The testing program will consist of the following stages:

School of Applied Sciences
ESSA Report 1: Susceptibility tests - Solution studies
27/02/01
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Background:
The minimum inhibitory concentration (MIC) of a chemical is the lowest concentration of the test agent that
will inhibit the growth of the test bacteria i.e. it measures the bacteriostatic activity of the test agent.

The minimum bactericidal concentration (MBC) of a chemical is the lowest concentration of the test agent
that will kill the test bacteria i.e. it measures the bacteriocidal activity of the test agent.
1. Susceptibility Testing of Bacteria Using a Standard Broth Dilution Method

Aim:
To determine the MIC and MBC of solutions of Tea Tree oil and Safe T Air gel on a wide range of airborne
bacteria.
Method:
Susceptibility tests were carried out on a range of common airborne bacteria. Bacterial species were chosen to
compare the results for Tea Tree oil and Safe T Air gel to previous published results (ESSA product
information sheet). Cultures were obtained from a standard reference collection of bacteria (Australian
Collection of Microorganisms).
Enteric bacteria (found in animal faeces)
Escherichia coli ATCC 25922
Klebsiella pneumoniae ATCC 13883
Proteus vulgaris ATCC 6280
Serratia marcescens NCTC 1377
Enterococcus faecalis ATCC 29212
Enterobacter aerogenes ATCC 13048
Pseudomonas putida ATCC 49128
Skin bacteria
Staphylococcus epidermidis ATCC 14990
Staphylococcus aureus NCTC 6571
Corynebacterium sp. ATCC 19409
Propionebacterium acnes ACM 821 (Australian Collection of Microorganisms)
Bacteria common in dust
Bacillus subtilis ATCC 11774
Bacillus cereus ATCC 10876A
Micrococcus luteus ATCC 10240
Environmental pathogens
Legionella pneumophila

Bacteria were tested against the following concentrations of tea tree oil/ Safe T Air gel:
1.0%, 0.75%, 0.5%, 0.4%, 0.3%, 0.2% and 0.1% mg/mL
The concentration of the Safe T Air gel was set up to obtain the same concentration of the oil component as
the pure oil. Each organism was tested against all concentrations of the oil taken from both 100% pure tea tree
oil and from the Safe T Air gel using the macrodilution method described in the NCCLS reference. For each
organism a full set of controls was also set up with equivalent amounts of 85% ethanol (used for dissolving
both the pure oil and the gel) to those that would have been present in each of the dilutions. A control with
neither tea tree oil nor ethanol was also included for each organism to check the viability of the inoculum.
Tea tree oil
MICs were determined by recording presence or absence of turbidity in the broth dilutions. After initial
reading of the tubes the last three dilutions showing no apparent growth were plated out onto a suitable agar
medium to determine the minimum bactericidal concentration (MBC).
Safe T Air gel
MICs of the gel-associated oil could not be read because the cloudiness of the diluted gel solutions made it
impossible to read turbidity changes. MBCs were determined by plating out the diluted gel solutions.
Results:
In the table below G = growth, N = no growth and NAG = no apparent growth. No distinction has been made
between levels of growth as this is difficult in broth cultures.

Organism
Chemical
Concentration of tea tree oil/ Safe T Air gel (w/v%)
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MIC = 0.2% MBC = 0.2%(oil), 0.2%(gel) Note that the gel-associated oil has a greater quantity of ethanol added with it, due to the low concentration of oil in the gel. This explains the difference between the results from the two sets of ethanol controls. MIC = 0.2% MBC = 0.2%(oil), 0.2%(gel) MIC = 0.5% MBC = 1.0% (oil), 0.75%(gel) MIC = 0.2% MBC = 0.3% (oil), 0.4% (gel) MIC = 0.1% MBC = 0.2% (oil), 0.2% (gel) The gel results are inconclusive because the ethanol appears to be inhibiting growth at the same concentration. MIC = 0.3% MBC = 0.3%(oil), <0.1%(gel) MIC = 0.3% MBC = >1.0% (oil), >1.0% (gel) MIC = 0.3% MBC = 0.3% (oil), 0.75%(gel) MIC = 0.2 - 0.4% MBC = >1.0% (oil), >1.0 % (gel) MIC = 0.1% MBC = 0.1% (oil), 0.2% (gel)
MIC = <0.1% MBC = <0.1% (oil), <0.1% (gel)

Summary:
Bacteria
Minimum inhibitory
Minimum bactericidal
concentration (MIC) % mg/mL
concentration (MBC) % mg/mL
Tea tree oil
Safe T Air gel#
Tea tree oil
Safe T Air gel#
# concentration of oil in the gel solution * not readable due to cloudiness of gel solution
Conclusion:

Minimum Inhibitory Concentration (MIC)
The growth of all bacteria tested was inhibited by tea tree oil at concentrations varying from 0.1 to 0.5%.
Minimum Bactericidal Concentration (MBC)
Both tea tree oil and Safe T Air gel were shown to have a bactericidal effect on most test bacteria at
concentrations of 0.1 to 0.4%. The results for tea tree oil confirm earlier studies published by ESSA.
Three species were shown be more resistant than the others:
Enterococcus faecalis MBC (oil) 1.0% MBC (gel) 0.75%
Bacillus subtilis
MBC (oil) >1.0% MBC (gel) >1.0%
The resistance of Bacillus subtilis and Bacillus cereus is probably related to their ability to form resistant
endospores.



2. Susceptibility Testing of Fungi Using a Standard Broth Dilution Method
Aim:

To determine the MIC and MFC of solutions of Tea Tree Oil on a wide range of airborne yeasts and moulds.

Test fungi:
Cultures were obtained from a standard reference collection of fungi (Australian Collection of
Microorganisms).
Yeasts
Trichophyton mentagrophytes ATCC 9533 Trichophyton rubrum ATCC 28188 Aspergillus niger ATCC 6275 Aspergillus flavus ATCC 15546
Each organism was tested against all concentrations of the oil taken from 100% pure tea tree oil using a
modified macrodilution method described by Arzeni et al(1998) and McGinnis & Pasarell (1998). For each
organism a full set of controls was also set up with equivalent amounts of 85% ethanol to those that would
have been present in each of the dilutions. A control with neither tea tree oil nor ethanol was also included for
each organism to check the viability of the inoculum.
After initial reading of the tubes the last three dilutions showing no apparent growth were plated out onto a
suitable agar medium to distinguish between the minimum inhibitory concentration (MIC) and the minimum
fungicidal concentration (MFC).
The tests with the gel dilutions could not be performed due to clouding of the broth caused by the gel.

Results:
In the table below G = growth, N = no growth and NAG = no apparent growth. No distinction has been made
between levels of growth, as this is difficult in broth cultures.
Organism
Chemical
Concentration of tea tree oil (w/v%)
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Summary:
Fungi
Minimum inhibitory
Minimum fungicidal
concentration (MIC) % mg/mL
concentration (MBC) % mg/mL
Tea tree oil
Tea tree oil
mentagrophytes Trichphyton rubrum

Conclusion:
The MIC of the chemical is the lowest concentration of the test agent that will inhibit the growth of the test
fungi i.e. it measures the fungiostatic activity of the test agent. The growth of all fungi tested was inhibited by
tea tree oil at concentrations varying from 0.1 to 0.5%.
The MFC of the chemical is the lowest concentration of the test agent that will kill the test fungi i.e. it
measures the fungicidal activity of the test agent. Tea tree oil was shown to have a fungicidal effect on the test
fungi at concentrations of 0.2 to 0.75%.
References:
Arzeni, D, Barchiesi, F., Compagnucci,P., Cellini, A., Simonetti, O. Offidani, A.M. & Scalise, G (1998). In
Vitro activity of terbinafine against clinical isolates of dermatophytes. Medical Mycology 36, 235-237
McGinnis & Pasarell, L. (1998). In vitro evaluation of terbinafine and itraconazole against dematiaceous
fungi. Medical Mycology 36, 243-246
NCCLS (2000). Methods for Dilution Antimicrobial Tests for Bacteria That Grow Aerobically; Approved
Standard – 5th Ed. NCCLS document M7-A5, NCCLS: Wayne, Pennsylvania, USA
T.H.JEAVONS & K.BURDETT
27/02/01

Source: http://www.pureroom.com.tw/images/MonashUniversityReport.pdf