Disk Diffusion Method
The disk diffusion method (also known the zone of inhibition method) is probably
the most widely used of all methods used for testing antibacterial activity. It uses
only small amounts of the test substance (10–30 μL), can be completed by research
staff with minimal training, and as such may be useful in field situations. The
method involves the preparation of a Petri dish containing 15–25 mL agar, bacteria
at a known concentration are then spread across the agar surface and allowed to es-
tablish. A paper disk (6 or 8 mm) containing a known volume of the test substance
is then placed in the center of the agar and the dish incubated for 24 h or more. At
this time the “cleared” zone (zone of inhibition) surrounding the disk is measured
and compared with zones for standard antibiotics or literature values of isolated
chemicals or similar extracts. Where the extract is viscous or a semi-solid (e.g. hon-
ey) a well can be created in the agar and the substance allowed to diffuse out of the
well rather than away from a disk.
Data from these assays are typically presented as mean size of zone of inhibition
(with or without standard deviation), although some authors employ a ranking sys-
tem of +, ++, and +++ to indicate levels of activity. Few authors provide any statis-
tical analysis of their data and levels of activity (slight, moderate, strong) are used
without any reference to standardized criteria.
One of the major criticisms of this method is that it relies on the ability of the ex-
tract to diffuse through agar and any component of the extract that does diffuse
away from the disk will create a concentration gradient, potentially creating a gra-
dient of active antibacterial compounds. All of the antibacterial testing methods
use an aqueous base for dispersion of the test substance, either via diffusion in
agar or dispersion within nutrient broth, consequently assays using extracts with
limited solubility in aqueous media (e.g. essential oils) may not reflect the true
antibacterial activity. This has been investigated by Griffin [18] and Southwell et al.
8.2 Antibacterial Assays 161
[27] who have demonstrated that many terpenoids, due to their poor water solubil-
ity, will not diffuse through aqueous media and hence essential oils high in these
terpenoids (e.g. linalool, linalyl acetate, p-cymene) will give a “false” result in these
assays.
There is also no consensus on the best agar to use for these assays. Oxoid’s Iso-
Sensitest agar is the media of choice for conventional antibiotic susceptibility test-
ing [14–16, 27], but several authors have noted that this may not be the case for
plant extracts, particularly essential oils. Pauli and Kubeczka [28] found when test-
ing eugenol that zone size varied according to agar used. Moon et al. [21] have also
demonstrated differences in zone of inhibition size between IsoSensitest and nu-
trient agar and have also shown that these differences are not consistent across or-
ganisms or essential oil used. Smith et al. [29] found increased sensitivity (i.e.
bigger zones of inhibition) when nutrient agar, rather than Difco brain heart infu-
sion agar, was used for a range of methanol plant extracts. These authors also dem-
onstrated that the size of the inoculum and temperature of incubation also affect
zone size. Indeed these authors suggest that inoculum density is the single most
important factor in the variability of zone size.
A further limitation that has not been directly addressed in the literature, but for
which evidence exists, is inference in the assay from vapors liberated from the ex-
tract during incubation. This is unlikely to be a major consideration in aqueous or
solvent extracts but may be a significant confounder in assays of essential oils. In-
ouye et al. [30] have shown that the volatile constituents of essentials oils can have
a good antibacterial activity; we have also demonstrated this with essential oils
from a range of Australian native plants and lavender. It is possible that the results
of antibacterial assays completed using a closed Petri dish will reflect the combined
actions of oil components diffusing through agar and exposure to gaseous compo-
nents liberated from the oil. Which is responsible for the majority of the antibacte-
rial activity is yet to be determined.
The disk diffusion method (also known the zone of inhibition method) is probably
the most widely used of all methods used for testing antibacterial activity. It uses
only small amounts of the test substance (10–30 μL), can be completed by research
staff with minimal training, and as such may be useful in field situations. The
method involves the preparation of a Petri dish containing 15–25 mL agar, bacteria
at a known concentration are then spread across the agar surface and allowed to es-
tablish. A paper disk (6 or 8 mm) containing a known volume of the test substance
is then placed in the center of the agar and the dish incubated for 24 h or more. At
this time the “cleared” zone (zone of inhibition) surrounding the disk is measured
and compared with zones for standard antibiotics or literature values of isolated
chemicals or similar extracts. Where the extract is viscous or a semi-solid (e.g. hon-
ey) a well can be created in the agar and the substance allowed to diffuse out of the
well rather than away from a disk.
Data from these assays are typically presented as mean size of zone of inhibition
(with or without standard deviation), although some authors employ a ranking sys-
tem of +, ++, and +++ to indicate levels of activity. Few authors provide any statis-
tical analysis of their data and levels of activity (slight, moderate, strong) are used
without any reference to standardized criteria.
One of the major criticisms of this method is that it relies on the ability of the ex-
tract to diffuse through agar and any component of the extract that does diffuse
away from the disk will create a concentration gradient, potentially creating a gra-
dient of active antibacterial compounds. All of the antibacterial testing methods
use an aqueous base for dispersion of the test substance, either via diffusion in
agar or dispersion within nutrient broth, consequently assays using extracts with
limited solubility in aqueous media (e.g. essential oils) may not reflect the true
antibacterial activity. This has been investigated by Griffin [18] and Southwell et al.
8.2 Antibacterial Assays 161
[27] who have demonstrated that many terpenoids, due to their poor water solubil-
ity, will not diffuse through aqueous media and hence essential oils high in these
terpenoids (e.g. linalool, linalyl acetate, p-cymene) will give a “false” result in these
assays.
There is also no consensus on the best agar to use for these assays. Oxoid’s Iso-
Sensitest agar is the media of choice for conventional antibiotic susceptibility test-
ing [14–16, 27], but several authors have noted that this may not be the case for
plant extracts, particularly essential oils. Pauli and Kubeczka [28] found when test-
ing eugenol that zone size varied according to agar used. Moon et al. [21] have also
demonstrated differences in zone of inhibition size between IsoSensitest and nu-
trient agar and have also shown that these differences are not consistent across or-
ganisms or essential oil used. Smith et al. [29] found increased sensitivity (i.e.
bigger zones of inhibition) when nutrient agar, rather than Difco brain heart infu-
sion agar, was used for a range of methanol plant extracts. These authors also dem-
onstrated that the size of the inoculum and temperature of incubation also affect
zone size. Indeed these authors suggest that inoculum density is the single most
important factor in the variability of zone size.
A further limitation that has not been directly addressed in the literature, but for
which evidence exists, is inference in the assay from vapors liberated from the ex-
tract during incubation. This is unlikely to be a major consideration in aqueous or
solvent extracts but may be a significant confounder in assays of essential oils. In-
ouye et al. [30] have shown that the volatile constituents of essentials oils can have
a good antibacterial activity; we have also demonstrated this with essential oils
from a range of Australian native plants and lavender. It is possible that the results
of antibacterial assays completed using a closed Petri dish will reflect the combined
actions of oil components diffusing through agar and exposure to gaseous compo-
nents liberated from the oil. Which is responsible for the majority of the antibacte-
rial activity is yet to be determined.
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