SCREENING METHODS IN THE STUDY OF ...3 Screening Methods in the Study of Antimicrobial Properties of Medicinal Plants deposited into well cut into the agar and can be used with large

International Journal of Biotechnology and Research (IJBTR) ISSN 2249-6858 Vol.2, Issue 1 June 2012 1-35 © TJPRC Pvt. Ltd.,

SCREENING METHODS IN THE STUDY OF ANTIMICROBIAL

PROPERTIES OF MEDICINAL PLANTS

SUJOGYA KUMAR PANDA

Department of Biotechnology, North Orissa University, Baripada, Odisha, India - 757003

ABSTRACT

The scientific communities have received attention on wide range of publication on various

techniques with special reference to antimicrobial activity. Much attention has been drawn to the

antimicrobial activity of plant extracts in crude form and their metabolites due to the challenge of

growing incidences of drug-resistant pathogens. Profuse use of commercial antibiotics, synthetic

pesticides develop multiple drug resistance among pathogens. Even some plants have shown the ability

to overcome resistance in certain organisms and this has led to researchers’ investigating their

mechanisms of action and isolating active compounds. Particular focus is on establishing the effect of the

plant extracts in terms of their microstatic and microcidal action and the spectrum of organisms affected.

This has enabled exploitation of plants for the treatment of microbial infections and in the development

of new antimicrobial compounds. However by simple isolating and characterizing phytochemicals

without bioactive properties is worthless. To achieve applied meaning and significance, extracts must

be screened for biological activity with antimicrobial and other bioassay. This paper reviews the current

methods used in the investigations of the efficacy of plants as antimicrobial agents and points out some

of the differences in techniques employed by different authors.

KEYWORDS: Plant extracts, antimicrobial activity, disk diffusion, agar cup, broth dilution, MIC,

MBC, TLC bioautography, SEM

ANTIMICROBIAL SUSCEPTIBILITY TESTING

Antimicrobial susceptibility test (AST) is used to determine the efficacy of potential antimicrobials

from natural products against a number of microorganisms. In clinical research, in vitro susceptibility

tests are important if an organism is suspected toward resistance to frequently used antimicrobial agents.

Today, numerous standard methods, approved by different organization like the National Committee for

Clinical Laboratory Science (NCCLS), British Society for Antimicrobial Chemotherapy (BSAC) and the

European Committee for Antimicrobial susceptibility testing (EUCAST), for antimicrobial susceptibility

testing of conventional drugs. A group of microorganisms such as bacteria, fungi, yeast, virus,

protozoan’s and helminthes are used for screening of plant extracts. Evaluation of the performance of a

susceptibility test should have ease to use, reproducibility, test sensitivity and specificity (Struelens et al.,

1995). AST standard tests can be conveniently divided into diffusion and dilution methods. Common

diffusion tests include agar well diffusion, agar disk diffusion and bioautography, while dilution methods

include agar dilution and broth micro or macrodilution. In addition to this, other commercial custom-

Sujogya Kumar Panda 2

prepared methods viz. the agar screen plate, Epsilometer test and the Vitek system are used as standard

reference methods but these are not common in routine AST for testing activity of plant extracts (Joyce

et al., 1992).

Antibacterial assays

Methods which are used to evaluate the activity of plant antimicrobial are divided into in vitro and in

vivo (application test). The former may be termed “screening methods” and might include any test in

which the compound is not applied directly to the product under use conditions. Generally, these tests

provide preliminary information to determined potential usefulness of the test compound. The second

type includes those tests in which an antimicrobial is applied directly to a product.

In vitro methods

In vitro screening methods are subdivided into endpoint and descriptive tests. Endpoint tests are

those in which a microorganism is challenged for an arbitrary period. The results reflect the inhibitory

power of a compound only for the time specified. In descriptive test, the microorganism is also

challenged, but periodic sampling is carried out to determine changes in viable cell number over time.

Endpoint screening methods

Agar Diffusion

Of the endpoint test, the agar diffusion test has probably been the most widely used throughout

history. It has often been referred to as the disk assay. However, this terminology is probably too narrow.

There are many variations of this method such as use of cylinder, well, the ditch plate, agar overlays etc.

Among the most common variations of the assay, the antimicrobial compound is applied to an agar plate,

using an impregnated filter paper disk, or placed in a well. The compound diffuses through the agar,

setting up a concentration gradient. The concentration is inversely proportional to the distance from disk

or area. Inhibition, which is the measure of activity, is indicated by a zone without growth of the

organism around the disk or well. The size of the zone is dependent on the rate of diffusion and growth

of the organism. The most widely used screening methods to measure the antimicrobial efficacy of

medicinal plants, spice, their essential oil, and their constituents are agar diffusion method (Kivanc and

Anguel, 1986; Deans and Ritchie, 1987; Farag et al., 1989; Aureli et al., 1992; Qamar et al., 1994; Bara

and Vanetti, 1995; Domokos et al., 1997; Lis-Balchin and Deans, 1997; Firouzi et al., 1998; Ilcim et al.,

1998; Lachowicz et al., 1998; Wei et al., 1998; Sun Kyung et al., 1999; Rauha et al., 2000; Yildirim et

al., 2000; Yong Seon et al., 2000; Elgayyar et al., 2001; Martins et al., 2001; Minija and Thoppil, 2001;

Okeke et al., 2001; Unal et al., 2001 Iscan et al., 2002; Rasooli et al., 2002; Velickovic et al., 2002;

Venturini et al., 2002; Aureli et al., 2003; Faleiro et al., 2003; Safak et al., 2003; Vilijoen et al., 2003;

Thiem and Goslinska, 2004; Panda et al., 2011a,d). With disk diffusion method, a paper disk soaked with

medicinal plant extract is laid on top of an inoculated agar plate. Factors such as the volume of extract

placed on the paper disk, the thickness of the agar layer and whether a solvent is used vary considerable

between studies. On the other hand in agar well test is a quantitative method in which the extract is

Screening Methods in the Study of Antimicrobial Properties of Medicinal Plants 3

deposited into well cut into the agar and can be used with large number of extracts and/or large number

of bacterial isolates (Dorman and Deans, 2000). To make bacterial growth easier to visualize, triphenyl

tetrazolium chloride may also added to the growth medium (Elgayyar et al., 2001; Mourey and Canillac,

2002). Microorganisms are generally termed susceptible, intermediate, or resistance, depending upon the

diameter of the inhibition zone. Quantitative results are possible with a high degree of standardization.

This method should not be used for anaerobic microorganisms.

Disk diffusion method

The disk diffusion method (also known the zone of inhibition method) is probably the most widely

used method because of its simplicity and low cost. It uses only small amounts of the test substance

(10-30 µL), can be completed by research staff with minimal training (plate-1). The method involves the

preparation of a Petridish containing 15-25 mL agar, bacteria at a known concentration are then spread

across the agar surface and allowed to establish. 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. On the other hands,

data from this assay is typically presented as mean size of zone of inhibition (with or without standard

deviation), or a ranking system such as “+”, “++”, and “+++” to indicate levels of activity. Few authors

also provide levels of activity (slight, moderate, strong) without any reference to standardized criteria.

One of the major criticisms of this method is that it relies on the ability of the extract 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 gradient 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. There is also no consensus on the

best agar to use for these assays.

Agar cup method

The principle of the agar cup or agar well diffusion is the same as that of the agar disk diffusion

method. A standardized inoculum culture is spread evenly on the surface of gelled gar plates. Wells of

between 6 and 8 mm are aseptically punched on the agar using a sterile cork borer allowing at least

30 mm between adjacent wells and the Petri dish. Fixed volumes of the plant extract are then introduced

into the wells (plate-2). The plates are then incubated at 37oC for 24 h for bacteria (Mbata et al., 2006,

Panda et al., 2011b,d,e).

Agar dilution methods

The agar dilution methods are relatively quick methods that do not involve the use of sophisticated

equipment. Any laboratory with basic microbiological facility can use this method. In this method, the

test substance is incorporated at known concentrations into the agar. Once the agar set, bacteria are


applied to its surface. Replicate dishes can be set up with a range of concentrations of the test substance

and by dividing the surface of the agar into wedges or squares. A number of bacterial species may be

applied to a single dish. In this way, a large number of bacteria may be screened within a single assay

run. The dishes are incubated for 24 h or more and the growth of the bacteria on the extract/agar mix is

scored either as present/absent or a proportion of the control (e.g. 0, 25%, 50%, 75%, 100%). During the

evaluation of antimicrobial activity of medicinal plant extracts by agar dilution method, several

researchers used different solvents (Prudent et al., 1995; Pintore et al., 2002), different volumes of

inoculums (Juven et al., 1994; Prudent et al., 1995), various inoculation techniques, e.g. dotting (Pintore

et al., 2002) or streaking (Farag et al., 1989). Despite these variations, the MICs of medicinal plant

extracts determined by agar dilution generally are in approximately the same order of magnitude (Farag

et al., 1989; Prudent et al., 1995; Pintore et al., 2002).

A criticism of this method is that when a scoring system is used it is difficult to compare one set of

results with another. This method suffers from several other limitations, including many that have been

discussed previously: (a) use of larger volumes of test substance than in other methods, (b) confounding

antibacterial actions from volatiles, (c) difficulty of achieving stable emulsions of essential oils in agar

and (d) restriction on the maximum concentration that can be used before the agar becomes too dilute to

solidify properly. However this technique is much more difficult with the extract deals with essential oil

and other hydrophobic plant extracts. Many researchers have thought they had incorporated their

essential oil into nutrient broth or other media but after an hour of the experiment, the oil had separated

out and was floating on top of the media. Griffin (2000), in their work on tea tree oil found that at

concentrations above 2% v/v the oil separated from the agar substrate and was seen as droplets on the

agar surface. The most commonly utilized method to overcome this problem is the use of surfactants

such as Tween-20, Tween-80, and alkyl dimethyl betaine (ADB). Several authors have described the use

of these products and the effect on antibacterial activity. The results of their studies show that surfactants

can interfere with calculation of MIC values and the growth of some test organisms (Hammer et al.,

1999). However, it has also been demonstrated that it is possible to use very small quantities of Tween

(<0.5% v/v) to emulsify the essential oil in media and thus avoid the effects on organism growth (Griffin,

2000; Hood et al., 2004). Hammer et al. (1999) also showed that inclusion of organic matter such as

bovine serum albumin in the agar also affected the antibacterial activity of tea tree oil.

Broth dilution methods

Difficulties with partitioning of hydrophobic compounds in agar a more accurate method was

developed known as broth dilution method. In this method, bacteria are grown in test-tubes in a liquid

media in the presence of the test substance (plate-3) (Panda et al., 2009a,b,c). At regular time intervals

(e.g. every 10 min or every hour) a sample is removed and the bacterial count (CFU) determined by

serial dilution. In contrast to the single data point (e.g. 24 h incubation) utilized in disk diffusion and agar

dilution assay, the broth dilution method allows much finer evaluation of the antibacterial events over

time. In addition to this, this method has other advantages includes recovery from the effects of the test

substance and proportion of organisms killed at a given time point can be determined. However, the


method is also time and resource intensive and can be impractical where very large numbers of test

substances are to be screened. As with other testing methods incorporation of hydrophobic compounds

and essential oils into the aqueous media is problematic, and as there is no solid phase to trap these

compounds they rapidly separate from the media and form a layer across the surface of the media. For

organisms sensitive to oxygen tension in the media this can present an additional problem as the oil can

inhibit gaseous exchange. Tween or ethanol may be used to enhance incorporation into the aqueous

media, however as previously discussed these compounds may interfere with the assay results. The broth

assay was used in both the macro-and microdilution versions. The microtube version allows an increase

in productivity through the use of microtiter plates. Critical control points or standardization of the assay

may be achieved at the following stages, medium type, pH, stock solutions, concentration range,

inoculums density, and incubation conditions.

Micro broth methods have also been developed, which utilize microtiter plates, thus reducing the

volume of extract needed, and have endpoints that can be determined spectrophotmetrically, either a

measure of turbidity or use of a cell viability indicator (e.g. resazurin, methylthiazoldiphenyltetrazolium

(MTT)) (Mann and Markham, 1998; Panda et al., 2010c, 2011a) (plate-4). They also propose that the cell

viability indicator is the best method of endpoint determination for experiments with essential oils

interface with turbidity measures. While these micro-broth methods generally work well for plant

extracts, problems arise when the extract is heavily colored as this can interfere with the measurement of

the indicator chemical. Further, as these methods use plastic microtiter plates, essential oils that have a

solvent action on plastics (e.g. Letospermum petersonii, Backhousia citriodora) cannot be used. Also the

addition of essential oils to media, changes its pH and this might be expected to be more significant in

small volumes, like the micro broth method (Hood et al., 2004). Micro broth methods are also less time

and resources intensive than other broth methods because bacterial count are eliminated.

With both the agar and broth dilution assays, the objective is to generate a single statistic to describe

the inhibition of a microorganism at a specific endpoint in time. The measurement of inhibition at a

specific time is termed the minimum inhibitory concentration (MIC). The MIC may be defined as the

lowest concentration at which no growth occurs in a nutrient medium. However, the definition of the

MIC differs between publications. In some cases, the minimum bactericidal concentration (MBC) or the

bacteriostatic concentration is stated, both terms agreeing closely with the MIC. A list of the most

frequently used terms in antimicrobial activity testing of crude medicinal plants extracts and essential

oils are presented in Table-1.

What is often found in antimicrobial assays is that concentrations at and above the MIC cause

reversible inhibition. Removing the inhibitory pressure of the antimicrobial will result in growth. This

can best be demonstrated with the broth dilution assay. An aliquot of medium from any tube which

demonstrates no growth in the MIC assay is transferred to fresh medium which contains no

antimicrobial. If no growth occurs in the fresh medium, lethality has occurred. This is defined as the

Minimum Lethal Concentration (MLC). In general, dilution tests should be used when quantitative data

are desired, when the strains tested have variable growth rates, when it is desirable to determine lethality,


to measure the effects of combinations of antimicrobials quantitatively, and when the test should be

performed with anaerobic or microaerophilic microorganisms. The agar dilution method was used to

determine the strength of the antimicrobial activity of medicinal plants extract, spices, and essential oils

by Banerjee et al., (1982), Kivanc and Akguel (1986), Bara and Vanetti (1995), Sekiyama et al. (1996),

Firouzi et al. (1998), Ward et al. (1998), Hammer et al. (1999), Elgayyar et al. (2001), Unal et al. (2001),

Iscan et al. (2002), Velickovic et al. (2002), Venturini et al. (2002), Alzoreky and Nakahara, (2003),

Aureli et al. (2003), Burt and Reinders (2003), Nguefack et al. (2004) and Tepe et al., (2004). The broth

dilution method was used by Bara et al. (1995), Carson (1995b), Cosentino et al. (1999), Hammer et al.

(1999), Jeong Jun et al. (1999), Unal et al. (2001), Delaquis et al. (2002), Jee Young et al. (2002),

Velickovic et al. (2002), Venturini et al. (2002), Araujo et al. (2003), Cosentino et al. (2003), Tepe et al.

(2004), Thiem and Goslinska (2004) and Yu et al. (2004). An alternation determination of the end point

of broth dilution assays is the measurement of conductance or impedance (Tassou et al., 1995; Wan

et al., 1998; Marino et al., 1999, 2001).

Gradient plates

A method similar to the agar dilution assay is the gradient plate technique. In this method, melted

agar is dispensed into a Petri dish with one edge elevated. The agar is allowed to solidify and form a

wedge. A second, overlay wedge which contains the antimicrobial is then poured. The result is a plate

which contains a gradient of antimicrobial concentrations from near 0 to the maximum in the overlay.

The plate may then be inoculated using a spread plate method or by parallel streaks of several strains of

microorganisms. The advantage of the analysis is that the microorganism is exposed to a continuous

gradient of concentrations, as opposed to the 2-fold or 10- fold dilutions used in the previous tests. This

technique was used to test the antimicrobial activity of medicinal plants, spices, and essential oils by

Ting and Deibel (1992) to study the sensitivity of Listeria monocytogenes to various extracts such as

clove, oregano and black pepper. A variation of the gradient plate technique utilizes a spiral plating

system. This assay uses the spiral plate to deposit a radial gradient of antimicrobial on an agar plate.

Because the volume of liquid deposited on any section of the plate is known, the concentration of

antimicrobial may then be determined as well. A bacterial suspension is inoculated onto the surface of

the plate to produce a radial streak. The MIC of the compound against the microorganism is determined

by a zone of clearence on the streak.

Descriptive screening methods

While endpoint methods are excellent for screening compounds, they give less information

concerning the effect of the compound on dynamic growth of microorganisms. Once the information has

been determined about the MIC for the microorganism, it is important to look at the microorganism’s

growth over time. There are several possible methods for evaluating the effect of an antimicrobial on the

growth of a microorganism over time. Two of the most popular are the turbidimetric assay and the

inhibition curve.


Turbidimetric assay

The simplest, most economical and productive growth measurement system is probably the

turbidimetric assay. It simply involves following the growth of a microorganism with a

spectrophotometer. One of the major problems with turbidimetric analysis is the range of detection.

Spectrophotometers generally require 106-107 CFU/ml for detection, thereby limiting cell concentrations

which can be successfully assayed with this method. This may create a situation in which no growth (i.e.,

no absorbance increase) is observed, when, in fact, undetectable growth is occurring at levels below 105

CFU/ml. An erroneous interpretation of ‘lethality’ could result (Davidson and Parish, 1989). There have

been many publications using this method to study the antimicrobial activity of medicinal plants, spices,

and their essential oils including Shelef et al. (1984), Meena et al. (1986), Syed et al. (1986a, 1986b),

Ismaiel and Pierson (1990), Kanemaru and Miyamoto (1990), Rajashekhara et al. (1990), Kim et al.

(1995a, 1995b), Sivropoulou et al. (1995, 1996), Chaibi et al. (1997), Wilson et al. (1997), Smith-Palmer

et al. (1998), Ultee et al. (1998), Jeong Jun et al. (1999), Pol and Smid (1999), Kokoska and Rada

(2001), Skandamis et al. (2001), Ultee and Smid (2001), and Mejlholm and Dalgaard (2002).

Inhibition or Time killing curve

In addition to turbidimetric analyses, there is the inhibition curve, also known as the ‘killing curve’

in clinical research. This test simply involves inoculation of a microorganism into a medium, addition of

an antimicrobial, followed by incubation and periodic sampling to determine growth of survival. It is a

more accurate analysis than the turbidimetric assay because of the wide detection range. Some of the

resulting curves are easy to interpret, other are not. Plate-5a shows some of possible situations one might

encounter in running this type of test. First (A) is growth level suppression. This is sometimes used with

a term called ‘percentage growth inhibition’ which may be misleading because it is a function of time.

Second (B) is a lag phase increase. Third (C) is a decrease in the growth rate with little effect on lag time.

Forth (D) is lethal effect, the time killing curve is the only test which will show this effect. Following an

antimicrobial test in which lethality occurs, every small percentage of the original population will often

remain viable. This method is versatile but has several disadvantages. No simple statistical method is

available to detect differences, no single statistic is produced to compare treatment such as MIC and it is

labour intensive and expensive. The time kill curve was used to evaluate the antimicrobial activity of

medicinal plants, spices, and essential oils by Beuchat (1976), Shelef et al. (1984), Ting and Deibel

(1992), Aureli et al. (1992), Stecchini et al. (1993), Tassou et al. (1995), Sivoropoulou et al. (1996),

Ultee et al. (1998), Wan et al. (1998), Pol and Smid (1999), Sun Kyung et al. (1999), Periago and

Moezelaar (2001), Skandamis et al. (2001), Sung Hwang and Young Rok (2001), Ulte and Smid (2001),

Mejlholm and Dalgaard (2002), Pintore et al. (2002), Young Rok and Sung Hwan (2002), Burt and

Reinders (2003), Cressy et al. (2003) and Nakamura et al. (2004). Screening methods should be used

together, one endpoint and one descriptive test. The endpoint test helps to determine the approximate

effective concentration, and the descriptive test evaluates the effect of a compound on growth over time.


Several factors may affect the suitability of these methods for use with plant extracts. These factors

include the type of organism being tested, concentration of inoculum, type of media and nature of the

extract being tested (pH, solubility etc.) (Griffin, 2000; Hood et al., 2004). 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 2 summarizes the limitations and advantages of these various

methods. All these methods are those most widely used for in vitro testing of plant extracts for

antibacterial activity, while some other methods are also have been used. For example, Garedew et al.

(2004) report on the use of a flow calorimetric method to assess antibacterial activity of honey and

demonstrated better sensitivity than other methods and Pitner et al. (2000) propose the use of high

throughput systems that measure bacterial respiration via a fluorescent signal. However, the practicality

of these methods for screening of plant extracts is yet to be determined.

Application methods

While in vitro tests can give a good deal of information antimicrobial performance, they cannot

necessarily duplicate all the variability which might exist in a plant. Therefore, once it has been

determined that the antimicrobial performs well in an in vitro situation, it should be applied to in vivo

system. Without the screening method data, it is difficult to determine starting concentrations.

Combination studies

Antimicrobial combinations are selected for various reasons

1) To attain broad-spectrum activity for empiric therapy in critically ill patients or when

polymicrobial infection is suspected.

2) To minimize drug toxicity by using the lowest possible doses of two or more agents

that have additive efficacies but independent toxicities, or to reduce the potential for

development of resistance to one agent.

Combined antimicrobial agents have been extensively studied in the pharmaceutical industry, and

methods have been developed to determine types of interactions between two antimicrobials (Barry,

1976; Krogstad and Moellering, 1986; Squires and Cleeland, 1985). When two antimicrobials are used in

combination, three things may occur. First, there may be an additive effect i.e. the combined effect is

equal to the sum of the effects observed with the two agents tested separately or equal to that of the most

active agent in combination (Barry, 1976). Additive effects occur when the antimicrobial activity of a

compound is neither enhanced nor reduced while in the presence of another agent. The second

occurrence can be synergistic i.e. the effect observed with a combination is greater than the sum of the

effects observed with the two agents independently. Synergism refers to an enhancement of overall

antimicrobial activity of a compound when in the presence of a second antimicrobial agent. Finally, one

may have antagonism between the pair. Antagonism occurs when the antimicrobial activity of one

compound is reduced in the presence of a second agent. According to Krogstad and Moellering (1986),

synergistic interactions are generally due to sequential inhibition of a common biochemical pathway e.g.,


use of compounds which inhibit enzyme that interactive antimicrobials, combinations of cell wall active

agent, and use of cell wall active compounds to increase uptake of other compounds. The mechanisms

for antagonism are much more complex and less well studied. Examples of causes for antagonism are

combinations of bacteriostatic and bactericidal agents, use of agents with same active site (e.g., the 50S

submit of the ribosome), and a chemical interaction (direct or indirect) between two agents (Krogstsad

and Moellering, 1986). Perhaps the most misused of the above term is synergism. This term has been

used to describe relationships between agents without regard to the overall concentration of antimicrobial

in combination. In other words, increased antimicrobial activity in a combination which contains

100 mM of compound A and 100 mM of A or B alone and would not necessarily constitute synergy.

Many reports exist where the activity of a single agent is reported along with the activity of a

combination and synergism is claimed by the investigator. A report of synergism requires that the

antimicrobial activity of each agent be reported with the combined effect and that the combined effect be

greater than the expected, based on the activity of the individual compounds.

Checkerboard method

Since day-to-day biological variability of MICs makes it difficult to compare confidently results

from different tests, the data may be transformed to produce what is called the fractional inhibitory

concentration, FIC (Barry, 1976). The FIC for an individual antimicrobial agent is the ratio of the

concentration of the antimicrobial in an inhibitory combination with a second agent to the concentration

of the antimicrobial by itself. In other words, the concentration of a given compound needed to inhibit

growth is given a value of 1, and the amount of the compound needed to inhibit growth when combined

with another antimicrobial agent at a given concentration is expressed as a fraction. Most researchers add

FICs for individual agents in a combination to produce a FIC Index (Barry, 1976). Calculation of a

combine FIC yields a single number which can be indicative of additive, synergistic, or antagonistic

effects. Theoretically, a combined FIC near 1 indicates additivity, <1 indicates synergy, and >1 indicates

antagonism. However, the degree to which a value must be greater than or less than 1 to suggest

antagonism or synergism is not clearly established (Plate-5).

Later, Orhan et al., (2005) represented a combination of antibiotic and crude extracts. The antibiotic

in combination with the crude drug was serially diluted along with antibiotic and crude drugs. An

inoculum with specific standard are prepared with a specific strain of Mycobacterium. The method is

same as broth microdilution but it will differentiate by treatment with combination of crude drug and a

specific antibiotic. Synergy is more likely to be expressed when the ratio of the concentration of each

antibiotic to the MIC of that antibiotic was same for all components of the mixture. The ∑FICs were

calculated as follows:∑FIC = FIC A + FIC B, where FIC A is the MIC of drug A in the

combination/MIC of drug A alone, and FIC B is the MIC of drug B in the combination/MIC of drug B

alone. The combination is considered synergistic when the ∑FIC is ≤0.5and antagonistic when the

∑FIC is ≥2 (Orhan et al., 2005; Panda et al., 2010a).


TLC-Bioautography

While the methods above are used to test whole extracts or extracts fractionated at another time there

is an increasing interest in bioassay guided fractionation, where the separation of extracts into fractions is

completed simultaneously with identification of bioactivity. In this method, TLC is performed using

crude extracts, extract fractions, or whole essential oils. The developed TLC plate is then sprayed with or

dipped into bacterial or fungal suspension (direct bioautography) or overlain with agar and the agar

seeded with the microorganism (overlay bioautography) (Hamburger and Cordell, 1987; Rahalison et al.,

1991, 1994; panda et al., 2010a, 2011c) (Plate-6). The later method has been particularly used for

determining the activity of extract against yeasts. This method has been used to screen a range of crude

and solvent prepared extracts with the activity observed dependent on both the method of extraction and

solvents used in the TLC process (Diallo et al., 2001; Nakamura et al., 1999; Sridhar et al., 2003). This

method has the advantage of combining both separation of extract constituents and simultaneous

identification of those fractions with bioactivity. However, it is not a suitable method for detecting

activity that is a product of synergy between two or more compounds. Further, the results will be affected

by the breakdown or alteration of compounds during the fractionation phase.

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 agricultural/horticultural applications. In general

these assays are quick, low cost, and do not involve access to specialized equipments. Activity of plant

extracts against the yeast Candida is typically assessed using the disk or well diffusion methods

described above, and many studies report anti-candidal activity with antibacterial activity rather than

with activity against fungi for this reason (Haraguchi et al., 1999; Rahua et al., 2000; Wilkinson and

Cavanagh, 2005; Panda et al., 2010b,d,e,f). Activity against filamentous fungi can be evaluated in well

diffusion, agar dilution, and broth/microbroth methods with many of the same limitations and advantages

as previously discussed for antibacterial assays (Inouye et al., 2001). 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 centre of an agar dish. A well is then bored in the centre

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 centre 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

mycelia 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

centre of the plate. The radial growth of the fungus after an appropriate time, depending on the growth

characteristics of the fungus, is then measured and compared with control samples. Sridhar et al. (2003)

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 de Aquino


Lemos et al. (2005). Antisporulation activity can be assessed by using scanning electron microscopy

(Inouye et al., 1998), 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 (Antonov et al., 1997). Additional observations of germinated conidia over a set period

will also allow evaluation of the effect of the plant extract on germ tube growth. All the methods have

their own advantages and disadvantages as describe above in testing of antibacterial activity. In addition

to these Inouye et al. (2001) 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 effects. Shahi et al. (1999) 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 Epidermophyton spp.).

Poison food technique

Generally antifungal activity is determined by poisoned food technique (Nene and Thapliyal, 2000,

Kiran et al., 2010). Five-day old fungal culture is punched aseptically with a sterile cork borer of

generally 6mm diameter. The fungal discs are then put on the gelled agar plate. The agar plates have

been prepared by impregnating desired concentration of plant extract at a temperature of 45-50 °C. The

plates are then incubated at temperature 26 ± 2°C for fungi. Colony diameter is recorded by measuring

the two opposite circumference of the colony growth. Percentage inhibition of mycellial growth is

evaluated by comparing the colony diameter of poisoned plate (with plant extract) and nonpoisoned plate

(with distilled water) and calculated using the formula given below (Verma and Kharwar, 2008);

Mycellial growth (control) -Mycellial growth (treatment) % Mycelial inhibition = X 100 Mycellial growth (control)

Spore germination assay

Spore germination assay is a slide technique used for testing of antifungal activity of plant extracts

(Nair et al., 1991). Plant extract of desired concentration and volume are added to the surface of dried

slides as a film or in a cavity of a cavity slide. Fixed volume and standard concentration of spore

suspension of test fungi are spread over the film whereas in controlled treatment, distilled water is added

in place of spore suspension. Slides are then placed on a glass rod in Petri dish under moistened

conditions and incubated for 26 ± 2°C. After incubation, slides are fixed in lacto phenol cotton blue and

observed microscopically for spore germination. Percentage spore germination is calculated according to

the following formula.

% Spore germination = Number of germinated spores / Total number of spores X 100


Specific recommendations on antibacterial and antifungal screening

Panel of test organisms

The choice of test organisms depends on the specific purpose of the investigation. In a primary

screening, drug-sensitive reference strains are preferably used and should represent common pathogenic

species of different classes. Various combinations are possible, but the panel should at least consist of

both Gram-positive and Gram-negative bacterium. It has been well-established that Gram-positive

bacteria are much more sensitive to drug action than Gram-negative bacteria, which is reflected by a

higher number of random ‘hits’ during a screening campaign. Extracts with prominent activity against

Gram-positive cocci and Gram-negative rods should preferentially also be tested against methicillin-

resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE) respectively as

these organisms are medically important with concern to antibiotic resistance. ATCC and MTCC strains

are well characterized and very popular for that purpose, but clinical field isolates may also be used if

fully characterized by antibiogram. Another challenging new area in the microbiological world is

biofilms (Mah and O’Toole, 2001). Although many bacteria grow in a free-living, ‘planktonic’ state, it is

quite common for them to adhere to surfaces by producing extracellular polymeric substances (EPS), e.g.

biofilms. Due to their higher resistance against antimicrobial agents, an interesting option in antibacterial

research is to include a bacterial biofilm model (e.g. Staphylococcus aureus ATCC6538).

Growth medium

Mueller-Hinton (MH) agar or broth and tryptic soy agar or broth (TSA or TSB) are general growth

media for bacteria, while Sabouraud (SAB) agar or broth is used for fungi. Growth of fastidious

micro-organisms, such as Streptococcus pneumonia and Legionella pneumophila, may require more

complex media, enrichment of the incubation atmosphere with 5% CO2 and/or extension of the

incubation time. Slight differences in the composition of the growth medium can greatly affect the

antibacterial activity of a compound. For example, addition of sheep blood to Mueller-Hinton medium

increases the MIC of flavomycin from 0.12 to 256 mg/l (Butaye et al., 2000). Consequently, a definite

choice of growth medium is essential to compare different antibacterial compounds or extracts. Mueller-

Hinton medium allows good growth of most non-fastidious bacteria and is generally low in antagonists.

It also meets the requirements of the NCCLS standard and is recommended as reference medium for agar

and broth-dilution tests (Anon, 2003).

Inoculum

The level of infection, i.e. inoculum concentration can have a profound influence on the antibacterial

and antifungal potency of a sample, endorsing the need for standardization of inoculates (Anon., 2003).

In dilution methods, an inoculum of about 105 CFU/ml is adequate for most bacterial species while for

yeasts and fungi between 103 and 104 CFU/ml is sufficient (Hadacek and Greger, 2000). A too low

inoculum size (e.g. 102 CFU/ml) will create many false-positives, while a too high inoculum size

(e.g. 107 CFU/ml) will hamper endpoint reading and increase the chances for false-negatives. Bacterial or

yeast inoculates can be prepared from overnight cultures or from existing biofreeze stocks. It is


recommended to collect from cultures during the logarithmic growth phase and always to take four or

five colonies of a pure culture on agar to avoid selecting an atypical variant (Anon., 2003).

In Vivo assessment of antibacterial and antifungal activity

The preceding discussion clearly demonstrates the similarity in methods used for in vitro

antibacterial and antifungal assays of plant extracts and there are many papers in the literature using one

of more of the methods. A smaller number of research groups have moved beyond the in vitro

environment and are investigating the in vivo efficacy of those extracts that show promise in the

laboratory. This is a more complex and costly activity as not only does the activity against the

microorganisms need to be evaluated, there must also be consideration of mammalian cell toxicity and

allergic reactions (Matura et al., 2005). To date most in vivo testing of plant extracts has involved the use

of essential oils against human skin infections, particularly fungal infections, and testing of extracts

follow standard clinical trial protocols. Perhaps the plant extract best known for its in vivo antibacterial

activity is honey, with a large number of studies demonstrating in vivo activity (Dunford et al., 2000;

Moore et al., 2001). It is important to note that demonstrated activity in vitro does not always translate to

activity in vivo. The best example of this is tea tree oil, which has been shown to have excellent activity

in vitro against the fungi responsible for various tinea’s (MIC 0.004-0.06%) (Hammer et al., 2002) yet

the results from clinical trials have been far from conclusive (Satchell et al., 2000). This illustrates the

caution with which researchers should view results from in vitro assays and reinforces the need for

clinical trials of plant extracts that show therapeutic promise.

In vivo antibacterial activity: Five groups of Albino mice were used in this experiment. The

selected test organism was cultured overnight at 37 °C on nutrient agar. The organism was harvested and

suspended in sterile saline and each mouse was challenged intraperitoneally with a single 0.5 mL portion

of bacterial suspension containing105 cfu of the desired bacterial sample per ml. This inoculum was

sufficient to cause 100% mortality in untreated mice with death occurring between 6 and 10 h after

challenge. 5.0, 10.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0, 100.0 and 200.0 mg/kg of plant extracts were

administered subcutaneously 15 min after bacterial challenge. The total number of mice surviving at each

dose level was recorded on day 7 after infection. The length of time before the death of each infected

animal was also noted.

Comparison between in vitro and in vivo method

In vitro assays are useful while working with crude extracts. However, with herbal materials several

issues come up. For example, what dose to use, what extract to use and what cut-offs to use? Doing

viability assays (using Trypan Blue exclusion or MTT assays) help in deciding upper cut-offs, however,

it is not fail-safe to lead to potentially active concentrations. Many herbal extracts are not water soluble

and therefore a solvent that is not toxic to cells have to be selected and this has to be used as a control.

Extrapolation from an in vivo dose helps in identifying the possibly effective dose - but with crude

extracts acceptable cut-offs must be predetermined. If the extract is coloured, then interpretation of

results when being estimated colourimetrically has to be done cautiously. Incorporation of appropriate


controls is also essential to confirm robustness of the assay system. If the substance is not soluble and we

use suspensions, this can seriously interfere with assays e.g. when we use herbo-mineral preparations.

Bioassays are expensive and need sophisticated instrumentation and expert personnel. Additionally,

de-differentiation and instability of cell lines adds to some uncertainties. Additional variables that affect

analysis include variability in media composition, temperature, viscosity, osmolarity, and buffering.

Naturally, therefore, in vitro bioassays cannot totally replace in vivo studies. However, the advantages of

in vitro studies are many. Thus, we can study cell interactions, cell-environment interactions,

intracellular activity, cell products, site and mechanism of action and genetic studies with the herbal

medicines are possible. The technology to use stem cells has opened new vistas. Apart from the

convenience, the lack of ethical dilemmas (unlike in human or animal studies), a control of the

experimental environment, the ability to characterize the sample and maintenance of homogeneity in the

procedure are other advantages. Up-scaling and mechanization are also major advantages, allowing

high-throughput screens. It must be emphasized here that in vivo bioassays are also a powerful tool used

in pharmacology for studying the effects of medicines, and are also of some use in herbal drug

development. Dose relationships and mechanistic studies are some of the classical examples. However,

the challenges with in vivo animal studies are also many, including what dose, route and for how long?

Extrapolation of data from animals to humans and vice versa is difficult.

In situ antifungal activity

Electron microscope has played an important role for visual inspection of fungal pathogens. Using

the electron microscopy techniques the effects of potential antifungal extracts from natural sources can

also be evaluated. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM)

allows visualizing morphological changes at the membrane and cell wall ultra structure. Electron

microscopy and atomic force microscopy (AFM) can give us a perspective at a molecular level to

understand each antimicrobial compound mechanism of action. Moreover, other complementary

microscopy techniques can be used to analyze the characteristic properties of each drug. With the

invention of confocal microscopy, an overall picture of the entire cell condition can be possible. Also it is

promising to assess the kinetics of the antimicrobials effect on fungal cultures and monitor cell

agglutination and other population behaviors.

Scanning electron microscopy

After treatment with plant extract, SEM observation will be carried out on fungal pathogens. The

method involves the preparation of a Petridish containing 15–25 ml potato dextrose agar, seeded with

1 ml of the fungal conidial spore suspension at a known concentration (105 spores per ml). 1 ml of the

extract at the concentration of IC50 (obtained from the hyphal growth inhibition test), was dropped onto

the inoculated agar and will be further incubated for another 4-7 days at 28 οC. A vehicle-treated culture

can be used as a control. Using cork borer (6-10 mm) segments will be cut from cultures growing on

potato dextrose plates at different time intervals from intial day to end of experiment for SEM

examination (Sasidharan et al., 2008) (plate-7). The specimen then placed on double-stick adhesive tabs


on a planchette and the planchette placed in a petri plate. In a fume hood, a vial cap containing 2%

osmium tetroxide in water will be placed in an unoccupied quadrant of the plate. After being covered, the

plate will be sealed with parafilm, and vapor fixation of the sample proceeded for 1 h. Once the sample is

vapor fixed, the planchette will be plunged into liquid nitrogen -210 οC and then transferred on to the

“peltier-cooled” stage of the freeze dryer, and freeze drying of the specimen will be kept for 10 h.

Finally, the freeze dried specimen will be sputter coated with 5–10 nm gold before viewing in the SEM.

Conventional SEM microscopy are frequently selected to visualize the ultrastructural damage on both

cell wall and cytoplamatic membrane of entire microbes when fixed material can be used. The SEM is

advantageous over several other microscopy methods as it is three-dimensional and almost the whole of

the specimen is sharply focused. Furthermore, besides having a combination of higher magnification,

larger depth of focus and greater resolution, the preparation of samples is also relatively easier, compared

to the TEM method (Sasidharan et al., 2010).

Transmission electron microscopy (TEM)

After SEM finding further confirmation can be obtained from TEM study. To study the antifungal

activity through TEM method the hyphal specimens (1×3 mm, with approximately 1 mm thickness of

underlying agar blocks) of test fungal strains will be excised from the margin of actively growing PDA

culture treated with plant extract using a sterilized razor blade. The specimens are then fixed with

modified Karnovsky’s fixative, consisting of 2% (v/v) glutaraldehyde and 2% (v/v) paraformaldehyde in

0.05 M sodium cacodylate buffer solution (pH 7.2) at 4 °C for overnight. Subsequently, the fixed

specimens are washed with the buffer solution three times for 10 min each. The specimens were then will

be post-fixed in the osmium tetroxide solution (1% w/v) at 4 °C for 2 h followed by washing with

millipore water twice each. The post fixed specimens will be enbloc stained with 0.5% (w/v) uranyl

acetate at 4 °C overnight and then will be dehydrated once in a graded ethanol series of 30, 50, 70, 80,

and 95% and three times in 100% ethanol for 10 min each. The specimens will be further treated with

propylene oxide twice for 30 min each as a transitional fluid and then will be embedded in Spurr’s resin.

Ultra-thin sections (approximately 50 nm in thickness) will be cut with a diamond/ glass knife using an

ultra-microtome. The sections will be mounted on copper grids and will be stained with 2% uranyl

acetate and Reynolds’ lead citrate (Reynolds, 1963) for 7 min each. After all these treatment finally the

stained sections will be observed with a transmission electron microscope. Ultrathin sections obtained by

conventional procedures, namely fixation with aldehydes, post-fixation with osmium tetraoxide,

dehydratation and embedding in Epoxy resin, allow the observation of membrane and cytoplasmatic

alterations. Treatment with AMPPs can induce several external and internal changes such as membrane

bleb, ruffling or detachment, the presence of electrodense dots or fibers, hypodense cytoplasmic release

and cell vacuolization. Most importantly From the TEM micrograph we can observe the changes caused

by the plant extract on fungal cytoplasm.


Confocal laser scanning microscopy (CLSM)

Although using SEM and TEM a detail ultrastracture can be obtained, it is also necessary to monitor

the overall integrity and viability of the fungal cells after exposure to antimicrobial compounds. Using

confocal microscopy, the kinetics and morphological evolution of microbial cell population can be

monitored at real time and compared with microorganism viability. To study the antifungal activity

through CLSM method the plant extract with MIC concentration will be prepared. Two days old fungal

culture will be developed by culturing the fungal strains on PDA with the treatment of plant extract either

by disc diffusion or agar cup method. Controls without the plant extract are also cultured. The 48 h

fungal culture will be gently transferred into a 12-well microtiter plate and rinsed with PBS for 15 s,

incubated at 28oC for 24 h. The viability of the fungal cells will be assessed by Molecular Probes

LIVE/DEAD BacLight Bacterial viability kit which comprises SYTO-9 and propidium iodide (PI). After

incubation with the dyes, the polymethylmethacrylate discs with biofilms will be placed on glass slides

and live/dead ratio of cells will be quantified using the CSLM system (Thein et al., 2007). The

advantages of confocal microscopy with SEM and TEM, is that it can be performed directly in solution,

thus the sample is not subject to artefacts i.e. sample fixation or drying.

CONCLUSIONS

Herbal medicines make an enormous contribution to primary health care and have shown great

potential in modern phytomedicine against numerous ailments and the complex diseases and ailments of

the modern world. Scientists from divergent fields are investigating plants anew with an eye to their

antimicrobial utility. All over the world thousands of phytochemicals have found which have inhibitory

effects on all types of microorganisms in vitro. There is still a need for more scientific evaluation of

Asian herbal medicines including their active constituents, synergistic interactions, formulation

strategies, herb drug interactions, standardization, pharmacological and clinical evaluation, toxicity,

safety and efficacy evaluation and quality assurance. Furthermore, more of these compounds should be

subjected to animal and human studies to determine their effectiveness in whole organism systems,

including in particular toxicity studies as well as an examination of their effects on beneficial normal

microbiota. It would be advantageous to standardize methods of extraction and in vitro testing so that the

search could be more systematic and interpretation of results would be facilitated. Also, alternative

mechanisms of infection prevention and treatment should be included in initial activity screenings.

Attention to these issues could accompany in a poorly needed new era of chemotherapeutic treatment of

infection by using plant derived principles. This review outline the main methods used in the evaluation

of antimicrobial activity of plant extracts; each method has advantages and limitations and all have been

widely cited in the literature. The question of which is the best one to use is essentially unanswerable as

preferred methods depend on a variety factors including access to specialized equipment and facilities,

the number of samples to be screened and the nature of the plant extract (e.g. volume, extract versus

essential oil, chemical composition). For large-scale screening of extracts for antibacterial and antifungal

activity disk and agar diffusion methods offer a fast, cost effective, low tech, and generally reliable


method of sorting those extracts worthy of further investigation from those unlikely to be of value. Broth

dilution methods provide more information but are more time and labour intensive and are best used as a

follow up to a large scale screening of plant extracts. Antiviral and antiparasitic assays are the most time

and labour intensive of the in vitro antimicrobial testing methods and often require access to cell culture

or other specialized laboratory facilities. These are used less frequently than antibacterial and antifungal

assays. Despite the limitations of many of the assay techniques, there is a vast amount of good data

demonstrating that some plant extracts possess strong to excellent antimicrobial activity. The next step is

to continue this work into the in vivo environment and to evaluate the activity of these extracts in the

treatment of infectious disease.

ACKNOWLEDGEMENTS

I wish to express my profound gratitude to Prof. S. K. Dutta, Dr. A. K. Bastia and Dr. G. Sahoo

(North Orissa University) for their cooperation and critical suggestion on the preparation of the

manuscript. Thanks are also to Laxmipriya Padhi and Susmita Mohapatra (North Orissa University) for

editing this manuscript.

REFERENCES

1. Alzoreky, N. S., & Nakahara, K. (2003). Antibacterial activity of extracts from some edible

plants commonly consumed in Asia. International Journal of Food Microbiology, 80, 223-230.

2. Anon. (2000). Determination of minimum inhibitory concentrations (MICs) of antibacterial

agents by agar dilution. Clinical Microbiology Infection, 6, 509-515.

3. Anon. (2003). Determination of minimum inhibitory concentrations (MICs) of antibacterial

agents by broth dilution. Clinical Microbiology Infection, 9, 1-7.

4. Antonov, A., Stewart, A., & Walter, M. (1997). Inhibition of condium germination and mycelial

growth of Botrytis cinerea by natural products. In: 50th Conference Proceeding of the New

Zealand Plant Protection Society, 159-164.

5. Araujo, C., Sousa, M. J., Ferreira, M. F., & Leao, C. (2003). Activity of essential oils from

Mediterranean Lamiaceae species against food spoilage yeasts. Journal of Food Protection, 66,

625-632.

6. Aureli, P., Costantini, A., & Zolea, S. (1992). Antimicrobial activity of some plant essential oils

against Listeria monocytogenes. Journal of Food Protection, 55, 344-348.

7. Aureli, P., Ferrini, A. M., Mannoni, V., Hodzic, S., Wedell-Weergaard, C., & Oliva, B. (2003).

Susceptibility of Listeria monocytogenes isolated from food in Italy to antibiotics. International

Journal of Food Microbiology, 83, 325-330.

8. Banerjee, A., Kaul, V. K., & Nigam, S. S. (1982). Antimicrobial activity of the essential oil of

Curcuma amada Roxb. Indian Journal of Microbiology, 22, 154-155.

9. Bara, M. T. F., & Vanetti, M.C.D. (1995). Antimicrobial effect of spices on the growth of

Yersinia enterocolitica. Journal of Herbs Spices Medicinal Plants, 3, 51-58.


10. Barry, A. L. (1976). The Antimicrobial Susceptibility Test: Principles and Practices. In Lorian,

V. (ed.). Antibiotics in Laboratory Medicine, 2nd ed. William & Wilkins, Baltimore.

11. Benli, M., Kaya, I., & Yigit, N. (2007). Screening antimicrobial activity of various extracts of

Artemisia dracunculus L. Cell Biochemistry and Function, 25, 681-686.

12. Beuchat, L. (1976). Sensitivity of Vibrio parahaemolyticus to spices and organic acids. Journal

of Food Science, 41, 899-902.

13. Burt, S. A., & Reinders, R. D. (2003). Antibacterial activity of selected plant essential oils

against Escherichia coli O157:H7. Letters in Applied Microbiology, 36, 162-167.

14. Butaye, P., Devriese, L. A., & Haesebrouck, F. (2000). Influence of different medium

components on the in vitro activity of the growth promoting antibiotic flavomycin against

enterococci. Journal of Antimicrobial Chemotherapy, 46, 713-716.

15. Canillac, N. & Mourey, A. (2001). Antibacterial activity of the essential oil of Picea excelsa on

Listeria, Staphylococcus aureus and coliform bacteria. Food Microbiology, 18, 261-268.

16. Carson, C. F., Cookson, B. D., Farrell, F. J. & Rilley, T.V. (1995a). Susceptibility of

methicillin-resistant Staphylococcus aureus to the essential oil of Melaleuca alternifolia.

Journal Antimicrobial Chemotherapy, 35, 421-424.

17. Carson, C. F., Cookson, B. D., Farrell, F. J., & Rilley, T. V. (1995b). Broth microdilution

method for determining the susceptibility of Escherichia coli and Staphylocuccus aureus to the

essential oil of Melaleuca alternifolia (tea tree oil). Microbios, 82: 181-185.

18. Chaibi, A., Ababouch, L. H., Belasri, K., Boucetta, S., & Busta, F. F. (1997). Inhibition of

germination and vegetative growth of Bacillus cereus T and Clostridium botulinum 62A spores

by essential oils. Food Microbiology, 14, 161-174.

19. Cosentino, S., Barra, A., Pisano, B., Cabizza, M., Piris, F. M., & Palmas, F. (2003).

Composition and antimicrobial properties of Sardinian juniperus essential oils against

foodborne pathogens and spoilage microorganisms. Journal of Food Protection, 66 (7), 1288-

1291.

20. Cosentino, S., Tuberoso, C. I. G., Pisano, B., Satta, M., Mascia, V., Arzedi, E., & Palmas, F.

(1999). In vitro antimicrobial activity and chemical composition of Sardinian thymus essential

oils. Letters in Applied Microbiology, 29, 130-135.

21. Davidson, P. M., & Parish, M. E. (1989). Methods for testing the efficacy of food

antimicrobials. Food Technology, 43, 148-155.

22. Davidson, P.M., & Parish, M.E. (1989). Methods for testing the efficacy of food antimicrobials.

Food Technology, 43, 148-155.

23. de Aquino Lemos, A., de Rosario Rodrigues, & Silva, M. (2005). Antifungal activity from

Ocimum gratissimum L. towards Cryptococcus neoformans. Memorias do Inscicuco Oswaldo

Cruz, 100, 55-58.

24. Deans, S. G., & Ritchie, G. (1987). Antibacterial properties of plant essential oils. International



25. Delaquis, P. J., Stanich, K., Girard, B., & Mazza, G. (2002). Antimicrobial activity of individual

and mixed fractions of dill, cilantro, coriander and eucalyptus essential oils. International


26. Diallo, D., Marston, A., Terreaux, C., Toure, Y., Smestad Paulsen, B., & Hostettmann, K.

(2001). Screening of Malian medicinal plants for antifungal, larvicidal, molluscicidal,

antioxidant and radical scavenging activities. Phytotherapy Research, 15, 401-406.

27. Domokos, J., Hethelyi, E., Palinkas, J., Szirmai, S., & Tulok, M. H. (1997). Essential oil of

rosemary (Rosmarinus officinalis L.) of Hungarian origin. Journal of Essential Oil Research, 9,

41-45.

28. Dorman, H. J. D., & Deans, S. G. (2000). Antimicrobial agents from plants: antibacterial

activity of plant volatile oils. Journal of Applied Microbiology, 88, 308-316.

29. Dunford, C., Cooper, R., Molan, P., & White, R. (2000). The use of honey in wound

management. Nurs Stand, 15, 63-68.

30. Elgayyar, M., Draughon, F. A., Golden, D. A., & Mount, J. R. (2001). Antimicrobial activity of

essential oils from plants against selected pathogenic and saprophytic microorganisms. Journal

of Food Protection, 64, 1019-1024.

31. Faleiro, M. L., Miguel, M. G., Ladeiro, F., Venancio, F., Tavares, R., J Brito, C., Figueiredo, A.

C., Barroso, J. G., & Pedro, L. G. (2003). Antimicrobial activity of essential oils isolated from

Portuguese endemic species of Thymus. Letters in Applied Microbiology, 36, 35-40.

32. Farag, R. S., Daw, Z. Y., Hewedi, F. M., & Baroty, G.S.A. (1989). Antimicrobial activity of

some Egyptian spice essential oils. Journal of Food Protection, 52, 665-667.

33. Firouzi, R., Azadbakht, M., & Nabinedjad, A. (1998). Anti-listerial activity of essential oils of

some plants. Journal of Applied Animal Research, 14, 75-80.

34. Garedew, A., Schnmolz, E., & Lamprecht, I. (2004) Microbiological and calorimetric

investigations on the antimicrobial actions of different propolis extracts: an in vitro, approach.

Thermochimica Acta 415, 99-106.

35. Griffin, S. (2000). Aspects of antimicrobial activity of terpenoids and the relationship to their

molecular structure (doctoral thesis). University of Western Sydney, Sydney, Australia.

36. Grover, R. K., & Moore, J. D. (1962). Toximetric studies of fungicides against brown rot

organisms, Sclerotia fructicola and S. laxa. Phytopathology, 52, 876-880.

37. Hamburger, M.O., & Cordell, A. G. (1987). Direct bioautographic TLC assay for compounds

possessing antibacterial activity. Journal of Natural Product, 50, 19-22

38. Hammer K. A., Carson C. F., & Riley T. V. (1999). Influence of organic matter, cations and

surfactants on the antimicrobial activity of Melaleuca alternifolia (tea tree) oil in vitro. Journal

of Applied Microbiology, 86(3), 446-452.


39. Hammer, K. A., Carson, C. F., & Riley, T. V. (2002) In vitro activity of Melaleuca alternifolia

(tea tree) oil against dermatophytes and other filamentous fungi. Journal of Antimicrobial

Chemotherapy, 50, 195-199.

40. Haraguchi, H., Kataoka, S., Okamoto, S., Hanafi, M., & Shibata, K. (1999). Antimicrobial

triterpenes from Ilex integra and the mechanism of antifungal action. Phytotherapy Research,

13, 151-156.

41. Homans, A. L., & Fuchs, A. (1970). Direct bioautographic on thin-layer chromatograms as a

method for detecting fungitoxic substances. Journal of Chromatography, 51, 327.

42. Hood, J. R., Cavanagh, H. M. A., & Wilkinson, J. M. (2004). Effects of essential oil

concentration of the pH of nutrient and Iso-sensitest broth. Phytotherapy Research , 18, 947-

949.

43. Ilcim, A., Digrak, M., & Bagci, E. (1998). The investigation of antimicrobial effect of some

plant extract. Turkish Journal of Biology, 22, 119-125.

44. Inouye, S., Tsuruoka, T., Uchida, K., & Yamaguchi, H. (2001). Effect of sealing and Tween 80

on antifungal susceptibility testing of essential oils. Microbiology & Immunology, 43, 201-208.

45. Iscan, G., Kirimer, N., Kurkcuoglu, M., Husnu Can Baser, K., & Demirci, F. (2002).

Antimicrobial screening of Mentha piperita essential oils. Journal of Agriculture & Food, 50,

3943-3946.

46. Ismaiel, A. A., & Pierson, M. D. (1990). Effect of sodium nitrite and origanum oil on growth

and toxin production of Clostridium botulinum in TYG Chemistry broth and ground pork.

Journal of Food Protection, 67, 958-960.

47. Jee Young, R., You Sun, M., Seung Hee, J., Kyue Yim, L., Su Yun, L., Chang Sub, S. & Won

Bong, P. (2002). Antimicrobial activities of combined extract of Aloe vera with propolis against

oral pathogens. Journal of Korean Society Food Science & Nutrition, 31, 899-904.

48. Jeong Jun, L., Sung Hoon, K., Byung Sik, C., Joong Bok, L., Chul Sung, H., Tae Jong, K., &

Young Jin, B. (1999). The antimicrobial activity of medicinal plants extracts against

Helicobacter pylori. Korean Journal of Food Science & Technology, 31, 764-770.

49. Joyce L. F, Downes J, Stockman K, & Andrew J. H. (1992). Comparison of five methods,

including PDM Epsilometer test (E-test), for antimicrobial susceptibility testing of

Pseudomonas aeruginosa. Journal of Clinical Microbiology, 30(10), 2709-2713.

50. Juven, B. J., Kanner, J., Schved, F., & Weisslowicz, H. (1994). Factors that interact with the

antibacterial action of thyme essential oil and its active constituents. Journal of Applied

Bacteriology, 76, 626-631.

51. Kanemaru, K., & Miyamoto, T. (1990). Inhibitory effects on the growth of several bacteria by

brown mustard and allyl isothiocyanate. Journal of Japan Society of Food Science &


Technology, 37, 823-829.

52. Kim, J. M., Marshall, M. R. Cornell, J. A., Preston III, J. F., & Wei, C. I. (1995b). Antibacterial

activity of carcacrol, citrol and geraniol against Salmonella typhimurium in culture medium and

on fish cubes. Journal of Food Science, 60, 1364-1374.

53. Kim, J. M., Marshall, M. R., & Wei, C. I. (1995a). Antibacterial activity of some essential oil

components against five food borne pathogens. Journal of Agricultural & Food Chemistry, 43,

2836-2845.

54. Kiran, B., Lalitha, V., & Raveesha, K.A. (2010). Screening of seven medicinal plants for

antifungal activity against seed borne fungi of maize seeds. Asian Journal of Basic and Applied

Sciences, 2(3-4), 99-103.

55. Kivanc, M., & Akguel, A. (1986). Antibacterial activities of essential oils from Turkish spices

and citrus. Flavour Fragrance Journal, 5, 179-175.

56. Kokoska, L., & Rada, V. (2001). Antibacterial activity of houttuynin sodium bisulphate (HSB).

Czech Journal of Food Science, 19, 37-40.

57. Krogstad, D. J., & Moellering, R.C. (1986). Combitions of antibiotic, mechanisms of interaction

against bacteria, p. 537. In V. Lorian, ed. Antibiotics in Laboratory Medicine, 2 nd. William &

Wilkins, Baltimore.

58. Lachowicz, K. J., Jones, G. P., Briggs, D. R., Bienvenu, F. E., Wan, J., Wilcock, A., &

Coventry, M. J. (1998). The synergistic preservative effects of the essential oils of sweet basil

(Ocimum basilicum L.) against acid-tolerant food microflora. Letters in Applied Microbiology,

26, 209-214.

59. Lambert, R. J. W., Skandamis, P. N., Coote, P. J., & Nychas, G. J. E. (2001). A study of the

minimum inhibitory concentration and mode of action of oregano essential oil, thymol and

carvacrol. Journal of Applied Microbiology, 91, 453-462.

60. Latté, K. P. & Kolodziej, H. (2000). Antifungal effects of hydrolysable tannins and related

compounds on dermatophyte, mould fungi and yeasts. Zeitschrift für Naturforschung C, 55,

467-472.

61. Lewis, W. H. & Elvin-Lewis, M. P. (1995). Medicinal plants as sources of new therapeutics.

Annals of the Missouri Botanical Garden, 82, 16-24.

62. Lis-Balchin, M., & Deans, S. G. (1997). Bioactivity of selected plant essential oils against

Listeria monocytogenes. Journal of Applied Microbiology, 82, 759-762.

63. Mah, T.F., & O’Toole, G. A. (2001). Mechanisms of biofilm resistance to antimicrobial agents.

Trends in Microbiology, 9, 34-39.

64. Mann, C. M., & Markham, J. L. (1998). A new method for determining the minimum inhibitory

concentration of essential oils. Journal of Applied Microbiology, 84, 538-544.

65. Marino, M., Bersani, C., & Comi, G. (1999). Antimicrobial activity of the essential oils of

Thymus vulgaris L. measured using a bioimpedometric method. Journal of Food Protection, 62,


1017-1023.

66. Marino, M., Bersani, C., & Comi, G. (2001). Impedance measurements to study the

antimicrobial activity of essential oils from Lamiaceae and Compositae. International Journal of

Food Microbiology, 67, 187-195.

67. Martins, A. P., Salgueiro, L. Goncalves, M. J. Proenca da Cunha, A. Vila, R. Canigueral, S.

Mazzoni, V. Tomi, F., & Casanova, J. (2001). Essential oil composition and antimicrobial

activity of three Zingiberaceae from S. tome Principe. Planta Medica., 67, 580-584.

68. Matura, M., Slkold, M., Borje, A., Andersen, K. E., Bruze, M., Frosch, P., Goosssens, A.,

Johansen, J. D., Svedman, C., White, I. R., & Karlberg, A. (2005). Selected oxidized fragrance

terpenes are common contact allergens. Contact Dermatitis, 52, 320-328.

69. Mbata, T. I, Debiao, L., & Saikia, A. (2006). Antibacterial activity of the crude extract of

Chinese Green Tea (Camellia sinensis) on Listeria minocytogenes. Internet Journal of

Microbiology. 2(2).

70. Meena, S., Rafique, M., Chaudhary, F. M., & Bhatty, M. K. (1986). Antimicrobial activity of

the essential oils of the Umbelliferae family. III. Pimpinella anisum, Pimpinella acuminata and

Pimpinella stewartii. Pakistan Journal of Science & Industrial Research, 29, 352-356.

71. Mejlholm, O., & Dalgaard, P. (2002). Antimicrobial effect of essential oils on the seafood

spoilage micro-organism Photobacterium phosphoreum in liquid media and fish products.

Letters in Applied Microbiology, 34, 27-31.

72. Minija, J., & Thoppil, J. E. (2001). Volatile oil constitution and microbicidal activities of

essential oils of Coriandrum sativum L. Journal of Natural Remedies, 1, 147-150.

73. Mishra, M., & Tiwari, S. N, (1992). Toxicity of Polyalthia longifolia against fungal pathogens

of rice. Indian Phytopathlogy, 45, 56-61.

74. Moore, O. A., Smith, L. A., Campbell, F., Seers, K., McQuay, H. J. & Moore, R. A. (2001).

Systematic review of the use of honey as a wound dressing. BMC Complementary Alternative

Medicine,1, 2.

75. Mourey, A., & Canillac, N. (2002). Anti-Listeria monocytogenes activity of essential oils

components of conifers. Food Control, 13, 289-292.

76. Nair, M. G., Safir, G.R., & Siqueira, J.O. (1991). Isolation and identification of vesicular

arbuscular mycorrhiza stimulatory compounds from clover (Tnifolium repens) roots. Applied

Environmental Microbiology, 57, 434-439.

77. Nakamura, C. V., Ueda-Nakamura, T., Bando, E., Melo, A. F. N., Cortez, D. A. G. & Filho, B.

P. D. (1999). Antibacterial activity of Ocimum gratissimum L. essential oil. Memorias do

Inscicuco Oswaldo Cruz, 94, 675-678.

78. Nene, Y. L., & Thapliyal, P. N. (2000). Poisoned food Technique. Fungicides in Plant Disease

Control. 3rd eds., Oxford and IBH publishing company, New Delhi, pp: 531-533.


79. Nguefack, J. V., Leth, P. H., Amvam Z., & Mathur, S. B. (2004). Evaluation of five essential

oils from aromatic plants of Cameroon for controlling food spoilage and mycotoxin producing

fungi. International Journal of Food Microbiology, 94, 329-34.

80. Nostro, A., Germano, M. P., D’Angelo, V.,Marino, A., & Cannatelli, M. A. (2000). Extraction

methods and bioautography for evaluation of medicinal plant antimicrobial activity. Letters in

Applied Microbiology, 30, 379-384.

81. Okeke, M. I., Iroegbu, C. U. Jideofor, C. O. Okoli, A. S., & Esimone, C. O. (2001).

Antimicrobial activity of ethanol extracts of two indigenous Nigerian spices. The Journal of

Herbs Spices & Medicinal Plant, 8, 39-46.

82. Onawunmi, G. O. (1989). Evaluation of the antimicrobial activity of citral. Letter Applied

Microbiology, 9, 105-108.

83. Orhan, G., Baykam, A., Zer, Y., & Balcky, Y. (2005). Synergy tests by E test & checkerboard

methods of antimicrobial combinations against Brucella melitensis, Journal of Clinical


84. Panda, S. K., Thatoi, H. N., Bastia, A. K., & Dutta, S. K. (2009a). In vitro evaluation of anti-

shigella activity of Cassia fistula L. leaf extracts. Plant Science Research, 31, 49-53.

85. Panda, S. K., Behera, B., & Dutta, S. K. (2009b). Anti-candidal activity of Vitex negundo L.:

An ethnomedicinal plant. Journal of Pure and Applied Microbiology, 3(2), 777-784.

86. Panda, S.K., Thatoi., H. N., & Dutta, S. K. (2009c). Antibacterial activity and phytochemical

screening of leaves and bark extracts of Vitex negundo L. Journal of Medicinal Plant Research,

3(4), 294-300.

87. Panda, S. K., Dutta, S. K., & Bastia, A. K. (2010a). Antibacterial activity of croton roxburghii

Balak. against the enteric pathogens. Journal of Advanced Pharmaceutical Technology and

Research, 1(4), 388-391.

88. Panda, S. K., Bastia, A. K., & Dutta, S. K. (2010b). Anticandidal activity of Croton

roxburghii Balak. International Journal of Current Pharmaceutical Research, 2(4), 55-59.

89. Panda, S. K., Bastia, A. K., & Dutta, S. K. (2010c). Anticandidal activity of Eleuthrine bulbosa

(Miller) Urban. Inventi Impact: Ethnopharamacology, 1(1), 44-46.

90. Panda, S. K., Polai, B. L., Bastia, A. K., & Dutta, S. K. (2010d). Anticandidal activity of

Terminalia arjuna Roxb. Bark from Similipal Biosphere Reserve, Orissa, India. Biohelica, 1(1),

45-51.

91. Panda, S. K., Brahma, S., & Dutta, S. K. (2010e). Selective anti-candidal action of crude

extracts of Cassia fistula L.: a preliminary study on Candida and Aspergillus species. Malaysian

Journal of Microbiology, 6(1), 62-68


92. Panda, S. K., Dubey, D., & Dutta, S. K. (2010f). Anticandidal activity of Diospyros

melanoxylon Roxb. bark from Similipal Biosphere Reserve, Orissa, India. International Journal

of Green Pharmacy, 4(2), 102-107.

93. Panda, S. K., Mohanty, G., Padhi, L. P., Sahoo, G., Dutta, S. K. (2011a). Phytochemical

analyses and Antimicrobial activities of different plant parts of Vitex negundo L. Journal of

Pharmaceutical Research. 4(9): 3184-3189.

94. Panda, S. K., Bastia, A. K., & Dutta, S. K. (2011b). Antidiarrheal activity of Terminalia arjuna

Roxb. from India. Journal of Biologically Active Products from Nature, 4, 236-247.

95. Panda, S. K., Padhi, L. P., & Mohanty, G. (2011c). Antibacterial activities and phytochemical

analysis of Cassia fistula (Linn.) leaf. Journal of Advanced Pharmaceutical Technology and

Research, 2(1), 62-67.

96. Padhi, L. P., Panda, S. K., Satapathy, S. N., & Dutta, S. K. (2011d). In vitro evaluation of

antibacterial potential of Annona squamosa L. and Annona reticulata L. from Similipal

Biosphere Reserve, Orissa, India. Journal Agricultural Technology, 7(1), 133-142.

97. Panda, S. K., & Dutta, S. K. (2011e). Antibacterial activity from bark extracts of Pterospermum

acerifolium (L.) Willd. International Journal of Pharmaceutical Science Research, 2(3), 584-

595.

98. Panda, S. K., Patra, N., Sahoo, G., Bastia, A. K., & Dutta, S. K. (2012). Anti-diarrheal activities

of medicinal plants of Similipal Biosphere Reserve, Orissa, India. International Journal of

Medicinal Aromatic Plants, 2(1), 123-134.

99. Periago, P. M., & Moezelaar, R. ( 2001). Combined effect of nisin and carvacrol at different pH

and temperature levels on the viability of different strains of Bacillus cereus. International

Journal of Food Microbiology, 68 (1-2), 141-148.

100. Pintore, G., Usai, M., Bradesi, P., Juliano, C., Boatto, G., Tomi, F., Chessa, M., Cerri, R., &

Casanova, J. (2002). Chemical composition and antimicrobial activity of Rosmarinus officinalis

L. oils from Sardinia and Corsica. Flavour and Fragrance Journal, 17, 15-19.

101. Pitner, J. B., Timmins, M. R., Kashdan, M., Nagar, M. Stitt, D. T.

(2000). High-throughput assay system for the discovery of antibacterial drugs. In AAPS

Proceedings, DB Biosciences.

102. Pol, I. E., & Smid, E. J. (1999). Combined action of nisin and

carvacrol on Bacillus cereus and Listeria monocytogenes. Letters in Applied Microbiology, 29,

166-170.

103. Prudent, D., Perineau, F., Bessiere, J. M., Michel, G. M., & Baccou, J. C. (1995). Analysis of

the essential oil of wild oregano from Martinique (Coleus aromaticus Benth.): Evaluation of its

bacteriostatic and fungistatic properties. Journal of Essential Oil Research, 7: 165-173.


104. Qamar, S., Rashid Khalid, M., & Chaudhry, F. M. (1994). Antimicrobial activity of Ageratum

conyzoides and Cymbopogon jawarancusa against yeast and mould. Science International, 6,

333-335.

105. Rahalison, L., Hamburger, M., Hostettmann, K., Monod M., & Frenk, E. (1991). A

bioautographic agar overlay method for the detection of antifungal compounds from higher

plants, Phytochem. Anal., 2, 199-203.

106. Rajashekhara, E., Tippannavar, C. M., Sreenivasa, M. N., &

Sharma, J. S. (1990). Inhibitory activity of papain on facultative pathogens. Zentralblatt fuer

Mikrobiologie, 145, 455-456.

107. Rasooli, I., Moosavi, M. L., Rezaee, M. B., & Jaimand, K. (2002). Susceptibility of

microorganisms to Myrtus communis L. essential oil and its chemical composition. Journal of

Agricultural Science & Technology, 4, 127-133.

108. Rauha, J. P., Remes, S. Heinonen, M. Hopia, A. Kahkonen, M. Kujala, T. Pihlaja, K. Vuorela,

H., & Vuorela, P. (2000). Antimicrobial effects of Finnish plant extracts containing flavonoids

and other phenolic compounds. International Journal of Food Microbiology, 56, 3-12.

109. Safak, S., Yuksekdag, Z. N., Aslim, B., & Beyatli, Y. (2003). Study on antimicrobial activities

of yeasts isolated from kombucha tea. Gida. 28 (1), 105-108.

110. Sasidharan, S., Yoga Latha, L., & Angeline, T. (2010). Imaging In vitro Anti-biofilm Activity to

Visualize the Ultrastructural Changes, In: Microscopy: Science, Technology, Applications and

Education A. Méndez-Vilas & J. Díaz, (Eds.), 622-626, Formatex, Spain.

111. Sasidharan, S., Zuraini, Z., Yoga Latha, L., & Suryani, S. (2008). Fungicidal effect and oral

acute toxicity of Psophocarpus tetragonolobus root extract. Pharmaceutical Biology, 46, 261-

265.

112. Sekiyama, Y., Mizukami, Y., Takada, A., Oosono, M., & Nishimura, T. (1996). Effect of

mustard extract vapor in fungi and spore-forming bacteria. Abstract. Journal of Antibacterial

and Antifungal Agents, 24, 171-178.

113. Shahi, S. K., Shukla, A. C., & Dikshit, A. (1999). Antifungal studies of some essential oils at

various ph levels for betterment of antifungal drug response Current Science, 77, 703-706.

114. Shelef, L. A., Jyothi, E. K., & Bulgarelli, M. A. (1984). Growth of

enteropathogenic and spoilage bacteria in sage-containing broth and foods. Journal of Food

Science, 49, 737-809.

115. Sivropoulou, A., Kokkini, S., Lanaras, T., & Arsenakis, M. (1995).

Antimicrobial activity of mint essential oils. Journal of Agricultural & Food Chemistry, 43,

2384-2388.

116. Sivropoulou, A., Papanikolaou, E., Nikolaou, C., Kokkini, S.,

Lanaras, T., & Arsenakis, M. (1996). Antimicrobial and cytotoxic activities of Origanum

essential oils. Journal of Agricultural & Food Chemistry, 44, 1202-1205.


117. Skandamis, P., Koutsoumanis, K., Fasseas, K., & Nychas, G. J. E. (2001). Inhibition of oregana

essential oil and EDTA on E.coli O157:H7. Italian Journal of Food Science, 13, 55-65.

118. Smith-Palmer, A., Stewart, J., & Fyfe, L. (1998). Antimicrobial

properties of plant essential oils and essences against five important food-borne pathogens.

Letters in Applied Microbiology, 26, 118-122.

119. Squires, E., & Cleeland, R. (1985). Methods of Testing Combinations of Antimicrobial Agents,

p. 153.

120. Sridhar, S., R.R, Rajagopal, R.V., Rajavel, R., Masilamani, S., & Narasimhan, S. Antifungal

activity of some essential oils. Journal of Agricultural & Food Chemistry, 2003, 51, 7596-7599.

121. Stecchini, M.L., Sarais, I., & Giavedoni, P. (1993). Effect of

essential oils on Aeromonas hydrophila in a culture medium and in cooked pork. Journal of

Food Protection, 56, 406-409.

122. Struelens, M. J., Nonhoff, C., van der, A. P., Mertens, R., & Serruys E. (1995). Evaluation of

Rapid ATB Staph for 5-hour antimicrobial susceptibility testing of Staphylococcus aureus.

Groupement pour le Depistage, L’Etude et la Prevention des Infections Hospitalieres-Groep ter

Opsporing, Studie en Preventie van Infecties in de Ziekenhuizen. Journal of Clinical


123. Sun Kyung, C., Jae Doo, J., & Sung Hwan, C. (1999). Antimicrobial activities of chopi

(Zanthoxylum piperitum DC) extract. The Korean Society of Food Science & Nutrition, 28,

371-377.

124. Sung Hwan, C., & Young Rok, K. (2001). Antimicrobial effects of Scutellariae radix extract

against Listeria monocytogenes. Journal of Korean Society of Food Science & Nutrition, 30

(5), 959-963.

125. Syed, M., Hanif, M., Chaudhary, F. M. & Bhatty, M. K. (1986b).

Antimicrobial activity of the essential oils of the Umbelliferae family. I. Cuminum cyminum,

Coriandrum sativum, Foeniculum vulgare and Bunium persicum oils. . Pakistan Journal Science

Industrial Research,29, 183-188.

126. Syed, M., Sabir, A. W., Chaudhary, F. M., & Bhatty, M. K.

(1986a). Antimicrobial activity of the essential oils of Umbelliferae family. II. Trachyspermum

ammi, Daucus carota, Anethum graveolens and Apium graveolens oils. Pakistan Journal

Science Industrial Research, 29, 189-192.

127. Tassou, C. C., Drosinos, E. H., & Nychas, G. J. E. (1995). Effects

of essential oil from mint (Mentha piperita) on Salmonella enteritidis and Listeria

monocytogenes in model food systems at 4° and 10°C. Journal of Applied Bacteriology, 78,

593-600.

128. Tepe, B., Donmez, E., Unlu, M., Candan, F., Daferera, D., Vardar Unlu, G., Polissiou, M., &


Sokmen, A. (2004). Antimicrobial and antioxidative activities of the essential oils and methanol

extracts of Salvia cryptantha (Montbret et Aucher ex Benth.) and Salvia multicaulis (Vahl).

Food Chemistry, 84 519-525.

129. Thein, Z. M., Samaranayake, Y. H., & Samaranayake, L. P. (2007). Dietary sugars, serum and

the biocide chlorhexidine digluconate modify the population and structural dynamics of mixed

Candida albicans and Escherichia coli biofilms. Acta Pathologica, Microbiologica et

Immunologica Scandinavica, 115, 1241-1251.

130. Thiem, B., & Goslinska, O. (2004). Antimicrobial activity of Rubus chamaemorus leaves.

Fitoterapia, 75, 93-95.

131. Unal, R., Fleming, H. P. McFeeters, R. F. Thompson, R. L. Breidt, F. Jr., & Giesbrecht, F. G.

(2001). Novel quantitative assays for estimating the antimicrobial activity of fresh garlic juice.

Journal of Food Protection, 64, 189-194.

132. Velickovic, D. T., Ristic, M. S., Randjelovic, N. V., & Smelcerovic, A. A. (2002). Chemical

composition and antimicrobial characteristic of the essential oils obtained from the flower, leaf

and stem of Salvia officinalis L. originating from southeast Serbia. Journal of Essential Oil

Research, 14, 453-458.

133. Venturini, M. E., Blanco, D., & Oria, R. (2002). In vitro antifungal activity of several

antimicrobial compounds against Penicillium expansum. Journal of Food Protection, 65, 834-

839.

134. Vilijoen, A., van Vuuren, S., Ernst, E., Klepser, M., Demirci, B., Baser, H., & van Wyk, B. E.

(2003). Osmitopsis asteriscoides (Asteraceae) the antimicrobial activity and essential oil

composition of a Cape-Dutch remedy. Journal of Ethnopharmacology, 88, 137-143.

135. Wan, J., Wilcock, A., & Coventry, M. J. (1998). The effect of essential oils of basil on the

growth of Aeromonas hydrophila and Pseudomonas fluorescens. Journal of Applied


136. Ward, S. M., Delaquis, P. J., Holley, R. A., & Mazza, G. (1998). Inhibition of spoilage and

pathogenic bacteria on agar and pre-cooked roast beef by volatile horseradish distillates. Food

Research Interanatinal, 31, 19-26.

137. Wei, L., Asada, Y., & Yoshikawa, T. (1998). Antimicrobial flavonoids from Glycyrrhiza glabra

hairy root cultures. Planta Medica, 64, 746-747.

138. Wilkinson, J. M., Cavanagh, H. M. A. (2005). Antibacterial activity of essential oils from

Australian native plants. Phytotherapy Research, 19, 643-646.

139. Yildirim, A., Mavi, A. Oktay, M. Kara, A. A. Algur, O. F., & Bilaloglu, V. (2000). Comparison

of antioxidant and antimicrobial activities of tilia (Tilia argentea Desf Ex DC), sage (Salvia

triloba L.) and black tea (Camellia sinensis L.) extracts. Journal of Agricultural & Food

Chemistry, 48, 5030-5034.

140. Yong Seon, A., Dong Hwa, S., & Nam In, B. (2000). Isolation and identification of

antimicrobial substance against Listeria monocytogenes from Ruta graveolens Linn. Korean


Journal of Agricultural Science & Technology, 32, 1379-1388.

141. Yu, J., Lei, J., Yu, H., Cai, X., & Zou, G. (2004). Chemical composition and antimicrobial

activity of the essential oil of Scutellaria barbata. Phytochemistry, 65, 881-884.

Table 1 : Terms used in antimicrobial activity testing

Term, definition, with

reference to

concentration of crude

extracts

Medicinal plants extract References

Minimum inhibitory

concentration (MIC)

Lowest concentration resulting in

maintenance or reduction of inoculums

viability

Lowest concentration required for complete

inhibition of test organism up to 48 hours

incubation

Lowest concentration inhibiting visible

growth of test organisms

Lowest concentration resulting in

significant decrease in inoculums viability

(>90%)

Carson et al., 1995a

Wan et al., 1998;

Canillac and Mourey,

2001

Barry, 1976; Hammer

et al., 1999a; Delaquis

et al., 2002

Cosentino et al., 1999

Minimum bactericidal

Concentration (MBC)

Concentration where 99% or more of the

initial inoculums are killed

Lowest concentration at which no growth is

observed after sub-culturing into fresh broth

Carson et al., 1995b;

Cosentino et al., 1999;

Canillac and Mourey,

2001; Barry, 1976;

Onawunmi, 1989

Bacteriostatic

concentration

Lowest concentration at which bacterial fail

to grow in broth, but are grow when broth is

plated onto agar in the absence of the

inhibitor

Smith-Palmer et al.,

1998

Bactericidial

concentration

Lowest concentration at which bacteria fail

to grow in broth and fail to grow when

broth is plated onto agar in the absence of

the inhibitor

Smith-Palmer et al.,

1998


Table 2 : Comparison of strengths and limitations of various assays for antimicrobial activity

Method Strength Limitation

Disk well diffusion

• Low cost

• Results available in within 1–2 days.

• Does not require specialized laboratory facilities.

• Uses equipment and reagents readily available in a microbiology laboratory.

• Can be performed by most laboratory staff.

• Data is only collected at one or two time points.

• Large numbers of samples can be screened.

• Results are quantifiable and can be compared statistically.

• Differential diffusion of extract components due to partitioning in the aqueous media.

• Inoculums size, presence of solubilizing agents, and incubation temperature can affect zone of inhibition.

• Volatile compounds can affect bacterial and fungal growth in closed environments.

Agar dilution • Low cost

• Does not require specialized laboratory facilities.

• Uses equipment and reagents readily available in a microbiology laboratory.

• Can be performed by most laboratory staff.

• Hydrophobic extracts may separate out from the agar.

• Inoculum size, presence of solubilizing agents and incubation temperature can affect zone of inhibition.

• Volatile compounds can affect bacterial and fungal growth in closed environments.

• Data is only collected at one or two time points.

• Use of scoring systems is open to subjectivity of the observer.

• Some fungi are very slow growing.

Broth dilution • Allows monitoring of activity over the duration.

• More accurate representation of antibacterial activity.

• Micro-broth methods can be used to screen large numbers of samples in a cost-effective manner.

• Essential oils may not remain in solution for the duration of the assay, emulsifier and solvent may interfere with accuracy of results.

• Labor and time intensive if serial dilution are used to determine cell count

• Highly colored extracts can interfere with colorimetric endpoints in micro broth methods.

TLC bioautograpy

• Simultaneously fractionation

and determination of bioactivity.

• Unsuitable where activity is due to component synergy

• Dependent on the extraction methods and TLC solvent used.


Plate 1: Photograph of disc diffusion method

a. Photograph with different zone of inhibition

b. Zone of inhibition of essential oil against Shigella species

c. Zone of inhibition of ethanol extract (3), positive control antibiotic-ciprofloxacin (1),

negative control-ethanol (2) against Pseudomonas aeruginosa

Plate 2: Photograph of agar diffusion method of various plants extract collected from Similipal

biosphere Reserve, Odisha, India (Source-Panda et al., 2012)

a. Zone of inhibition against V. cholerae

b. Zone of inhibition against S. typhi


Plate 3: Photograph of broth dilution method (A-G: decreasing concentration of extract,

H-control)


Plate 4 : Photograph of broth dilution method i. model of 96 well microtiter plate;

ii. MIC result of plant extract by microdilution me thod using TTC (A-G: decreasing

concentration of extract, H-control)


Plate 5: Graph showing time kill kinetics and combination studies

a. Isobolograms displaying the three types of results possible with combination of

antimicrobials

b. Type of growth and inhibition curves possible when a microorganism is Exposed to an

plant extract/antimicrobials over time (Source-Davidson and Parish, 1989)

c. Time kill kinetics against S. epidermidis (Panda et al., 2011)


Plate 6 : Photograph of TLC and bioautography (Source-Panda et al., 2011)


Plate 7 : Scanning electron microscope images of P. aeruginosa cells after treatment with

methanol extract (suspended with 5ml distilled water) of A. dracunculus. (a) The region

of inhibition zone (arrows) and shrinking and degradation of the cells. (b) The damaged

cells of P. aeruginosa (arrows) in inhibition zone (Source-Benli et al., 2007)

Documents

SCREENING METHODS IN THE STUDY OF ...3 Screening Methods in the Study of Antimicrobial Properties of Medicinal Plants deposited into well cut into the agar and can be used with large