to
give a final concentration of tea tree oil of 125% (v/v). Tubes
were incubated at 37 C and checked hourly for any signs of
oil separation. The results indicated that the oilwater mixture separated within 3 h in the presence of 05% Tween 20
or Tween 80, 2% ethanol or 02% DMSO, but no separation
was evident for 19 h when 015% agar was added as a stabilizer. Thus, this was incorporated in all further tests.

Inoculum densities for the microtitre assay. In order to measure MIC as the lowest concentration of oil at which inoculum viability is maintained or reduced (Carson et al. 1995b),
it was necessary to determine minimum microbial densities
needed to produce a visible colour change through reduction
of resazurin. Serial dilutions of each organism were incubated
with the resazurin reagent as described in Materials and
Methods, and the number of viable cells from adjacent blue,
mauve and pink wells determined by the pour plate method
(Table 1). The inoculum density subsequently used in the
assay was 1 log cycle lower than that required for a change
of the reagent to mauve. Resazurin reduction kinetics are
genus-dependent, so reduction densities were determined for
each organism.

First incubation time. This time represents the major period

Agar dilution MIC assay

Using a modification of the assay described by Southwell

et al. (1993), tea tree oil was added to molten ISA/Tween 20
medium at 48 C, to give oil concentrations ranging from
0 to 062% (v/v). Plates, prepared in triplicate, were spot
inoculated with 3 ml aliquots of culture in NB or BHIB
adjusted to yield a density within 205 log10 of those used in
the resazurin assays. Plates were incubated at 37 C for 18 h
and the MIC was determined as the lowest concentration of
oil to result in no growth of the inoculum on two of three
plates.
RESULTS
Optimization of the assay

Emulsion stability. In order to maintain contact between the

oil and the micro-organism throughout the test period, a

stable emulsion is necessary. Previously, use of 01% Tween
20 resulted in vigorous bacterial growth for the higher oil
concentrations because contact between the oil and the bac-

of inhibition of the bacteria by the test oil. It must be sufficient

to allow bacterial densities in non-inhibited wells to increase
at least one log cycle thereby reaching their resazurin
reduction density. The majority of published MIC methods
involve a contact time of 1824 h, but a more rapid assay
would have many advantages. Incubation times of 35 and

Table 1 Microbial resazurin reduction densities (log10 cfu ml1)

Organism
Blue
Mauve
Pink

Escherichia coli
72
82
86
Staphylococcus aureus
53
63
73
MRSA
50
60
71
Group A Streptococcus
50
59
71
Group B Streptococcus
59
70
79
Proteus vulgaris
58
71
81

MRSA, Methicillin resistant Staphylococcus aureus

1998 The Society for Applied Microbiology, Journal of Applied Microbiology 84, 538544

D ET ER M IN IN G MI C O F ES SE N TI AL O IL S 541

18 h were compared and the results are shown in Table 2.

MIC values for four of the five organisms tested were the
same following the short or the long incubation time.
Second incubation time. This period represented the
maximum time taken for metabolically active cells of 12
organisms at or close to their resazurin reduction densities to
effect reduction, which occurred within 15105 min, depending on the organism (data not shown). The second incubation
was standardized at 2 h, as this allowed detection of organisms
which reduced resazurin more slowly.
Comparison of visual and photometric determination of end
point. The method depends on an easily recognized colour

change from blue (indicating inhibition of the test organism)

to mauve or pink (no inhibition). Immediately after visual
determination of the endpoint, the absorbance at 570 nm was
recorded. At this wavelength absorbance increases as the
colour changes from blue, through mauve, to pink (Figs 1, 2,
3). However, absorbance readings alone were limited by the
fact that, in some cases, irreversible reduction to pink resorufin is rapidly followed by reversible reduction to colourless
dihydroresorufin with a concomitant decline in A570 (Fig. 1).
Whilst spectrophotometric measurements provided a useful
comparison during method development, plates can be more
conveniently read visually during routine use of the method.

Fig. 1 Comparison of methods of determining minimum

inhibitory concentration (MIC) values of tea tree oil against
Escherichia coli. Inoculum density for E. coli was 54 log10 cfu
ml1 and incubation time 18 h. E, A570 nm; , log10 cfu ml1. Visual
readings: B, blue; M, mauve; P, pink; PP, pale pink or colourless.
The MIC was determined from plate count data as the percentage
oil at which the inoculum density was maintained or reduced

Validation of assay

Aliquots (100 ml) from microwells were plated to determine

the concentration of viable cells. Although for the purposes
of visual and spectrophotometric reading, eight replicates of
each dilution were prepared, only one well from each dilution
was plated. These results were compared with visual colour
assessments (recorded as blue, mauve, pink or pale pink) and
A570 (Figs 1,2,3). Data are presented for three of the six

Table 2 Comparison of incubation times in minimum inhibitory

concentration (MIC) assay (n  8)

MIC (% v/v)

Organism
35 h incubation
18 h incubation

Escherichia coli
008
008
Staphylococcus aureus
004
004
Group A Streptococcus
008
008
Group B Streptococcus
031
031
Proteus vulgaris
008
016

Fig. 2 Comparison of methods of determining minimum

inhibitory concentration (MIC) values of tea tree oil against
Staphylococcus aureus. Inoculum density was 52 log10 cfu ml1
and incubation time 18 h. See Fig. 1 for explanation of symbols

organisms tested, but results for all six showed good agreement as determined by all three methods of assessing the
MIC. It was found that, for five of the six organisms, the
microwell containing the highest oil concentration to remain
blue after incubation contained, upon plating, a cell density
at or below that of the original inoculum density. For Staph.

1998 The Society for Applied Microbiology, Journal of Applied Microbiology 84, 538544

542 C .M . M A NN AN D J. L. M AR KH A M

DISCUSSION

Fig. 3 Comparison of methods of determining minimum

inhibitory concentration (MIC) values of tea tree oil against MRSA.

Inoculum density was 54 log10 cfu ml1 and incubation time 18
h. See Fig. 1 for explanation of symbols

aureus, the remaining blue well was within 1 dilution of this

value.
Comparison of resazurin assay with agar dilution
assay

The resazurin method was compared with the agar dilution

assay. The incubation times for both assays were similar. The
sensitivity patterns of the organisms are similar by the two
methods, but MIC values determined by the resazurin
method are lower for most organisms (Table 3), indicating
that the resazurin method is more sensitive than the agar
dilution method.

Table 3 Comparison of minimum inhibitory concentrations

(MICs) obtained by resazurin and agar dilution methods

MIC (% v/v)

Organism
Resazurin
Agar dilution

Escherichia coli
008
031
Staphylococcus aureus
004
008
MRSA
004
008
Group A Streptococcus
008
008
Group B Streptococcus
031
031
Proteus vulgaris
008
031

MRSA, Methicillin resistant Staphylococcus aureus.

The MIC of the essential oil of M. alternifolia was reliably

predicted using the resazurin microdilution method. Determination of MICs for essential oils has hitherto been problematic due to the immiscibility of the oil with broth cultures.
Broth MIC methods have relied mainly on surfactants such
as Tween, although emulsion stability and interaction with
the oil have been cited as problems. In a study examining the
stability of emulsions of Tween 20 and 80 and thyme essential
oil, Allegrini et al. (1973) found that a concentration of 5%
of either Tween 80 or Tween 20 was required to maintain a
stable emulsion of 5% oil in water over a 24-h period. The
present study concurred with this finding, noting that none
of the dispersing agents trialed were capable of maintaining
adequate contact between the oil and the test organism. Use
of 015% agar as a chemically and microbially inert stabilizer
was found to overcome the problem of emulsion stability for
tea tree oil.
Resazurin has a number of advantages as an MIC endpoint
indicator. Reduction results in an easily identified colour
change occurring at cell densities meaningful for MIC testing.
Resazurin undergoes a colour change when the surrounding
medium is reduced as a result of bacterial depletion of dissolved oxygen and acid production (Frandsen 1958). For this
reason it is necessary to assess reduction densities for each
organism used. Reduction occurs in two stages. In the first
stage, blue resazurin is irreversibly reduced to pink resorufin
(Amax 580 nm). In the second stage, resorufin is reversibly
reduced to colourless dihydroresorufin.
TTC reduction occurs due to its function as an artificial
terminal electron acceptor in respiration (Bitton and Koopman 1986). However, the end-point is less easily determined
than that of resazurin, which involves a more distinct colour
change and it has been reported that TTC reduction does not
correlate exactly with MIC (Carson et al. 1995a). A further
advantage of resazurin over dyes such as TTC or FDA is
that the dye reduction does not involve cellular uptake. In a
study on the use of FDA as a vital stain to detect metabolically
active bacteria in soil, Lundgren (1981) noted that fewer
organisms hydrolysed FDA if they were grown in laboratory
medium. He postulated that changes in growth medium may
result in changed membrane properties and altered enzyme
substrate affinities. This prompted choice of a dye which
did not depend on cellular uptake or reduction directly by
bacterial enzymes.
The majority of bacteria tested in this study reduced resazurin and gave clearly defined end-points. However, the assay
was not suitable for Pseudomonas aeruginosa which has slow
reduction kinetics (Holley et al. 1977), or for Candida albicans.
Other indicators used to detect metabolically active cells share
this problem. For example, Lundgren (1981) reports that
20% of the isolates tested did not stain with FDA.

1998 The Society for Applied Microbiology, Journal of Applied Microbiology 84, 538544

D ET ER M IN IN G MI C O F ES SE N TI AL O IL S 543

The validity of the resazurin method to predict MIC is

borne out by the correlation of visible colour change with
bacterial densitites in microwells at the completion of the
assay. Further validation was obtained by comparing MICs
determined using the agar dilution assay of Southwell et al.
(1993). A comparison showed that some MICs were lower by
the resazurin method; however, MICs were relatively the
same between organisms. This lowering of MICs indicates
the enhanced sensitivity of the new method.
MICs encountered in the essential oil literature often vary
with regard to contact time between oil and test organism
and may not always specify inoculum densities. Rapid assays,
where contact time is typically less than 8 h, should be validated by a prolonged exposure to ensure that apparent inhibition is not due to the test bacteria entering an extended lag
phase preceding eventual adaptation. It has been shown here
that, for five of the six organisms trialed, the rapid method
agrees well with results obtained over the 18 h contact time.
Only one organism, Pr. vulgaris, demonstrated a rise in MIC
of one dilution as incubation was extended from 55 to 18 h.
The rapid assay is well suited to screening organisms or
oils for the purpose of comparing microbial responses or oil
bioactivities and will provide valuable preliminary information on bioactivity. However, it is recommended that the
18 h procedure be used in MIC determinations. Microtitre
methods are more economical of time and resources than
alternative methods. Whilst rigorous asepsis is needed for the
18 h MIC procedure, the use of sterile microtitre trays and
tips is unimportant for the screening method, as the short
incubation period precludes the growth of background levels
of contaminants to densities sufficient for resazurin reduction.
There is considerable interest from the tea tree industry
in the development of reliable methods for assessing the
comparative antimicrobial properties of various oil blends so
that plant breeding and product formulation can be optimized. It is postulated that current methods, which mostly
rely on the use of Tween, underestimate the MIC of tea tree
oil (Altman 1991). Registration of the oil as a therapeutic
agent relies in part on the oils activity, underscoring the
need of the industry for a reliable method which will not
compromise MIC measurement. The resazurin microdilution
method offers a suitable alternative to currently used
methods.

ACKNOWLEDGEMENTS

This study was supported by a grant from the Australian Tea

Tree Industry Association and Rural Industries Research and
Development Corporation (DAN 104 A). Dr Ian Southwell
from Wollongbar Agricultural Institute is thanked for providing the samples of tea tree oil used in this work.

REFERENCES
Allegrini, J., de Buochberg, M.S. and Maillols, H. (1973) Emulsions
dhuiles essentielles fabrication et applications en microbioloige.
Travaux de la Societe de Pharmacie de Montpellier 33, 7386.
Altman, P. (1991) Australian tea tree oil. Cosmetics and Toiletries
Manufacture 12, 2324.
Beylier, M.F. (1979) Bacteriostatic activity of some Australian essential oils. Perfumer and Flavorist 4, 2325.
Biondi, D., Cianci, P., Geraci, C. and Ruberto, G. (1993) Antimicrobial activity and chemical composition of essential oils from
Sicilian aromatic plants. Flavour and Fragrance Journal 8, 331
337.
Bitton, G. and Koopman, B. (1986) Use of resazurin in toxicity
testing. In Toxicity Testing using Micro-organisms. Vol. 1 ed Bitton,
G. & Dutka, B.J., pp. 4142. Florida: CRC Press.
Brophy, J.J., Davies, N.W., Southwell, I.A., Stiff, I.A. and Williams,
L.R. (1989) Gas chromatographic quality control for oil of Melaleuca terpinen-4-ol type (Australian tea tree). Journal of Agricultural and Food Chemistry 37, 13301335.
Carson, C.F., Cookson, B.D., Farrelly, H.D. and Riley, T.V.
(1995a) Susceptibility of methicillin-resistant Staphylococcus
aureus to the essential oil of Melaleuca alternifolia. Journal of
Antimicrobial Chemo 35, 421424.
Carson, C.F., Hammer, K.A. and Riley, T.V. (1995b) Broth microdilution method for determining the susceptibility of Escherichia
coli and Staphylococcus aureus to the essential oil of Melaleuca
alternifolia (tea tree oil). Microbios 82, 181185.
Chand, S., Luzunzi, I., Veal, D.A., Williams, L.R. and Karuso, P.
(1994) Rapid screening of the antimicrobial activity of extracts
and natural products. Journal of Antibiotics 47, 12951304.
Deans, S.G., Kennedy, A.I., Gundidza, M.G., Mavi, S., Waterman,
P.G. and Gray, A.T. (1994) Antimicrobial activities of the volatile
oil of Heteromorpha trifoliata (Wendl.) Eckl. & Zeph. (Apiaceae).
Flavour and Fragrance Journal 9, 245248.
Dellar, J.E., Cole, M.D., Gray, A.I., Gibbons, S. and Waterman,
P.G. (1994) Antimicrobial sesquiterpenes from Prostanthera aff.
melissifolia and P rotundifolia. Phytochemistry 36 (4), 957960.
Frandsen, J.H. (1958) (ed.) Dairy Handbook and Dictionary.
Pennsylvania. p. 731.
Hammer, K.A., Carson, C.F. and Riley, T.V. (1996) Susceptibility
of transient and commensal skin flora to the essential oil of Melaleuca alternifolia (tea tree oil). American Journal of Infect Cont 24
(3), 186189.
Holley, R.A., Smith, S.M. and Kempton, A.G. (1977) Rapid
measurement of meat quality by resazurin reduction. I. Factors
affecting test validity. Canadian Institute Food Science and Tech
Journal 10, 153157.
Janssen, A.M., Scheffer, J.J.C. and Baerheim Svendsen, A. (1986)
Antimicrobial activity of essential oils: A 19761986 literature
review. Aspects of the test methods. Planta Medica 53, 395398.
Kazmi, S.J.A. and Mitchell, A.G. (1978) Preservation of solubilised
emulsion systems. II. Theoretical development of capacity and
its role in antimicrobial activity of chlorocresol in cetamacrogolstabilised systems. Journal of Pharm Sc 67, 12661271.
Lundgren, B. (1981) Flurorescein diacetate as a stain of metabolically active bacteria in soil. Oikos 36, 1722.
Patkar, K.L., Usha, C.M., Shetty, H.S., Paster, N. and Lacey, J.

1998 The Society for Applied Microbiology, Journal of Applied Microbiology 84, 538544

544 C .M . M A NN AN D J. L. M AR KH A M

(1993) Effect of spice essential oils on growth and aflatoxin B1

production by Aspergillus flavus. Letters in Applied Microbiology
17, 4951.
Penfold, A.R. and Grant, R. (1925) The germicidal values of some
Australian essential oils and their pure constituents. Together
with those for some essential oil isolates, and synthetics. Part III.
Journal of Proceedings of the Royal Society of NSW 59, 346350.
Remmal, A., Bouchikhi, T., Rhayour, K. and Ettayebi, M. (1993)
Improved methods for the determination of antimicrobial activity
of essential oils in agar medium. Journal of Essential Oil Research
5, 179184.
Schmolka, I.R. (1973) The synergistic effects of nonionic surfactants
upon cationic germicidal agents. Journal of Society of Cosmet
Chemistry 24, 577592.
Scortichini, M. and Rossi, M.P. (1991) Preliminary in vitro evalu-

ation of the antimicrobial activity of terpenes and terpenoids

towards Erwinia amylovora (Burrill) Winslow et al. Journal of
Applied Bacteriology 71, 109112.
Southwell, I.A., Hayes, A.J., Markham, J. and Leach, D.N. (1993)
The search for optimally bioactive Australian tea tree oil. Acta
Horticulturae 334, 256265.
Van Doorne, H. (1990) Interactions between preservatives and pharmaceutical components. In Denyer, S. & Baird, R (eds) Guide
to Microbiological Control in Pharmaceuticals. New York: Ellis
Horwood.
Villar, A., Recio, M.C., Rios, J.L. and Zafra-Polo, M.C. (1986)
Antimicrobial activity of essential oils from Sideritis species.
Pharmazie 41, 298299.
Walsh, L.J. and Longstaff, J. (1987) The antimicrobial effects of an
essential oil on selected oral pathogens. Periodontology 8, 1115.

1998 The Society for Applied Microbiology, Journal of Applied Microbiology 84, 538544