Methods for Testing the Antimicrobial Activity of Extracts
Jenny M. Wilkinson
Summary
There is increasing interest in the use of plant extracts as therapeutic agents, par-
ticularly the capacity for these extracts to inhibit the growth of pathogenic microor-
ganisms. In this chapter the main methods for the in vitro assessment of antimi-
crobial activity are discussed and the strengths and limitations of each method
highlighted. Methods for the assessment of antibacterial, antifungal, antiviral, and
antiparasitic activity are discussed with key issues illustrated by reference to the lit-
erature. The aim is to provide an overview of the available methods and to allow the
reader to choose the method that best suits their needs.
8.1
Introduction
Over the past decade there has been an explosion of interest in the antimicrobial,
particularly antibacterial and antifungal, activity of natural products [1–5]. This is
driven by a number of factors including increasing antibiotic resistance and fear of
development of even more infectious “superbugs,” the impact of infectious diseas-
es on mortality and morbidity, and increasing interest in “natural” therapies and a
move to more self-care. Traditional communities also wish to retain their ethnoph-
armacological heritage and exploration of traditional treatments for a variety of dis-
eases has the potential to empower these communities and improve both their
health and economy. This is particularly important in developing nations where
the use of conventional antibiotics may be limited due to cost or other factors. In
addition these communities often have a rich tradition of use of herbal and other
plant products for endemic infections; this serves as a starting point for research-
ers interested in finding treatments for these diseases.
Recommendations for the use of various natural products (e.g. essential oils,
honey, plant extracts) for infectious diseases is widespread and appears in a num-
ber of popular and other easily obtainable texts [6–10]. However, despite these nu-
157
merous claims few products have been comprehensively evaluated for their anti-
microbial activity.
One of the difficulties for researchers in this area has been the absence of a sin-
gle validated and standardized method of testing plant extracts and, in general,
methods used for in vitro testing of antimicrobial activity have been adopted from
the testing of conventional pharmaceuticals. This has several limitations; for exam-
ple, unlike conventional pharmaceuticals, natural products are complex mixes of
tens or hundreds of compounds that may or may not act as expected in the test sys-
tem. These constituents may also have limited solubility in the aqueous media that
is the typical base of many assays. Further, there are no standardized methods for
extraction or distillation of products, consequently, the exact composition of the ex-
tract being tested may be unknown with some researchers lacking access to fund-
ing or equipment to perform gas chromatography–mass spectroscopy (GC-MS)
and other chemical analyses of the extracts they are screening.
Essential oils present additional difficulties in that they may interact with dispos-
able laboratory plastics, rendering use of plastics impossible. For example the Aus-
tralian native essential oil of Backhousia citriodora (lemon myrtle) has a high per-
centage of citral (~95% or higher [11]) and direct contact with the oil, or contact via
oil volatiles, can turn standard laboratory plastics into a sticky mess (unpublished
observations). As a result, all assays with this oil must be carried out in glass equip-
ment – an additional expense that adds significantly to assay costs.
A survey of the published literature shows that there are a number of different
methods used for the assessment of antimicrobial activity; however, there is no one
method that is used by all researchers and no comprehensive study to determine
which is the best method for in vitro assays. This chapter will describe the main
methods of testing of antimicrobial activity in plant extracts and highlights the ad-
vantages and limitations of each method. Although several plants have been identi-
fied as having antibacterial or antifungal activity (e.g. cranberry juice, garlic cloves),
the most widely used plant extracts for antibacterial and antifungal activity are the es-
sential oils [12, 13], hence this discussion draws heavily on the essential oil literature.
An important consideration in this discussion is the cost (both financial and
time) and need for specialized equipment to complete some assays. Investigations
of antimicrobial activity of plant extracts, particular in the early stages of an inves-
tigation, may involve the screening of large numbers of extracts and/or large num-
bers of organisms, screening may also need to be carried out in the field or in loca-
tions where laboratory facilities are rudimentary. With these factors in mind there
may not be one “best” method, rather a selection of good methods, each best suit-
ed to a different circumstance.
8.2
Antibacterial Assays
Perhaps the most common in vitro assay used for plant extracts is the assessment
of antibacterial activity, with the majority of researchers using one of the three fol-
lowing assays: disk diffusion, agar dilution, or broth dilution/microdilution. These
158 8 Methods for Testing the Antimicrobial Activity of Extracts
methods are based on those described for standardized testing of antibiotics
[14–17]; however several factors may affect the suitability of these methods for use
with plant extracts. These factors include the type of organism being tested, con-
centration of inoculum, type of media (e.g. IsoSensitest versus nutrient agar) and
nature of the extract being tested (pH, solubility) [18–21]. The methods can be used
to simply determine whether or not antibacterial activity is present or can be used
to calculate a minimum inhibitory concentration (MIC). Table 8.1 summarizes the
limitations and advantages of these various methods.
8.2 Antibacterial Assays 159
Table 8.1 Comparison of strengths and limitations of various assays for antimicrobial activity.
Method Strengths Limitations
Antibacterial and antifungal assays
Disk well diffusion Low cost Differential diffusion of extract
components due to partitioning in the
aqueous media
Results available in 1–2 days Inoculum size, presence of solubilizing
agents, and incubation temperature
can affect zone of inhibition
Does not require specialized Volatile compounds can affect
laboratory facilities bacterial and fungal growth in closed
environments
Uses equipment and Data is only collected at one or two
reagents readily available in time points
a microbiology laboratory
Can be performed by most
laboratory staff
Large numbers of samples
can be screened
Results are quantifiable and
can be compared statistically
Agar dilution Low cost Hydrophobic extracts may separate out
from the agar
Does not require specialized Inoculum size, presence of solubilizing
laboratory facilities agents and incubation temperature can
affect zone of inhibition
Uses equipment and Volatile compounds can affect bacterial
reagents readily available in and fungal growth in closed
a microbiology laboratory environments
Can be performed by most Data is only collected at one or two
laboratory staff time points
Use of scoring systems is open to
subjectivity of the observer
Some fungi are very slow growing
160 8 Methods for Testing the Antimicrobial Activity of Extracts
Table 8.1 (Continued)
Jenny M. Wilkinson
Summary
There is increasing interest in the use of plant extracts as therapeutic agents, par-
ticularly the capacity for these extracts to inhibit the growth of pathogenic microor-
ganisms. In this chapter the main methods for the in vitro assessment of antimi-
crobial activity are discussed and the strengths and limitations of each method
highlighted. Methods for the assessment of antibacterial, antifungal, antiviral, and
antiparasitic activity are discussed with key issues illustrated by reference to the lit-
erature. The aim is to provide an overview of the available methods and to allow the
reader to choose the method that best suits their needs.
8.1
Introduction
Over the past decade there has been an explosion of interest in the antimicrobial,
particularly antibacterial and antifungal, activity of natural products [1–5]. This is
driven by a number of factors including increasing antibiotic resistance and fear of
development of even more infectious “superbugs,” the impact of infectious diseas-
es on mortality and morbidity, and increasing interest in “natural” therapies and a
move to more self-care. Traditional communities also wish to retain their ethnoph-
armacological heritage and exploration of traditional treatments for a variety of dis-
eases has the potential to empower these communities and improve both their
health and economy. This is particularly important in developing nations where
the use of conventional antibiotics may be limited due to cost or other factors. In
addition these communities often have a rich tradition of use of herbal and other
plant products for endemic infections; this serves as a starting point for research-
ers interested in finding treatments for these diseases.
Recommendations for the use of various natural products (e.g. essential oils,
honey, plant extracts) for infectious diseases is widespread and appears in a num-
ber of popular and other easily obtainable texts [6–10]. However, despite these nu-
157
merous claims few products have been comprehensively evaluated for their anti-
microbial activity.
One of the difficulties for researchers in this area has been the absence of a sin-
gle validated and standardized method of testing plant extracts and, in general,
methods used for in vitro testing of antimicrobial activity have been adopted from
the testing of conventional pharmaceuticals. This has several limitations; for exam-
ple, unlike conventional pharmaceuticals, natural products are complex mixes of
tens or hundreds of compounds that may or may not act as expected in the test sys-
tem. These constituents may also have limited solubility in the aqueous media that
is the typical base of many assays. Further, there are no standardized methods for
extraction or distillation of products, consequently, the exact composition of the ex-
tract being tested may be unknown with some researchers lacking access to fund-
ing or equipment to perform gas chromatography–mass spectroscopy (GC-MS)
and other chemical analyses of the extracts they are screening.
Essential oils present additional difficulties in that they may interact with dispos-
able laboratory plastics, rendering use of plastics impossible. For example the Aus-
tralian native essential oil of Backhousia citriodora (lemon myrtle) has a high per-
centage of citral (~95% or higher [11]) and direct contact with the oil, or contact via
oil volatiles, can turn standard laboratory plastics into a sticky mess (unpublished
observations). As a result, all assays with this oil must be carried out in glass equip-
ment – an additional expense that adds significantly to assay costs.
A survey of the published literature shows that there are a number of different
methods used for the assessment of antimicrobial activity; however, there is no one
method that is used by all researchers and no comprehensive study to determine
which is the best method for in vitro assays. This chapter will describe the main
methods of testing of antimicrobial activity in plant extracts and highlights the ad-
vantages and limitations of each method. Although several plants have been identi-
fied as having antibacterial or antifungal activity (e.g. cranberry juice, garlic cloves),
the most widely used plant extracts for antibacterial and antifungal activity are the es-
sential oils [12, 13], hence this discussion draws heavily on the essential oil literature.
An important consideration in this discussion is the cost (both financial and
time) and need for specialized equipment to complete some assays. Investigations
of antimicrobial activity of plant extracts, particular in the early stages of an inves-
tigation, may involve the screening of large numbers of extracts and/or large num-
bers of organisms, screening may also need to be carried out in the field or in loca-
tions where laboratory facilities are rudimentary. With these factors in mind there
may not be one “best” method, rather a selection of good methods, each best suit-
ed to a different circumstance.
8.2
Antibacterial Assays
Perhaps the most common in vitro assay used for plant extracts is the assessment
of antibacterial activity, with the majority of researchers using one of the three fol-
lowing assays: disk diffusion, agar dilution, or broth dilution/microdilution. These
158 8 Methods for Testing the Antimicrobial Activity of Extracts
methods are based on those described for standardized testing of antibiotics
[14–17]; however several factors may affect the suitability of these methods for use
with plant extracts. These factors include the type of organism being tested, con-
centration of inoculum, type of media (e.g. IsoSensitest versus nutrient agar) and
nature of the extract being tested (pH, solubility) [18–21]. The methods can be used
to simply determine whether or not antibacterial activity is present or can be used
to calculate a minimum inhibitory concentration (MIC). Table 8.1 summarizes the
limitations and advantages of these various methods.
8.2 Antibacterial Assays 159
Table 8.1 Comparison of strengths and limitations of various assays for antimicrobial activity.
Method Strengths Limitations
Antibacterial and antifungal assays
Disk well diffusion Low cost Differential diffusion of extract
components due to partitioning in the
aqueous media
Results available in 1–2 days Inoculum size, presence of solubilizing
agents, and incubation temperature
can affect zone of inhibition
Does not require specialized Volatile compounds can affect
laboratory facilities bacterial and fungal growth in closed
environments
Uses equipment and Data is only collected at one or two
reagents readily available in time points
a microbiology laboratory
Can be performed by most
laboratory staff
Large numbers of samples
can be screened
Results are quantifiable and
can be compared statistically
Agar dilution Low cost Hydrophobic extracts may separate out
from the agar
Does not require specialized Inoculum size, presence of solubilizing
laboratory facilities agents and incubation temperature can
affect zone of inhibition
Uses equipment and Volatile compounds can affect bacterial
reagents readily available in and fungal growth in closed
a microbiology laboratory environments
Can be performed by most Data is only collected at one or two
laboratory staff time points
Use of scoring systems is open to
subjectivity of the observer
Some fungi are very slow growing
160 8 Methods for Testing the Antimicrobial Activity of Extracts
Table 8.1 (Continued)
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