Antifungal Assays
Antifungal assays are regularly used to determine whether plants extracts will have
potential to treat human fungal infections (e.g. tinea) or have use in agricultu-
ral/horticultural applications. In general these assays are quick, low cost, and do
not involve access to specialist equipment. Activity of plant extracts against the
yeast candida is typically assessed using the disk or well diffusion methods de-
scribed above, and many studies report anti-candida activity with antibacterial ac-
tivity rather than with activity against fungi for this reason (see, for example, refs
[45–48]). Activity against filamentous fungi can be evaluated in well diffusion, agar
dilution, and broth/micro-broth methods with many of the same limitations and
advantages as previously discussed for antibacterial assays [49, 50].
When the well diffusion and disk diffusion techniques are used, fungal plugs are
removed from an actively growing colony and placed at a predetermined distance
(typically 2 cm) from the center of an agar dish. A well is then bored in the center
of the agar and test substance added to the well, or the test substance is added to a
paper disk and the disk placed in the center of the agar. (The specific agar to be
used, and temperature and time of incubation, will be determined by the fungi to
be used.) The growth of the fungi is monitored and any inhibition of mycelial
growth noted. This inhibition of growth is then expressed as a percentage of the
growth of control colonies. In the agar dilution method (also known as the poison
food technique) the test substance is incorporated into the agar substrate and then
a sample of actively growing fungus is placed at the center of the plate. The radial
growth of the fungus after an appropriate time, depending on the growth charac-
teristics of the fungus, is then measured and compared with control samples.
Sridhar et al. [44] used this method to show the activity of essential oils against a
range of fungi of agricultural and medical importance.
Alternatively a fungal cell suspension may be inoculated onto the plate and the
MIC determined by the lowest concentration of test substance that prevents visible
fungal growth [51]. Antisporulation activity can be assessed by using scanning elec-
tron microscopy [52], while effects on conidium germination can be evaluated by
exposing the conidia to the test substance and subsequently counting the number
of conidia with germ tubes equal to 1–1.5 times conidium length [53]. Additional
observations of germinated conidia over a set period will also allow evaluation of
the effect of the plant extract on germ tube growth.
Inouye and co-workers have investigated the susceptibility of fungi to several es-
sential oils and have shown that MIC values can be calculated using a range of
methods [50, 52, 54, 55] . Most significantly, they have shown that when assays are
done under closed conditions (i.e. the Petri dish is sealed) the MICs are significant-
ly lower than when performed under open conditions [50]. The action of essential
oil and plant extract volatiles on fungal growth has been demonstrated for a range
of fungi [55–57] and has important implications for the screening of plant extracts
for antifungal activity. Results in these assays will depend not only on the antifun-
gal activity mediated by direct contact with the test substance but also on the vol-
8.3 Antifungal Assays 165
ume of the experimental chamber and whether it is open or closed (and hence the
presence and concentration of extract or oil volatiles). The method for evaluating
the antifungal activity of extract volatiles is straightforward and involves the place-
ment of a paper disk with test substance on the inverted lid of a Petri dish and sub-
sequent evaluation of fungal growth; however, this is rarely considered in antifun-
gal screening assays. Given the impact that volatiles can have on fungal growth it
is recommended that this be included as a standard part of antifungal assessment
of plant extracts.
Inouye et al. [50] also showed that the inclusion of Tween-80 resulted in weaker
bioactivity in agar dilution assays and the size of the original fungal inoculum had
a significant effect with larger inoculums being more resistant to antifungal ef-
fects. Shahi et al. [58] in their study of the antifungal activity of essential oils found
that the antifungal response was altered by modifying the pH of the fungal growth
media. As the media pH become more alkaline the eucalyptus essential oils had a
greater inhibitory effect on the fungi (Trichophyton spp., Microsporum spp, and Epi-
dermophyton spp.).
Antifungal assays are regularly used to determine whether plants extracts will have
potential to treat human fungal infections (e.g. tinea) or have use in agricultu-
ral/horticultural applications. In general these assays are quick, low cost, and do
not involve access to specialist equipment. Activity of plant extracts against the
yeast candida is typically assessed using the disk or well diffusion methods de-
scribed above, and many studies report anti-candida activity with antibacterial ac-
tivity rather than with activity against fungi for this reason (see, for example, refs
[45–48]). Activity against filamentous fungi can be evaluated in well diffusion, agar
dilution, and broth/micro-broth methods with many of the same limitations and
advantages as previously discussed for antibacterial assays [49, 50].
When the well diffusion and disk diffusion techniques are used, fungal plugs are
removed from an actively growing colony and placed at a predetermined distance
(typically 2 cm) from the center of an agar dish. A well is then bored in the center
of the agar and test substance added to the well, or the test substance is added to a
paper disk and the disk placed in the center of the agar. (The specific agar to be
used, and temperature and time of incubation, will be determined by the fungi to
be used.) The growth of the fungi is monitored and any inhibition of mycelial
growth noted. This inhibition of growth is then expressed as a percentage of the
growth of control colonies. In the agar dilution method (also known as the poison
food technique) the test substance is incorporated into the agar substrate and then
a sample of actively growing fungus is placed at the center of the plate. The radial
growth of the fungus after an appropriate time, depending on the growth charac-
teristics of the fungus, is then measured and compared with control samples.
Sridhar et al. [44] used this method to show the activity of essential oils against a
range of fungi of agricultural and medical importance.
Alternatively a fungal cell suspension may be inoculated onto the plate and the
MIC determined by the lowest concentration of test substance that prevents visible
fungal growth [51]. Antisporulation activity can be assessed by using scanning elec-
tron microscopy [52], while effects on conidium germination can be evaluated by
exposing the conidia to the test substance and subsequently counting the number
of conidia with germ tubes equal to 1–1.5 times conidium length [53]. Additional
observations of germinated conidia over a set period will also allow evaluation of
the effect of the plant extract on germ tube growth.
Inouye and co-workers have investigated the susceptibility of fungi to several es-
sential oils and have shown that MIC values can be calculated using a range of
methods [50, 52, 54, 55] . Most significantly, they have shown that when assays are
done under closed conditions (i.e. the Petri dish is sealed) the MICs are significant-
ly lower than when performed under open conditions [50]. The action of essential
oil and plant extract volatiles on fungal growth has been demonstrated for a range
of fungi [55–57] and has important implications for the screening of plant extracts
for antifungal activity. Results in these assays will depend not only on the antifun-
gal activity mediated by direct contact with the test substance but also on the vol-
8.3 Antifungal Assays 165
ume of the experimental chamber and whether it is open or closed (and hence the
presence and concentration of extract or oil volatiles). The method for evaluating
the antifungal activity of extract volatiles is straightforward and involves the place-
ment of a paper disk with test substance on the inverted lid of a Petri dish and sub-
sequent evaluation of fungal growth; however, this is rarely considered in antifun-
gal screening assays. Given the impact that volatiles can have on fungal growth it
is recommended that this be included as a standard part of antifungal assessment
of plant extracts.
Inouye et al. [50] also showed that the inclusion of Tween-80 resulted in weaker
bioactivity in agar dilution assays and the size of the original fungal inoculum had
a significant effect with larger inoculums being more resistant to antifungal ef-
fects. Shahi et al. [58] in their study of the antifungal activity of essential oils found
that the antifungal response was altered by modifying the pH of the fungal growth
media. As the media pH become more alkaline the eucalyptus essential oils had a
greater inhibitory effect on the fungi (Trichophyton spp., Microsporum spp, and Epi-
dermophyton spp.).
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