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INTRODUCTION
D ET ER M IN IN G MI C O F ES SE N TI AL O IL S 539
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
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.
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
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
Validation of assay
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
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
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
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
ACKNOWLEDGEMENTS
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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
1998 The Society for Applied Microbiology, Journal of Applied Microbiology 84, 538544