Table of Contents

Page Number
Acknowledgements
Abstracts 1
1. Introduction
1.1. The Honey 2
1.2. Antibacterial Effects of Honey 3-4
1.3. Methods Used in the Investigation 4-5
1.4. The Bacteria Species 5
1.5 The Aim 5
2. Materials and Methods
2.1. Media 6
2.2. Sources of Isolates 6
2.3. Culture Incubation and Storage Conditions 6
2.4. Detection of Antibacterial Activity 7-8
2.5. Detection of The Minimum Inhibitory Concentration 8-9
3. Results
3.1. Analysis of Results 10
3.2. Disc Diffusion Assay 11-15
3.3. Dose Response Curves 16-20
4. Discussion 21-24
5. Further Discussion and Conclusion 24-25
Appendix 26-30
References 31-33

ABSTRACT
The antibacterial effects and the minimum inhibitory concentration (MIC) of honey, were tested
using five honeys which were Manuka Honey, Turkish Clover Honey, Forest Honey, Royale Honey
and Organic Flower Honey. These were selected to examine and compare their ability to inhibit the
growth of the four bacteria species. Escherichia coli, Bacillus subtilis, seudomonas flourescens and
Staphylococcus epidermidis which were isolated from pure cultures. This study was conducted using
the disc diffusion assay method and showed that these honeys exhibit a level of antibacterial activity.
The most inhibition obtained was with the manuka honey against Escherichia coli and
Staphylococcus epidermidis with a peak in inhibition against Staphylococcus epidermidis at 30%
concentration. The most inhibition was also obtained by the Royale honey against Pseudomonas
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flourescens and by the organic flower honey against Bacillus subtilis. The Turkish Clover honey
exhibited an inhibitory peak against Staphylococcus epidermidis at 40% and against Pseudomonas
flourescens at 30%. The Minimum Inhibitory Concentration was found to lie between 10 and 20%
concentration for most of the honeys depending on the organism. A change of the dominant
antibacterial mechanism may have taken place at the concentrations of honey between 20 and 40%
and this needs to be investigated further.
The antibacterial activity of the Organic Flower and Turkish Clover honeys at low concentration is
dominated by hydrogen peroxide activity. But the antibacterial activity of the Forest honey but is
thought to be dominated by the lower pH and lower water activity in pure honey.
1.INTRODUCTION

1.1. The Honey

Honey is a sweet, viscous fluid produced by bees from the collection of nectar, primarily
from flowers. It is considered to be a natural syrup. The Nectar is gathered by the bees and is slowly
transformed into honey, through a long process involving the addition of enzymes and the gradual
reduction of moisture. Honey is a rich source of carbohydrates mainly Fructose and Glucose. The
chemical composition of honey varies depending on the plant source, season and production
methods. Therefore the Colour, Concentration and Compounds vary depending on the floral sources.
Other compounds which can be found in Honey include Proteins and acids such as Gluconic Acid
(C6H11O7, also known as 2,3,4,5,6- pentahydroxyhexanoic Acid), Minerals and Anti-Oxidants such
as Hydrogen Peroxide (H202) and Vitamins (B6 and B12), (BD. Yates et. al. 1996). Honey has a
low pH and a low moisture content, which is usually on average about 17 percent. The Gluconic
Acid in honey is produced when bees secrete Glucose Oxidase, while processing the nectar, this give
honey a low pH. There are many varieties of honey from around the world which come in three main
types which are liquid, whipped and comb honey (Yates B.D. et.at. 1996; Office Complementary
Medicine, 1998). In this study five honey’s were chosen which are Manuka Honey, Turkish Clover
Honey, Flower Honey, Royale Honey and Organic Forest Honey. Each of these honey’s were
chosen because they are organic and readily available in health food stores.

1.2. The Antibacterial Effects of Honey

1.2.1. The Five Honeys

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 Honey has been shown to have antibacterial properties, in particular Manuka honey. Manuka
Honey has had extensive research done on it. It has been shown in many studies that Manuka Honey
has antimicrobial effects (Barret J., et. al, 2005; Coumbes A. L., et. al. 2004; Mundo, MA. 2004). In
this study the antibacterial activity of the five honeys were compared, the Manuka honey was used
as a positive control. This is because it is known to have antibacterial properties. This would be
compared with the other honeys to determine if the other honeys have antibacterial activity and how
much of an effect they have, also which bacteria they effect.

1.2.2. The Mechanism of Antibacterial Properties

Honey has many natural properties which enable it to inhibit bacteria. These properties
include, a low pH which is in the range of pH 3.2 to 4.5, approximately 3.9 which is due to its
content of acidic compounds mainly Gluconic acid as stated above (Shin H. et. al. 2005, Office
Complementary Medicine, 1998). A low pH is inhibitory to most bacteria. Since most bacteria live
in environments around pH 7, the pH of honey could inhibit the bacteria (Barrett, J et. al. al 2005).
This is because pH affects the way large proteins such as enzymes work. Which causes the shape of
enzymes to change, which then alters the overall charge, this causes the protein to denature. Honey
contains small amounts of Hydrogen peroxide (H202) this varies depending on the honey, it is
produced as a result of the enzyme glucose oxidase activity in producing Gluconic acid Mundo,
MA. et. al. 2004). Hydrogen peroxide is a powerful oxidising agent (Free Radical) which has the
ability to damage cells. In an aqueous solution hydrogen peroxide acts like an acid and can oxidise a
variety of compounds, by accepting free unpaired electrons. This allows the formation of other free
radicals, which then causes a cascade effect. Therefore altering biological structures and therefore
damaging cells. The honey has high levels of carbohydrates in the form of monosaccharides, which
gives honey a low water activity (aw) which is in the range of 0.562 to 0.62. Water activity means
the amount of water which is available in an environment is low in comparison with the level of
solutes. The low water activity of the honey inhibits bacteria through the effect of osmosis. The high
sugar levels causes the water inside of a cell to diffuse out through the cell membrane and wall, by
creating a concentration gradient, So water travels from a region of high water concentration to a
region of low concentration. This causes the cell to dehydrate through losing water and shrinking
will occur. The process by which the cell is dehydrated through osmosis is called plasmolysis, the
result is that the cell becomes plasmolyzed. This could cause the cells to die, because water is

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needed in many reactions since most reactions occur in solution and if there is no water available
then these reaction would stop.

1.3. Method Used in the Investigation

There are three main ways to determine the antibacterial effects of honey. These three
methods which can be used to measure the amount of antibacterial activity are the Disc Diffusion
Assay, Well Diffusion Assay and the Spectrometric Assay. These methods can also be used to
determine the Minimum Inhibitory Concentration, the most widely used methods are the Disc
Diffusion Assay and the Well Diffusion Assay. The main problem with these two methods is that
they can be unreliable, this would be mainly because these methods rely on visual measurement and
determination. So therefore measurements can be more subjective than objective, this in itself can
account for some of the human errors made with these types of methods (Barret, J. et. al. 2005). For
this investigation the method which was employed in the experiments was the Disc Diffusion Assay
method, it was chosen because it was the easiest and the simplest method to use.

1.4 The Bacteria Species

Four different species of bacteria will be used in this study to explore the effectiveness of
each honey on the inhibition of growth; the bacteria I have chosen for this study are both Gram-
Positive and Gram- Negative Bacteria, Aerobic and all four bacteria Genera have significance with
interaction with humans (Homo sapiens) The four bacterial species, which would be used in this
study are: Escherichia coli, Bacillus subtilis, Pseudomonas flourescens and Staphylococcus
epidermidis. These microbes are widely distributed in nature. Escherichia coli are facultative
anaerobic Gram-negative, rod shaped bacteria (Garrity G M 2001), which normally inhabit the
Gastro-Intestinal tracts of Human and most Animals. Staphylococcus epidermidis (also known as
Staphylococcus albus) are Gram-positive, spherical shaped bacteria (Garrity G M 2001), which are
part of the microbial flora of the nose and skin of most humans. Bacillus subtilis, are Gram-positive,
facultative aerobic, endospore forming, rod-shaped, bacteria (Garrity G M 2001), usually found in
soil or extreme environments. Pseudomonas flourescens are Gram-negative, non-spore forming,

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straight rod shaped bacteria (Garrity G M 2001), which are abundant in nature, normally living in
soil, water and marine environment.

2. THE AIM
The purpose of this study is to determine if honey is an antibacterial agent, which inhibits the
growth of microorganisms if so, then what would be the Minimum Inhibitory Concentration. It is
also to determine which honey has the most amount of Inhibition.

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2. Material and Methods
2.1. Media
In this investigation Nutrient agar (Oxoid) and Nutrient Broth (Oxoid) were used to culture
four different bacteria species. The nutrient agar was used to isolate colonies and to observe the zone
of inhibition around sterile absorbent discs. The nutrient broth was used in making liquid cultures
from isolated colonies from the agar plates. The liquid cultures were then used in the disc diffusion
assay, the maximum recovery diluent (Oxoid) was used to dilute the honeys to make up the serial
dilutions.
2.2. Sources of Isolates
Pure cultures of Escherichia coli, Bacillus subtilis, Pseudomonas flourescens and
Staphylococcus epidermidis were used to separately inoculate four different nutrient agar plates
using an inculum loop to streak the bacteria onto each plate.
2.3. Culture Incubation and Storage Conditions
All the organisms used in the investigation were of level 1 classification, the inoculated
culture plates were incubated at the temperatures which are stated in table 2.1, for up to 48 hours.

Table 2.1. Incubation Temperatures

Organism Incubation Temperature (˚C)

Escherichia coli 37
Bacillus subtilis 37
Staphylococcus epidermidis 37
Pseudomonas flourescens 25

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*These bacteria cultures were then stored at 4˚C.
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2.4. Detection of Antibacterial Activity
2.4.1. Culture Preparation
Four Universal bottles containing 9ml each of nutrient broth were inoculated separately with
Escherichia coli, Bacillus subtilis, Pseudomonas flourescens and Staphylococcus epidermidis using
an inoculum loop. The nutrient broth solutions which were inoculated with Escherichia coli, Bacillus
subtillis and Staphylococcus epidermidis were then incubated at 37˚C for up to 48 hours. The
nutrient broth solution which was inoculated with Pseudomonas flourescens was incubated at 25˚C
for up to 48 hours.
2.4.2. Honey Preparation
10 ml of Royale Honey, Manuka Honey Forest Honey, Organic Flower Honey and the
Turkish Clover Honey were each poured into five separate flasks and were covered with foil. These
were stored at room temperature out of direct sunlight.
2.4.3. Disc Diffusion Assay
Five sets of four Nutrient Agar plates were set out, each agar plate in every setwas inoculated
separately with the bacteria Escherichia coli, Bacillus subtilis, Pseudomonas flourescens and
Staphylococcus epidermidis, by pipetting 100Cl of each bacteria directly onto the agar surface of
each plate of every set. Using the spread plate technique, the bacteria samples were then spread
across the surface using a glass spreader. The plates were left to dry for 15 minutes, whilst sterile
absorbent discs were placed into each honey flask. The absorbent discs were left in the honey for 10
minutes to absorb the honey. An absorbent disc from each honey was placed on every agar plate in
each set.
The plates which were inoculated with Escherichia coli, Bacillus subtilis and Staphylococcus
epidermidis with then incubated at 37˚C for up to 48 hours. The plates which were inoculated with
Pseudomonas flourescens was then incubated at 25˚C for up to 48 hours.

2.4.4. Detection of Antibacterial Activity

After the plates had been incubated the inhibition of the bacteria was determined by the
visual
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 confirmation of a zone of inhibition. A zone of inhibition is a clear area surrounding the
absorbent disc.

2.5. Detection of The Minimum Inhibitory Concentration

2.5.1. Honey Preparation

Each of the five honeys was diluted using maximum recovery diluent (MRD), in which six
dilutions were prepared. The concentration of each dilution was measured using weight in grams of
honey against the volume in cm3 of MRD, grams/volume (g/vol.). Using universal bottles, the honey
concentrations wereprepared using the following measurements of honey and MRD as seen in table
2.2.
Table 2.2: Honey Dilutions
Percentage (%) Weight in grams, of Volume in cm3 of
Concentration: honey Maximum Recovery Diluent
0 0 10
10 1 9
20 2 8
30 3 7
40 4 6
50 5 5

Each honey dilution was kept at room temperature out of direct sunlight.

2.5.2. Disc Diffusion Assay

In this method, for each honeys, four sets of six nutrient agar plates were set out, each set
was theninoculated with one species of bacteria. In each set of nutrient agar plates, each agar plate
was inoculated with bacteria by pipetting 100Cl of nutrient broth bacterial culture, directly onto the
agar surface. Using the spread plate technique, the bacteria samples were then spread across the
surface of the agar using a glass spreader. The plates were left to dry for 15 minutes, whilst sterile
absorbent discs were placed into each honey concentration in of the five honeys. The absorbent discs
were left in the honey dilutions for 10 minutes to absorb the honey. An absorbent disc from each

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honey dilution series was placed directly onto the surface of every agar plate in each set, this was
done for each honey.The plates which were inoculated with Escherichia coli, Bacillus subtilis and
Staphylococcus epidermidis were then incubated at 37˚C for up to 48 hours. The plates which were
inoculated with Pseudomonas flourescens was then incubated at 25˚C for up to 48 hours. The 0
percent honey dilution for each honey, which contained only MRD as stated in table 2.1 is a negative
control for inhibition. The amount of inhibition was recorded by measuring the diameter of the zone
of inhibition, in millimetres (mm), this was measured using a ruler. The measurement included the
diameter of the absorbant disc.

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