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
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)
Page 2 of 38
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Biology Extended Essay
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 Page 3 of 38
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Biology Extended Essay
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
Page 4 of 38
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Biology Extended Essay
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.
Figure 1.1.1 (from left to right): Propolis, Commercial honey, Active Manuka honey 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. Page 5 of 38
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Biology Extended Essay
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
Glucose oxidase
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.
Figure 1.2.1 [3] : Staphylococcus aureus under microscope
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Biology Extended Essay
2. Materials and Methods 2.1
Apparatus list • • • • • • • • • • • • • • • • • • • • • • • • •
2.2
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
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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Biology Extended Essay
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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Biology Extended Essay
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).
Black sugar paper / fibreglass cloth Plastic board
Wooden block
Figure 2.4.1: Incubator
2.5
Figure 2.4.2: Heat shield
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.
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Ng Siang Hang 2.7
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Biology Extended Essay
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.
Figure 2.7.1: Cooling the nutrient agar plates
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.
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Biology Extended Essay
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 sterile distilled water used / ml (± 0.01 ml)
Total volume / ml (± 0.02 ml)
75
Volume of unexposed/sunlightexposed Active Manuka honey solution used / ml (± 0.01 ml) 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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Biology Extended Essay
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.
Figure 3.1.1: Comparing turbidity
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
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Biology Extended Essay
The tables show the component of the honey samples:
Table 3.1.2: Component of Unexposed Active Manuka honey samples Component Unexposed Active Manuka honey samples
Volume of Unexposed Active Manuka honey solution
Volume of Mueller Hilton nutrient broth solution
Volume of Staphylococcus aureus solution
Total volume / ml
/ ml / ml
/ 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 Component Sunlight-exposed Active Manuka honey samples
Volume of Sunlight-exposed Active Manuka honey solution
Volume of Mueller Hilton nutrient broth solution
Volume of Staphylococcus aureus solution
Total volume
/ ml
/ 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
/ ml
/ ml
(Concentration of added Sunlight-exposed Active Manuka honey)
Page 13 of 38
Ng Siang Hang 3.2
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Biology Extended Essay
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.
Page 14 of 38
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Biology Extended Essay
Procedure
0.01 ml of honey sample
0.01 ml of 10-2 diluted sample
0.01 ml of 10-4 diluted sample
9.99 ml of sterile distilled water 10-2 dilution
Honey sample
0.99 ml of sterile distilled water
10-4 dilution
10-5 dilution
Diagram 3.2.1.1: Procedure of diluting the honey samples
Table 3.2.1.2: Dilution table of honey samples Dilution
Volume of honey sample used
Volume of sterile distilled water
Total volume / ml
/ ml
/ 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.
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Biology Extended Essay
4. Improvised Method of Testing Method in section 3.1 was repeated. 4.1
Improvised method of quantification: Spectrophotometry [9]
Initial light intensity
>
End light intensity
Light
Staphylococcus aureus
Figure 4.1.a: Illustration of the theory behind spectrophotometry
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.
Page 16 of 38
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Biology Extended Essay
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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Biology Extended Essay
5. Data Collection and Processing 5.1
Observations of Unexposed and Sunlight-exposed Active Manuka honey
2
Figure 5.1.1: Unexposed Active Manuka honey (left) and Sunlight-exposed Active Manuka honey (right)
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.
Page 18 of 38
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Results from Standard Plate Count Method 5.2.1
*
Dilution plates of Unexposed Active Manuka honey samples
**
~
Biology Extended Essay
*
~~
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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*
Biology Extended Essay
**
~
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~~
Figure 5.2.1.5: MH12.5% Dilution Plates
*
**
~
~~
Figure 5.2.1.6: MH6.25% 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
5.2.2
*
~
Dilution plates of Sunlight-exposed Active Manuka honey samples
**
*
~~
Figure 5.2.2.1: E100% Dilution Plates
~
**
~~
Figure 5.2.2.2: E75% 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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*
~
Biology Extended Essay
**
*
**
~~
~
~~
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
*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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Biology Extended Essay
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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Ng Siang Hang 5.3
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Biology Extended Essay
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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Biology Extended Essay
10-5
76
Calculations 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 = 7.6 10 10 = 7.6 10 CFU/ml
dilution factor
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,
Calculations,
OD600
. dilution factor
Staphylococcus aureus concentration / CFU/ml
Undiluted
0.283
-
7.600
1:2
0.132
Total
7.6
10
3.800
10
1.900
10
0.950
10
0.475
2 1:4
0.068
7.6 4
1:8
0.006
7.6 8
1:16
-0.040*
7.6 16
*Results for 1:16 dilution were not plotted in the standard curve because ODλ cannot be negative.
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Biology Extended Essay
Graph 5.3.1.3: Standard curve for Staphylococcus aureus (Optical Density at 600 nm wavelength vs. Staphylococcus aureus concentration) 9
Concentration of Staphylococcus aureus (108 CFU/ml)
8 7 6 5 4 3 2 1 0 0
0.05
0.1
0.15
0.2
0.25
0.3
Optical Density at 600 nm wavelength, OD600
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Ng Siang Hang 5.3.2
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Biology Extended Essay
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 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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Biology Extended Essay
Bar chart 5.3.2.3: Comparison of OD600-NB between Unexposed and Sunlight-exposed Active Manuka honey samples 0.16
E100%
0.14
OD600 minus OD600 of nutrient broth, OD600-NB
E75% 0.12 MH100% 0.1
0.08
Unexposed Active Manuka honey samples
E50% MH75%
0.06
E25%
MH50%
Sunlight-exposed Active Manuka honey samples
0.04 MH25% 0.02
0 100.00%
75.00%
50.00%
25.00%
Concentration of Unexposed/Sunlight-exposed Active Manuka Honey in the 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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Biology Extended Essay
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 Page 28 of 38
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Biology Extended Essay
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 sunlightexposed 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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Biology Extended Essay
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 Concentration of unexposed/sunlightexposed 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
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
(d = xb-xa)
*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
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Biology Extended Essay
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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Biology Extended Essay
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 OD600NB
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 Page 34 of 38
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Biology Extended Essay
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. Page 37 of 38
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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, onetailed test).
N 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
One Tailed Significance levels: 0.025 0.01 Two Tailed significance levels: 0.05 0.02 0 2 0 4 2 6 3 8 5 11 7 14 10 17 13 21 16 25 20 30 24 35 28 40 33 46 38 52 43 59 49 66 56 73 62 81 69 89 77
0.005 0.01 0 2 3 5 7 10 13 16 20 23 28 32 38 43 49 55 61 68
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