ib ee on antimicrobial inhibition of manuka honey on the growth of e coli and staph a

International Baccalaureate Diploma Program

Sri KDU Smart School

Extended Essay

-Biology-

In vitro study of the effect of sunlight exposure on the bacteriostasis

of Active Manuka Honey against Staphylococcus aureus

3914 words

By

Ng Siang Hang

002206-029 cqn461

Ng Siang Hang 002206-029 cqn461 Biology Extended Essay

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Abstract

This extended essay is an in vitro study of the effects of sunlight exposure on the bacteriostasis

(inhibition of bacteria growth) of Active Manuka honey against Staphylococcus aureus.

Active Manuka honey was exposed to sunlight for 8 hours. Staphylococcus aureus was inoculated and

grown in honey samples (nutrient broths solution containing unexposed or sunlight-exposed Active

Manuka Honey solutions at various concentrations) for 24 hours. First, the standard plate count

quantifying method was adopted. 10.0 µl of each sample was diluted and inoculated onto nutrient agar

plates. The Staphylococcus aureus concentration of each sample was to be determined by counting the

colony-forming units (CFU). However, the results were inconclusive due to contamination.

Therefore the spectrophotometry quantifying method was adopted. A standard curve for

Staphylococcus aureus was plotted. Yet, optical densities at 600 nm wavelength (OD600) of

Staphylococcus aureus in the honey samples could not be obtained using the spectrophotometer. The

Staphylococcus aureus concentration could not be quantitatively measured. Nonetheless, OD600 minus

OD600 of nutrient broth (OD600-NB) of the samples was obtained and compared qualitatively. It was

observed that from honey concentration 100 % – 25 %, OD600-NB of all sunlight-exposed honey

samples were greater than unexposed honey samples. The difference was supported to be significant

by the Wilcoxon signed-rank test at 2.5 % significance level. Since the OD600 of unexposed and

sunlight-exposed Active Manuka honey should be similar at every concentration, the difference in

OD600-NB between the samples must be due to difference in Staphylococcus aureus growth. Therefore,

based on Beer-Lambert’s law, higher OD600-NB indicated that sunlight-exposed honey samples had

more Staphylococcus aureus. Sunlight-exposed Active Manuka honey showed lower inhibition effect

towards Staphylococcus aureus.

The conclusion was that sunlight exposure decreases the bacteriostasis of Active Manuka honey

against Staphylococcus aureus.

(288 words)


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Contents

i. Cover Page………………………………………………………………………………. 1

ii. Abstract………………………………………………………………………………… 2

iii. Contents………………………………………………………………………..………. 3

1. Introduction…………………………………………………………………............….. 5

1.1 Rationale of Study………………………........................................………….. 5

1.2 Active Manuka Honey………………………………………………... ………. 6

1.3 Staphylococcus aureus………………………………………………………… 6

2. Materials and Methods………………………………………………………..……….. 7

2.1 Apparatus list…………………………………………………….. ………. 7

2.2 Materials and Chemicals list……………………………………………… 7

2.3 Sterilization……………………………………………………….………. 8

2.4 Incubator…………………………………………………………. ………. 9

2.5 Bacterial Culture…………………………………………………. ………. 9

2.6 Preparation of McFarland 0.5 Standard Solution………………........…… 9

2.7 Preparation of Nutrient agar plates………………………………. ………. 10

2.8 Preparation of Nutrient broth solution…………………………… ………. 10

2.9 a Plating/Inoculation………………………………………………..………. 10

2.9 b Preparation of Unexposed and Sunlight-exposed

Active Manuka Honey at different concentrations………………. ………. 11

2.9.1 Sunlight-exposed Active Manuka Honey………………….. ………. 11

2.9.2 Dilution of Manuka honey…………………………………. ………. 11

3. Method of Testing……………………………………………………………………… 12

3.1 Preparing the honey samples……………………………………………... 12

3.2 Method of Quantification: Standard Plate Count Method……….. ………. 14

3.2.1 Procedure………………………………………..………………….. 15

4. Improvised Method of Testing………………………………………..……………….. 16

4.1 Improvised Method of Quantification: Spectrophotometry……………… 16

4.1.1 Procedure…………………………………………............... ………. 17

I) Plotting a Standard Curve for Staphylococcus aureus…………………. 17

II) Determining the number of Staphylococcus aureus

in the honey samples…………………………………….………. 17


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5. Data Collection and Processing………………………………………………..……… 18

5.1 Observation of Unexposed and

Sunlight-exposed Active Manuka Honey………………………………… 18

5.2 Results of Standard Plate Count Method……………………………................ 19

5.2.1 Dilution plates of

Unexposed Active Manuka honey samples……………...………. 19

5.2.2 Dilution plates of

Sunlight-exposed Active Manuka honey samples………. ………. 20

5.2.3 Summarized observations of the dilution plates

of Unexposed and Sunlight-exposed

Active Manuka honey samples…………………………..………. 22

5.3 Results from Spectrophotometry………………………………………...……. 23

5.3.1 Standard curve…………………………………………….......……. 23

5.3.2 Optical densities of honey samples………………………....………. 26

6. Data Analysis…………………………………………………………………………… 28

6.1 Observation of Unexposed and Sunlight-exposed

Active Manuka honey…………………………………………………….. 28

6.2 Results from Standard Plate Count Method………………….……………….. 28

6.3 Results from Spectrophotometry……………………………………………… 28

6.4 Statistical analysis: Wilcoxon Signed-Rank Test………………………..……. 30

6.4.1 Workings…………………………………………………....………. 30

7. Conclusion……………………………………………………………………................ 33

8. Evaluation……………………………………………………………………… ………. 34

8.1 Limitation of Wilcoxon Signed-Rank Test & Improvement.………....……… 34

8.2 Limitation of Spectrophotometry & Improvements…………………………... 34

8.3 Limitation of Standard Plate Count Method & Improvements………..………. 35

9. References……………………………………………………………….……………… 37

10. Appendix…………………………………………………………………........………. 38

10.1 Wilcoxon Test Critical Values table………………………………… ………. 38


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1. Introduction

1.1 Rationale of Study

This extended essay is an in vitro study of the effect of sunlight exposure on the bacteriostasis of

Active Manuka honey against Staphylococcus aureus.

I was interested to study the antibacterial activity of honey because my family consumes honey every

day, from sweetener in fruit juices to baking. Active Manuka honey was chosen because its

antibacterial property is one of the greatest. Antibacterial property is divided into killing and

bacteriostasis. Bacteriostasis is the inhibition of bacterial growth [1]. This study was narrowed down

to Active Manuka Honey bacteriostasis because since ancient times, the native Maoris in New

Zealand have been using Manuka honey as traditional wound dressing to prevent infection [2]. This

fact had also prompted me to study particularly the bacterium – Staphylococcus aureus because it is

the most common cause for wound infections [3]. Staphylococcus aureus is also very sensitive

towards Active Manuka honey [4]. Therefore, the bacteriostasis of Manuka honey will be easier to

detect.

The variable of sunlight exposure was studied because Active Manuka Honey has black tinted

container. Other honeys have light transparent containers (See figure 1.1.1). The opaque container

may be to protect Active Manuka honey from sunlight as its antibacterial property can be induced by

enzymes. Some enzymes are sensitive to heat and light. Heat and ultraviolent rays from the sun can

denature the enzymes in Manuka honey, thus affecting its bacteriostasis.

I hope my study can discover the best storage condition like types of containers, either translucent or

opaque, for Active Manuka Honey as to preserve its antibacterial property.

Figure 1.1.1 (from left to right): Propolis, Commercial honey, Active Manuka

honey


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1.2 Active Manuka Honey

The antibacterial property of Active Manuka honey is due to 2 factors: hydrogen peroxide, H2O2 (a

proven antiseptic) and UMF [5]. When bees produce honey, they insert an enzyme called glucose

oxidase [6], which catalyses the oxidation of glucose to produce hydrogen peroxide:

Glucose + H2O + O2 Gluconic Acid + H2O2 [6]

UMF is the abbreviation for Unique Manuka Factor. The mechanism of the UMF-induced

antibacterial property is still unknown. Nevertheless, it is known that UMF and hydrogen peroxide

has a synergistic effect which enhances the antibacterial property of Active Manuka honey. Therefore

Manuka honey is now used for medicinal purposes, like treating ulcers and wounds. It has been

proven to inhibit the growth of pathogenic bacteria like Helicobacter pylori (stomach ulcer) and

Methilicin-resistant Staphylococcus aureus (MRSA) [4].

1.3 Staphylococcus aureus

Staphylococcus aureus is a Gram positive bacterium. These bacteria live in the nose or on the skin of

a living person [3]. It is spherical and can be seen as grape-like clusters under the microscope.

Glucose oxidase

Figure 1.2.1 [3] : Staphylococcus aureus under microscope


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2. Materials and Methods

2.1 Apparatus list

• Pressure cooker • Visible spectrophotometer (400 -700 nm) • Electronic weigh balance (± 0.001 g) • Sterile Petri dishes (Diameter: 90 mm) • 1.00 ml Pipette • Micropipette (55 – 60 µl) • Sterile pipette tips • 100 W Light bulbs • 10.0 cm3

calibrated measuring cylinders • Beakers • Glass bottles • Pill box • Sterile culture bottles • Sterile cuvette • Sterile metal stirrer • Sterile cotton swaps • Aluminium foil • Marker pen • Black sugar paper • Fibreglass cloth • Plastic board • Wooden block • Metal cupboard • Bunsen burner • Tripods

2.2 Materials and Chemicals list

• Staphylococcus aureus (Strain: ATCC 25923) • Raw Unblended Active Manuka honey (UMF 10; Brand: Caremark SDN BHD) • Mueller Hilton nutrient broth powder • Mueller Hilton nutrient agar powder • Sterile distilled water • McFarland 0.5 standard solution


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2.3 Sterilization

All solutions and instruments were sterilized using the heat sterilization method. I improvised by

using a pressure cooker as an autoclave. Liquids were poured into glass bottles. The bottle caps were

not tightly screwed to avoid pressure accumulation within the bottles. Meanwhile, apparatuses like

cotton swaps were put into a dry beaker. The beaker top was enclosed with aluminium foil. Next, the

beaker was put into a larger beaker with glass weights (to stabilise the beaker). Water was added into

the pressure cooker. The water level was below the containers to avoid tumbling. The pressure valve

was closed. Lastly, the containers were put into the pressure cooker and let cook for 15 minutes. Once

completed, the pressure valve was opened to release steam. Then, the containers were taken out for

cooling.

Figure 2.3.1: Condition of the pressure cooker before heat sterilizing


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2.4 Incubator A metal cupboard was improvised as an incubator (see figure 2.4.1). A space was left as the

incubation site with two light bulbs, one at each side. The temperature inside was approximately

37.0 °C. During incubation, the glass door was shut. Heat shields were set up around the incubation

site to avoid direct overheating. The heat shield was built by gluing a wooden block to a plastic board.

The unglued part of the board was wrapped with black sugar paper or fibreglass cloth (See figure

2.4.2).

2.5 Bacterial Culture Staphylococcus aureus (strain ATCC 25923) was used and prepared by the lab teacher. 2.6 Preparation of McFarland 0.5 standard solution [7] 0.05 ml of 1.0 % barium chloride solution (BaCl) and 9.95 ml of 1.0 % sulphuric acid solution (HCl)

were mixed in a glass bottle.

Black sugar paper / fibreglass cloth

Wooden block

Plastic board

Figure 2.4.1: Incubator Figure 2.4.2: Heat shield


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2.7 Preparation of Nutrient agar plates 9.500 g of Mueller Hilton nutrient agar powder was dissolved in approximately 250.0 cm3 of distilled

water in a beaker. The solution was heated and constantly stirred until all nutrient agars had

completely dissolved. Then, it was heat sterilized.

After that, approximately 10.0 cm3 of the nutrient agar solution were poured into each Petri dish and

let to cool until hardened. Once hardened, the lids were covered. This process was repeated until there

were sufficient plates for the experiment.

2.8 Preparation of Nutrient broth solution 5.250 g of Mueller Hilton nutrient broth powder was dissolved in approximately 250.0 cm3 of distilled

water. The solution was heated and stirred until all nutrient broth had dissolved. Then, it was heat

sterilized.

2.9 a Plating/Inoculation [8] This is an aseptic transfer of Staphylococcus aureus from a liquid medium to nutrient agar plates. For

this experiment, 10 µl of nutrient broth or honey samples containing Staphylococcus aureus was first

micropipette onto the agar plate. I improvised by adding 30 µl of sterile distilled water to the plate to

ease inoculation. A sterile cotton swap was used to disperse the bacteria solution evenly by streaking

the swap over the entire plate surface at approximately 45° from the plate. Finally, the rim of the plate

was swapped.

Figure 2.7.1: Cooling the nutrient agar plates


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2.9 b Preparation of Unexposed and Sunlight-exposed Active Manuka Honey at different concentrations

2.9.1 Sunlight-exposed Active Manuka Honey

20.000 g of Manuka honey was weighed into a pill box. It was put under direct sunlight for 8

hours, from 8.00 a.m. until 6.00 p.m.

2.9.2 Dilution of Manuka honey

100%

The mass/volume (m/v) percentage concentration was used because an accurate volume of

honey was difficult to obtain using pipette. Honey is very viscous.

For 100 % honey solution, 10.000 g of unexposed Manuka Honey was dissolved in 10.00 ml

of distilled water. Then, a dilution and serial dilution were conducted according to table

2.9.2.1. This procedure was repeated using sunlight-exposed Active Manuka Honey.

Table 2.9.2.1: Dilution table of Manuka honey

Honey Concentration

/ %

Volume of unexposed/sunlight-

exposed Active Manuka honey solution used

/ ml (± 0.01 ml)

Volume of sterile distilled water used

/ ml (± 0.01 ml)

Total volume / ml (± 0.02 ml)

75 2.25 (100%) 0.75 3.00

50 3.00 (100%) 3.00 6.00

25 3.00 (50%) 3.00 6.00

12.5 3.00 (25%) 3.00 6.00

6.25 3.00 (12.5%) 3.00 6.00

(Concentration of used Active Manuka honey)


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3. Method of Testing

3.1 Preparing the honey samples

Staphylococcus aureus was cultured in nutrient broth solution for 24 hours by the lab teacher. The

turbidity of the Staphylococcus aureus solution was visually compared with a McFarland 0.5 standard

solution 1 against a white board with lines. The bacteria solution was diluted until the turbidity was the

same as the standard solution to avoid too many bacteria per µl.

Honey samples = Nutrient broth + Staphylococcus aureus + unexposed / sunlight-exposed Active Manuka honey at different concentrations

Sterile culture bottles were arranged and labelled. 4.00 ml of nutrient broth solution were pipette into

each culture bottle. 1.00 ml of unexposed and sunlight-exposed Manuka honey solutions at different

concentrations were pipette into the culture bottles according to Table 3.1.2 and Table 3.1.3 (page 13).

Then, 10.0 µl (0.01 ml) of Staphylococcus aureus solution was added into the culture bottles. The

bottles were shaken well.

Lastly, the samples were incubated for 24 hours.

1 It represents cell density of 1 10 CFU/ml [7] , CFU = colony-forming unit

Figure 3.1.1: Comparing turbidity


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The tables show the component of the honey samples:

Table 3.1.2: Component of Unexposed Active Manuka honey samples

Unexposed Active Manuka honey samples

Component

Volume of Unexposed

Active Manuka honey solution

/ ml

Volume of Mueller Hilton nutrient broth

solution

/ ml

Volume of Staphylococcus aureus solution

/ ml

Total volume

/ ml

MH100% 1.00 (100%) 4.00 0.01 5.01

MH75% 1.00 (75%) 4.00 0.01 5.01

MH50% 1.00 (50%) 4.00 0.01 5.01

MH25% 1.00 (25%) 4.00 0.01 5.01

MH12.5% 1.00 (12.5%) 4.00 0.01 5.01

MH6.25% 1.00 (6.25%) 4.00 0.01 5.01

(Concentration of added Unexposed Active Manuka honey)

Table 3.1.3: Component of Sunlight-exposed Active Manuka honey samples

Sunlight-exposed Active Manuka honey samples

Component

Volume of Sunlight-exposed Active Manuka honey solution

/ ml

Volume of Mueller Hilton nutrient broth

solution

/ ml

Volume of Staphylococcus aureus solution

/ ml

Total volume

/ ml

E100% 1.00 (100%) 4.00 0.01 5.01

E75% 1.00 (75%) 4.00 0.01 5.01

E50% 1.00 (50%) 4.00 0.01 5.01

E25% 1.00 (25%) 4.00 0.01 5.01

E12.5% 1.00 (12.5%) 4.00 0.01 5.01

E6.25% 1.00 (6.25%) 4.00 0.01 5.01

(Concentration of added Sunlight-exposed Active Manuka honey)


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3.2 Method of Quantification: Standard Plate Count Method [9]

This method measures the bacteriostasis of Active Manuka honey by counting the viable colonies

(colony-forming unit, CFU) formed on the agar plates. The CFU has to be distinguishable and

countable, within the range from 30 – 300. The Staphylococcus aureus concentration in each honey

sample was obtained by multiplying the total CFU per ml with dilution factor.

If Staphylococcus aureus concentrations in sunlight-exposed samples are greater than unexposed

samples, the bacteriostasis of sunlight-exposed Active Manuka honey has decreased. Because more

bacteria growth shows that sunlight-exposed Manuka honey can inhibit less bacteria.


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3.2.1 Procedure

Table 3.2.1.2: Dilution table of honey samples

Dilution Volume of honey sample used

/ ml

Volume of sterile distilled water

/ ml

Total volume

/ ml

10-2 0.01 9.99 10.00

10-4 0.01 (10-2) 9.99 10.00

10-5 0.01 (10-4) 0.99 1.00

(Dilution of honey sample added)

The incubated honey samples obtained from section 3.1 were diluted to 10-4 and 10-5 according to

diagram 3.2.1.1 and table 3.2.1.2. For 10-4 dilution, the samples were first diluted to 10-2.

Then, 10 µl (0.01 ml) of 10-4 and 10-5 dilutions of each sample were inoculated onto nutrient agar

plates. Each dilution was plated in two replicates. The plates were incubated for 24 hours.

Only 10-4 and 10-5 dilutions were plated because it was assumed that there would be rapid growth in

the samples after 24 hours of incubation. The colonies formed at these high dilutions would be less

and countable.

0.01 ml of honey sample

9.99 ml of sterile distilled

water

0.01 ml of 10-2 diluted sample

0.01 ml of 10-4 diluted sample

0.99 ml of sterile distilled

water

Honey sample 10-2 dilution 10-4 dilution 10-5 dilution

Diagram 3.2.1.1: Procedure of diluting the honey samples


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4. Improvised Method of Testing

Method in section 3.1 was repeated.

4.1 Improvised method of quantification: Spectrophotometry [9]

This method is based on Beer-Lambert’s Law - there is a direct relationship between absorbance and

concentration of an absorbing species [10]. Absorbance is the light intensity loss rate. Optical density

(ODλ) is absorbance at certain wavelength [11].

Staphylococcus aureus absorbs light. When light is shown towards a medium with Staphylococcus

aureus, there will be loss of light intensity. Spectrophotometer measures the light intensity loss and

translates it into ODλ. ODλ reflects the concentration of bacteria in the medium.

Light

Staphylococcus aureus

Initial light intensity > End light intensity

Figure 4.1.a: Illustration of the theory behind spectrophotometry


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4.1.1 Procedure

I) Plotting a Standard Curve for Staphylococcus aureus

Firstly, the optical density at 600 nm (OD600) of the Staphylococcus aureus solution cultured by

teacher was obtained. The bacteria solution was diluted to 10-3, 10-4 and 10-5, and inoculated onto agar

plates. The plates were incubated for 24 hours and then observed.

The number of CFU formed on the 10-5 dilution plate was recorded. The total Staphylococcus aureus

concentration in the bacteria solution was determined as below:

Total concentration Total CFU per ml Dilution factor

Next, the bacteria solution was diluted to 1:2, 1:4, 1:8 and 1:16. OD600 at each dilution was obtained.

Staphylococcus aureus concentrations in the dilutions were calculated as follows:

concentration Total concentration

dilution factor

A standard curve was plotted, with Staphylococcus aureus concentration as the y-axis and OD600 as

the x-axis.

II) Determining the Staphylococcus aureus concentration in the samples [12]

OD600 of Staphylococcus aureus in the samples were obtained by “blanking” OD600 of nutrient broth

and Manuka honey. This was done by first preparing blank buffers. Blank buffers are solutions

identical to the honey samples (in terms of volume of nutrient broth, volume and concentration of

Manuka honey) except that the blanks do not contain Staphylococcus aureus.

OD600 of blank buffers were first measured and zeroed. Then, OD600 of the honey samples were taken.

The OD600 obtained would be the OD600 of Staphylococcus aureus. Staphylococcus aureus

concentrations of the samples were determined by referring the OD600 of Staphylococcus aureus to

the standard curve.


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5. Data Collection and Processing

5.1 Observations of Unexposed and Sunlight-exposed Active Manuka honey

Observations

1. Sunlight-exposed honey was less viscous than unexposed honey.

2. Many air bubbles were trapped in the sunlight-exposed honey.

3. Sunlight-exposed honey was darker and more translucent.

Figure 5.1.1: Unexposed Active Manuka honey (left) and Sunlight-exposed Active Manuka honey (right)

2


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5.2 Results from Standard Plate Count Method 5.2.1 Dilution plates of Unexposed Active Manuka honey samples

Figure 5.2.1.1: MH100% Dilution Plates Figure 5.2.1.2: MH75% Dilution Plates

Figure 5.2.1.3: MH50% Dilution Plates Figure 5.2.1.4: MH25% Dilution Plates

* **

~ ~~

* **

~ ~~

* **

~ ~~

* **

~ ~~

*10-4 dilution plate, replicate 1 **10-4 dilution plate, replicate 2 ~10-5 dilution plate, replicate 1 ~~10-5 dilution plate, replicate 2


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5.2.2 Dilution plates of Sunlight-exposed Active Manuka honey samples

Figure 5.2.1.5: MH12.5% Dilution Plates Figure 5.2.1.6: MH6.25% Dilution Plates

Figure 5.2.2.1: E100% Dilution Plates Figure 5.2.2.2: E75% Dilution Plates

* **

~ ~~

* **

~ ~~

* **

~ ~~

* **

~ ~~




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Figure 5.2.2.3: E50% Dilution Plates Figure 5.2.2.4: E25% Dilution Plates

Figure 5.2.2.5: E12.5% Dilution Plates Figure 5.2.2.6: E6.25% Dilution Plates

* **

~ ~~

* **

~ ~~

* **

~ ~~

* **

~ ~~



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5.2.3 Summarized observations of the dilution plates of Unexposed and Sunlight-exposed honey samples

1. Most plates had no bacterial growth.

2. Despite high dilution, some plates were covered with thick layer of bacterial growth.

(Example: Figure 5.2.1.1)

3. Two different colonies were found on the plates.

- Some were grape-like shaped. (Example: Figure 5.2.1.6)

- Some were slime-pool shaped. (Example: Figure 5.2.2.2)


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5.3 Results from Spectrophotometry

5.3.1 Standard curve

Table 5.3.1.1: Dilution plates of Staphylococcus aureus solution

Dilution of Staphylococcus aureus solution

Dilution Plate Number of CFU

(per 10 µl of dilution plated)

10-2

-

10-3

-

10-4

-

-Too many CFU to count


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10-5

76

Calculations

Table 5.3.1.2: The optical densities and bacterial concentration of Standard Curve

Dilution of

Staphylococcus aureus solution

Optical density at 600 nm wavelength,

OD600

Calculations,

Total . dilution factor

Staphylococcus aureus

concentration / CFU/ml

Undiluted 0.283 - 7.600

1:2 0.132 7.6 102

3.800

1:4 0.068 7.6 104

1.900

1:8 0.006 7.6 108

0.950

1:16 -0.040* 7.6 1016

0.475

Number of CFU at 10-5 dilution plate = 76 colonies (per 0.01 ml) Staphylococcus aureus concentration at 10-5 dilution = 7.6 10 CFU/ml Total Staphylococcus aureus concentration = Staphylococcus aureus concentration at 10-5 dilution dilution factor = 7.6 10 10 = 7.6 10 CFU/ml

*Results for 1:16 dilution were not plotted in the standard curve because ODλ cannot be negative.


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0

1

2

3

4

5

6

7

8

9

0 0.05 0.1 0.15 0.2 0.25 0.3

Con

cent

ratio

n o

f Sta

phyl

ococ

cus a

ureu

s (10

8C

FU/m

l)

Optical Density at 600 nm wavelength, OD600

Graph 5.3.1.3: Standard curve for Staphylococcus aureus (Optical Density at 600 nm wavelength vs. Staphylococcus aureus

concentration)


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5.3.2 Optical densities of honey samples

Table 5.3.2.1: OD600-NB (OD600 minus OD600 of nutrient broth) of Unexposed Active Manuka honey samples

Unexposed Active Manuka

honey samples OD600 minus OD600 of

nutrient broth, OD600-NB

MH100% 0.104

MH75% 0.061

MH50% 0.058

MH25% 0.029

MH12.5% -0.005*

MH6.25% 0.070

*This OD600-NB reading was rejected because ODλ cannot be negative.

Table 5.3.2.2: OD600-NB of Sunlight-exposed Active Manuka honey samples


OD600 minus OD600 of nutrient broth,

OD600-NB

E100% 0.151

E75% 0.123

E50% 0.074

E25% 0.059

E12.5% 0.024

E6.25% 0.015*

*This OD600-NB was rejected due to inaccuracy. (Refer section 6.3, page 29)


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MH100%

MH75%MH50%

MH25%

E100%

E75%

E50%

E25%

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

100.00% 75.00% 50.00% 25.00%

OD

600

min

us O

D60

0of

nut

rien

t bro

th,

OD

600-

NB

Concentration of Unexposed/Sunlight-exposed Active Manuka Honey in the honey samples (%)

Bar chart 5.3.2.3: Comparison of OD600-NB between Unexposed and Sunlight-exposed Active Manuka honey samples

Unexposed Active Manuka honey samples


Comparison for honey concentration at 12.5 % and 6.25 % were omitted because OD600-NB for MH12.5% and E6.25% were invalid.


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6. Data Analysis 6.1 Observations of Unexposed and Sunlight-exposed Active Manuka honey

The decreased viscosity of sunlight-exposed Manuka honey might be because sunlight had activated

glucose oxidase, which produced hydrogen peroxide. Hydrogen peroxide then decomposed to water

and oxygen. The water diluted the honey and oxygen was trapped as air bubbles.

Meanwhile, the colour change of sunlight-exposed honey might be due to release of chemicals like

pigment by ultraviolet ray from sunlight, changing the chemical component in honey.

6.2 Results from Standard Plate Count Method

Based on the agar plates, bacteriostasis of Manuka honey was unable to be assessed.

One reason was because some plates had no growth. No colonies could be counted. This might be due

to slow growth or mistakes in transferring or diluting the honey samples. However, the factor of slow

growth was weak to explain the absence of growth because some plates were covered with bacteria

(Refer figure 5.2.1.1, page 19). Another reason was because the existence of slime-pool-like colonies

on some plates indicated there was another bacterium - contamination. Hence, the grape-like colonies

in some plates might not be Staphylococcus aureus.

6.3 Results from Spectrophotometry

Due to limitation of the spectrophotometer, the optical densities of Manuka honey in the honey

samples were unable to be ‘blanked’. The sole OD600 of Staphylococcus aureus could not be

determined; therefore the standard curve could not be used to determine the Staphylococcus aureus

concentration in the samples.

Nevertheless, I improvised by obtaining the OD600-NB (OD600 minus OD600 of nutrient broth) of both

honey sample and assessed the results qualitatively. This was done by ‘blanking’ the nutrient broth –

using blank buffers without honey and bacteria.

Firstly, result for MH6.25% (0.070) was anomalous. Its OD600-NB was higher than unexposed honey

samples at higher honey concentrations (> {MH75%, MH50%, MH25%}). Optical density of low

concentrated honey should be lower than high concentrated honey because of the difference in honey

density. This could be an error of the spectrophotometer after being switched on for too long [12].

Secondly, result for MH12.5% (-0.005) was erroneous because optical density cannot be negative.

Thirdly, OD600-NB of E6.25% was inaccurate because the spectrophotometer is not reliable in giving


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absorbance reading below 0.020 [13]. Therefore, OD600-NB of MH12.5%, MH6.25% and E6.25% were

omitted from analysis and comparison.

Based on bar chart 5.3.2.3 (page 27), it was observed that OD600-NB of both unexposed and sunlight-

exposed Active Manuka honey samples decreased as honey concentration decreased. This was

because honey solutions are dense at high concentrations and less dense at low concentrations.

Bar chart 5.3.2.3 also showed that overall from honey concentration 100 % – 25 %, OD600-NB of all

sunlight-exposed honey samples were higher than unexposed honey samples. At any given

concentrations, both unexposed and sunlight-exposed Active Manuka honey solution should have

similar optical densities. In fact, sunlight-exposed Active Manuka honey should have a lesser optical

density as it appeared less viscous (more diluted) as observed in section 5.1. Since ODλ of both

sunlight-exposed and unexposed Active Manuka honey should be similar, the difference in OD600-NB

between the two honey samples must be accounted by the difference in Staphylococcus aureus growth

in the samples. Referring back to Beer Lambert’s law, greater OD600-NB indicated there was greater

Staphylococcus aureus growth in sunlight-exposed honey samples. Sunlight-exposed Active Manuka

honey showed decrease inhibition towards Staphylococcus aureus.

The decrease in bacteriostasis of Active Manuka honey after sunlight exposure might be due to the

denaturation of enzymes which release active ingredients responsible for its antibacterial property.

Ultraviolet ray from sunlight can denature the enzymes because enzymes are sensitive to heat and

light. Inhibition of honey could also happen due to osmotic effect – when high concentrated honey

leaves little water available for bacteria to grow [6]. Another possible explanation for the decreased

bacteriostasis was that sunlight had decreased the viscosity of Manuka honey, thus its osmotic effect.

Before it could be concluded that sunlight exposure decreases the bacteriostasis of Active Manuka

honey, strong evidence was needed to support that the difference in OD600-NB between the samples

was significant because it could have happened due to random chance. Therefore, a statistic test was

conducted.


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6.4 Statistical analysis: Wilcoxon Signed-Rank Test [14] (One-tailed test at 2.5% significance level)

The Wilcoxon Signed-Rank test was used because the data were matched and could not be assumed to

be normally distributed due to small sample sizes. This test compares the medians of the OD600-NB of

both honey samples.

Although OD600-NB of MH12.5%, MH6.25% and E6.25% were inaccurate and unreliable, I included

the data into the test to increase test power and mainly because the minimum sample size required for

the test is 6. Some adjustments were made. I adjusted the OD600-NB of MH12.5% (-0.005) and MH6.25%

(0.070) to 0 to compensate the invalidity of the results most likely caused by error from

spectrophotometer. OD600-NB of E6.25% (0.015) was accepted as it was because the test emphasizes on

the qualitative difference, not quantitative.

6.4.1 Workings

First, two hypothesises were constructed:

• Null hypothesis, H0:

Median of OD600-NB of sunlight-exposed samples = median of OD600-NB of unexposed samples

• Alternative hypothesis, H1:

Median of OD600-NB of sunlight-exposed samples > median of OD600-NB of unexposed samples


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Next, the difference and signed ranks were calculated:

Table 6.4.1.1: Difference in OD600-NB between sunlight-exposed and unexposed Active Manuka honey

samples

*Adjusted values

Table 6.4.1.2: Signed rank of the differences

Difference, d Sorted differences Signed Rank, rd

+ 0.047 + 0.015 + 1

+ 0.062 + 0.016 + 2

+ 0.016 + 0.024 + 3

+ 0.030 + 0.030 + 4

+ 0.024 + 0.047 + 5

+ 0.015 + 0.062 + 6

Concentration of unexposed/sunlight-

exposed Manuka honey in the samples (%)

xa: OD600-NB of Unexposed Active

Manuka honey samples

xb: OD600-NB of Sunlight-exposed Active Manuka honey samples

Difference, d

(d = xb-xa)

100 0.104 0.151 + 0.047

75 0.061 0.123 + 0.062

50 0.058 0.074 + 0.016

25 0.029 0.059 + 0.030

12.5 0.000* 0.024 + 0.024

6.25 0.000* 0.015 + 0.015


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Let

P – Sum of positive ranks, ∑

Q – Sum of negative ranks, ∑

T – Smallest of P and Q

Therefore,

1 2 3 4 5 6 21

0

0

At 2.5 % significance level and sample size – N = 6, critical value of T is 0 (Refer Appendix 10.1).

As T was equal to critical value, the null hypothesis was rejected and the alternative hypothesis was

accepted [14]. Median of OD600-NB of sunlight-exposed honey samples was greater than the median of

OD600-NB of unexposed honey samples. This supported that the difference in OD600-NB between the

samples was significant.


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7. Conclusion

This extended essay investigated (in vitro) the effect of sunlight exposure towards the bacteriostasis of

Active Manuka Honey against Staphylococcus aureus.

From the improvised method, results showed that overall from honey concentrations 100% to 25%,

OD600-NB of all sunlight-exposed Active Manuka honey samples were greater than unexposed honey

samples. Wilcoxon signed-rank test supported that the difference in OD600-NB was significant. Based

on the argument in section 6.3, the conclusion was that sunlight exposure decreases the

bacteriostasis of Active Manuka honey against Staphylococcus aureus. However, this conclusion

was inconclusive due to many weaknesses in methodology (See section 8).

Some new questions arose from this investigation, like whether the decrease in bacteriostasis of

Active Manuka honey will stay true for low concentrations (< 25%). Also, the exact reason for the

decreased bacteriostasis was unknown. Possible explanations were that enzymes in Manuka honey

were denatured by sunlight or osmotic effect of honey had decreased.

Sunlight-exposed Manuka honey changed colour and was less viscous. Maybe the colour change was

due to change in honey chemical compound. The decreased viscosity might be because glucose

oxidase was activated by sunlight. These unresolved questions can be answered with an investigation

into the change in chemical component of Manuka honey after sunlight exposure.

Another interesting question was that whether the decreased bacteriostasis was only against

Staphylococcus aureus or also other bacteria strains. This was asked because gram negative bacteria

have different cell walls than gram positive bacteria, and therefore will have different susceptibility

towards Manuka honey. Perhaps the experiment can be conducted on a gram negative bacterium.

More can be revealed about the antibacterial mechanism of Manuka honey.


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8. Evaluation

8.1 Limitation of Wilcoxon Signed Rank Test & Improvement Despite low confidence level (2.5 %), the Wilcoxon test was unreliable for determining the

significance of the difference in OD600-NB. One reason was because the sample size was very small.

Another reason was because results were adjusted to compensate the error of spectrophotometer.

What if the data were accurate? Then the adjustment was inappropriate and had distorted the data. The

difference in OD600-NB of the samples might be insignificant after all.

One way to improve is to use a larger sample size.

8.2 Limitation of Spectrophotometry & Improvements

The spectrophotometer was unable to “blank” the OD600 of Manuka honey in the samples because

honey is very dense. OD600 of bacteria and its concentration could not be determined. This

investigation could not quantitatively study the extent of the effect of sunlight on the Active Manuka

Honey bacteriostasis.

Plus, the improvised measurements (OD600-NB readings) were highly inaccurate and unreliable due to

limitation of spectrophotometer. The spectrophotometer would give inaccurate ODλ readings after it

was switched on for a long time [12]. Low ODλ readings (> 0.020) were also unreliable [13]. Another

cause for the results inaccuracy was that the spectrophotometer was not calibrated. The OD600-NB

results were also unreliable because it was taken only once and at one wavelength. Many random

errors were possibly involved.

To improve, the spectrophotometer should be switched off from time to time. It should be calibrated

to increase the accuracy. To reduce the random errors, ODλ readings should be taken several times

and at several wavelengths to obtain the mean.

Meanwhile, the evaluation of bacteriostasis of Active Manuka honey based on the difference in OD600-

NB might be invalid because at high concentrations, there might be deviation from Beer Lambert’s law

[10]. The OD600-NB of high concentrated honey samples might not reflect the Staphylococcus aureus

concentration.

Even if ODλ does reflect the bacteria concentration, ODλ shows the concentration of both living and

killed bacteria. So, the spectrophotometry method can only show the general antibacterial activity, but

not the specific bacteriostasis of Manuka honey. Also, ODλ does not indicate that the bacteria were


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Staphylococcus aureus. The samples could be contaminated with other bacteria. This can be improved

by examining the samples under microscope.

The spectrophotometry method introduced many uncertainties and inaccuracies to this investigation.

Hence, the conclusion was inconclusive. If this investigation is to be reproduced, the Standard Plate

Count method will be more appropriate. It can accurately reflect the Manuka honey bacteriostasis

because colonies can only be formed by living bacteria. It does not show dead bacteria. It also allows

confirmation of the bacteria strain by examining the colonies under microscope. However, certain

limitations of the Standard Count method should be first evaluated and overcome.

8.3 Limitation of Standard Plate Count Method & Improvements

One major limitation was contamination. There were several possible factors. Firstly, some culture

bottles were not completely clean (See figure 8.3.1). The stains could be living bacteria from previous

experiments as the bottles had been used many times. Another factor was leakage in the pressure

cooker (See figure 8.3.2). Steams escaped although the pressure valve was closed. This leakage might

have caused the pressure cooker to fail to create a high temperature and pressure condition. Bacteria

could not be completely killed. Therefore, possible sources of contamination could come from the

distilled water, cotton swaps and culture bottles, which might not be sterile due to the leakage.

To improve, an autoclave should be used. Plus, the apparatus can be double-sterilized with alcohol

after heat sterilization.

Figure 8.3.1: A glass culture bottle with a white ring stain after

cleaning

Figure 8.3.2: Steam leaking from pressure cooker


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Another limitation was that most agar plates had no growth. This might be due to mistakes in diluting

and transferring the honey samples, and faulty method of dilution. 0.01 ml sample/bacteria in 9.99 ml

water was too diluted. The bacteria might not be distributed evenly in such large volume. Also using

such small volume might have caused some samples containing Staphylococcus aureus to remain in

the pipette tips. Improvements for these are to correctly label the apparatuses and use larger volume of

sample while diluting. Another possible source for the limitation was using cotton swaps for

inoculation. The cotton swap could have absorbed the sample/bacteria solution, which was already

very little (0.01 ml). A glass rod should be used instead.


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9. References

1. The American Heritage® Dictionary of the English Language, Fourth Edition. Houghton Mifflin Company; 2004. Available at: http://www.answers.com/topic/bacteriostasis?cat=technology. Accessed January 28, 2008.

2. Manukahoney.co.uk. Available at: http://www.manukahoney.co.uk/therapeuticuses.html. Accessed January 28, 2008.

3. Staphylococcus aureus. Wikipedia. Available at: http://en.wikipedia.org/wiki/Staphylococcus_aureus. Accessed January 30, 2008.

4. Carr AC. Therapeutic properties of manuka honey. Tea Tree and Their Therapeutic Properties. Available at: http://lpi.oregonstate.edu/f-w98/teatrees.html. Accessed January 30, 2008.

5. UMF Active Manuka Honey. Available at: http://www.umfactivemanukahoney.com/index.htm. Accessed January 28, 2008.

6. Honey as an Antimicrobial Agent. Waikato Honey Research Unit. Available at: http://bio.waikato.ac.nz/honey/honey_intro.shtml#Potential. Accessed January 28, 2008.

7. McFarland standards. Wikipedia. Available at: http://en.wikipedia.org/w/index.php?title=McFarland_standard. Accessed February 15, 2008.

8. Wikler MA, Cockerill FR, Craig WA et al. Performance Standards for Antimicrobial Disk Susceptibility Tests; Approved Standard-Ninth Edition. Vol. 26 No. 1. January 2006.

9. Quantification of Bacteria. Available at: http://biology.fullerton.edu/biol302/302labf99/quant.html. Accessed February 13, 2008.

10. Beer-Lambert Law. Molecular Spectroscopy. Available at: http://hplc.chem.shu.edu/NEW/Undergrad/Molec_Spectr/Lambert.html. Accessed March 24, 2008.

11. Optical Density. Wikipedia. Available at: http://en.wikipedia.org/wiki/Optical_density. Accessed February 13, 2008.

12. Spectrophotometer Comprising Two Detectors For Overlapping Waelength Ranges. Available at:http://www.wipo.int/pctdb/en/wo.jsp?IA=WO2007068919&WO=2007068919&DISPLAY=DESC. Accessed April 25, 2008.

13. Protocols Altman Laboratory at the Emory Vaccine Center. Altman Lab Home Page. Available at: http://www.microbiology.emory.edu/altman/f_protocols/f_instruments/spectrophotometer. Accessed March 20, 2008.

14. McGill F, McLennan S, Migliorini S. Statistics: Complete Advanced Level Mathematics. Cheltenham: Stanley Thornes; 2000: 435-436.

15. Table of critical values for the Wilcoxon test. Available at: http://www.sussex.ac.uk/Users/grahamh/RM1web/WilcoxonTable2005.pdf. Accessed April 1, 2008.


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10. Appendix

10.1 Wilcoxon Test Critical Values Table [15]

Table of critical values for the Wilcoxon test: To use this table: compare your obtained value of Wilcoxon's test statistic to the critical value in the table (taking into account N, the number of subjects). Your obtained value is statistically significiant if it is equal to or SMALLER than the value in the table. e.g.: suppose my obtained value is 22, and I had 15 participants. The critical value in the table is 25: my obtained value is smaller than this, and so I would conclude that the difference between the two conditions in my study was unlikely to occur by chance (p<.05 two-tailed test, or p<.025, one-tailed test).

One Tailed Significance levels:

0.025 0.01 0.005 Two Tailed significance levels:

N 0.05 0.02 0.01 6 0 - - 7 2 0 - 8 4 2 0 9 6 3 2

10 8 5 3 11 11 7 5 12 14 10 7 13 17 13 10 14 21 16 13 15 25 20 16 16 30 24 20 17 35 28 23 18 40 33 28 19 46 38 32 20 52 43 38 21 59 49 43 22 66 56 49 23 73 62 55 24 81 69 61 25 89 77 68

ib ee on antimicrobial inhibition of manuka honey on the growth of e coli and staph a

Documents

sunlightexposed honey

honey concentration

dilution of manuka honey

unexposed honey samples

staphylococcus aureus

od600 of unexposed

effect of sunlight exposure

effects of sunlight