224
R. Tesser et al.
The effectiveness of essential oil is demonstrated by the extension of the zone of
microorganism growth inhibition around the disc or well, and it is usually expressed
as the diameter of this zone. The main drawbacks of the agar diffusion method are:
(i) the volatile components of EOs are likely to evaporate with the dispersing solvent
during the incubation time; (ii) poorly soluble EOs components do not diffuse well
in the agar broth. Notwithstanding these rather serious drawbacks, this test is still the
most common technique for the antibacterial and antifungal EOs activity assessment,
it is easy to implement and requires only very low amounts of essential oil. It can be
recommended for a preliminary screening when a large number of EOs samples have
to be tested and, among them, the selection of the most active will be then submitted
to more sophisticated methods [77].
The second test method considered here, consists of a sequence of dilutions (dilution method), widely employed for bacteria and fungi. The broth dilution method
has recently become popular [36] also in the EOs assessment test. The test consists
of putting Agar broth cultures in Petri dishes or tubes, while liquid broth cultures are
cultivated in conical flasks filled with culture medium [122]. For the liquid broth in
conical flasks the results, expressed as inhibitory growth index, are computed as a
percent of the variation in mould’s biomass compared to the control culture [122].
The inhibitory effect of essential oil in the test tubes cultures and microtiter plates
is instead measured turbidimetrically [101] or with the count plate method (Kester
et al. 1996). Both in agar and liquid broth, the tested microorganisms can settle onto
the membrane [107], which strongly simplifies the procedure and give an accurate
estimation of EO activity. As a summary, the result of this inhibition test, can be
expressed in the two following ways:
• The growth inhibition index defined as the percentage ratio to the control growth
culture without essential oil.
• Minimal inhibitory concentration (MIC) restraining microorganism growth or
minimal lethal concentration (MLC).
In addition to the two described assessment tests, it is worth to cite at last also
the turbidimetry method, also a commonly used technique, where the changes in the
optical density (OD) of the growing culture is measured [122].
Fungicides
Pesticides play an important role in the agriculture sectors. Pesticides are mainly in the
form of aqueous concentrates (AC) for herbicides, as emulsifiable concentrates (EC)
for insecticides and as wettable powders (WP) for fungicides. Customers increasingly require safer and more convenient pesticide formulations such as suspension
concentrates (SC), water-dispersible granules (WDG) and emulsions in water. Generally, the preparation of pesticide formulations will include two main components,
i.e. the active ingredient(s) and inert ingredients. Active ingredient(s) is the chemical
or chemicals responsible for preventing, destroying, repelling or mitigating insects,
fungi, weeds, rodents, etc., whereas, inert ingredients or supplements are inactive
ingredients that have no pesticide action but function as wetting agents, dispersing
agents, emulsifiers, spray adjuvants, solvents, carrier/diluents, etc. At present, there
R. Tesser et al.
The effectiveness of essential oil is demonstrated by the extension of the zone of
microorganism growth inhibition around the disc or well, and it is usually expressed
as the diameter of this zone. The main drawbacks of the agar diffusion method are:
(i) the volatile components of EOs are likely to evaporate with the dispersing solvent
during the incubation time; (ii) poorly soluble EOs components do not diffuse well
in the agar broth. Notwithstanding these rather serious drawbacks, this test is still the
most common technique for the antibacterial and antifungal EOs activity assessment,
it is easy to implement and requires only very low amounts of essential oil. It can be
recommended for a preliminary screening when a large number of EOs samples have
to be tested and, among them, the selection of the most active will be then submitted
to more sophisticated methods [77].
The second test method considered here, consists of a sequence of dilutions (dilution method), widely employed for bacteria and fungi. The broth dilution method
has recently become popular [36] also in the EOs assessment test. The test consists
of putting Agar broth cultures in Petri dishes or tubes, while liquid broth cultures are
cultivated in conical flasks filled with culture medium [122]. For the liquid broth in
conical flasks the results, expressed as inhibitory growth index, are computed as a
percent of the variation in mould’s biomass compared to the control culture [122].
The inhibitory effect of essential oil in the test tubes cultures and microtiter plates
is instead measured turbidimetrically [101] or with the count plate method (Kester
et al. 1996). Both in agar and liquid broth, the tested microorganisms can settle onto
the membrane [107], which strongly simplifies the procedure and give an accurate
estimation of EO activity. As a summary, the result of this inhibition test, can be
expressed in the two following ways:
• The growth inhibition index defined as the percentage ratio to the control growth
culture without essential oil.
• Minimal inhibitory concentration (MIC) restraining microorganism growth or
minimal lethal concentration (MLC).
In addition to the two described assessment tests, it is worth to cite at last also
the turbidimetry method, also a commonly used technique, where the changes in the
optical density (OD) of the growing culture is measured [122].
Fungicides
Pesticides play an important role in the agriculture sectors. Pesticides are mainly in the
form of aqueous concentrates (AC) for herbicides, as emulsifiable concentrates (EC)
for insecticides and as wettable powders (WP) for fungicides. Customers increasingly require safer and more convenient pesticide formulations such as suspension
concentrates (SC), water-dispersible granules (WDG) and emulsions in water. Generally, the preparation of pesticide formulations will include two main components,
i.e. the active ingredient(s) and inert ingredients. Active ingredient(s) is the chemical
or chemicals responsible for preventing, destroying, repelling or mitigating insects,
fungi, weeds, rodents, etc., whereas, inert ingredients or supplements are inactive
ingredients that have no pesticide action but function as wetting agents, dispersing
agents, emulsifiers, spray adjuvants, solvents, carrier/diluents, etc. At present, there