The intent of this experiment was to find the correlativity between the concentration of manuka honey against the growing of Staphylococcus aureus and at the same clip finding the minimal repressive concentration of manuka honey against S.aureus. The manuka honey was assorted with the broth solution along with the bacteriums to be cultured in specimen tubings. These tubings were incubated for 24 hours and the turbidness of the solutions which represents the growing of bacterium was determined by utilizing a spectrophotometer. The minimal repressive concentration was determined by the lowest concentration of manuka honey that prevented bacterial growing wholly, that is, a clear broth solution ( low turbidness ) .A statistical trial utilizing the Pearson product-moment coefficient which was carried out at 5 % significance degree showed that there was a important negative correlativity between concentration of honey and optical density which indicates growing of bacteriums. The deliberate R value ( 0.7706 ) was larger than the critical value ( 0.3172 ) and this showed a important negative correlativity, hence, the void hypothesis was rejected. The minimal repressive concentration of honey to suppress bacterial growing is about 50 % harmonizing to the informations obtained.
Honey was one time used as an antibacterial agent a long clip ago but was phased out after the find of antibiotics. Recently, due to the prevalence of assorted antibiotic resistant bacterium at that place has been a resurgence of the usage of honey, particularly for surface infections such as lesions. Honey contains antibacterial activities because of its high sugar content, sourness and presence of H peroxide. [ 1 ] [ 2 ]
a-?Figure 1: Manuka honey
hypertext transfer protocol: //www.amazon.com/Manuka-Spirits-Bay-Active-Standard/dp/B002JEAAHI
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Manuka honey is more powerful than other types of honey and is peculiarly effectual in destructing bacteriums like E.coli and S.aureus ( wound infecting bacteriums ) . Manuka honey besides contains phytochemical constituents which are non peroxide antibacterial factors present in the honey. When catalase is used to handle these honeys to take the H peroxide, these honeys with the presence of phytochemical constituents still showed important antibacterial activity. The honey used in this experiment, manuka honey has been found to hold exceeding degrees of non peroxide activity. [ 1 ] [ 2 ] The extra phytochemical constituent of honey has been known as the Unique Manuka Factor ( UMF ) or more specifically methylglyoxal [ 7 ] and due to this belongings, this honey can be used to kill many bacteriums. [ 1 ] [ 2 ] [ 11 ]
a-?Figure 2: Structure of methylglyoxal
hypertext transfer protocol: //en.wikipedia.org/wiki/Methylglyoxal
Wounds in research lab have been found to mend more quickly when treated with manuka honey compared to other types of honey. [ 1 ] Many bacteriums even resistant bacteriums which have become immune to antibiotics are susceptible to the antibacterial activity of manuka honey. Manuka honey has become the lone solution to kill bacteriums when antibiotics can no longer be used in certain instances. Manuka honey is besides stable to heat, visible radiation and aging unlike other honey. [ 1 ] [ 2 ] Recent surveies besides have shown that Manuka honey is able to suppress enzymes called cysteine peptidases which is responsible for muscular dystrophy, viral reproduction and others. [ 1 ] [ 2 ]
Staphylococcus aureus is a really common wound infecting bacteriums found in many types of lesions. Staphylococcus aureus can do serious infections when they get in through the lesion, these include infections in the lungs, castanetss, articulations and many others, but skin infections are most common if they do n’t acquire into the organic structure. [ 3 ] Staphylococcus is a Gram positive bacteria and are observed either in bunchs, separately or in braces. The cell wall of these bacteriums contain peptidoglycan and teichoic acid. Furthermore, these bacteriums can last at temperatures up to 50A°C, high salt concentrations and even drying. The ability of this bacterium to coagulum plasma has served as a common standard for the designation of these bacteriums. [ 4 ]
a-?Figure 3: Staphylococcus aureus
hypertext transfer protocol: //184.108.40.206/fzjx/wsw/newindex/tuku/MYPER/zxj/92589a.htm
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The purpose of this experiment was to happen out the relationship between the concentration of honey and the growing of bacteriums and therefore find the minimal repressive concentration of honey to be used on lesions infected with bacteriums alternatively of conventional antibacterial pick or antibiotics. The dependance on antibacterial picks and antibiotics like this will merely increase the choice force per unit area on bacteriums and promote the production of more immune strains which are more hard to destruct.
The consequences from this experiment clearly portray that even though honey was diluted to a certain extent still had important antibacterial activity. The minimal repressive concentration was besides found so that it would salvage cost for infirmaries if they were to utilize honey to handle lesions. Manuka honey costs a batch, so if infirmaries were to thin the honey and at the same clip accomplish bacterial suppression or important bacterial suppression, this would salvage a batch of money and at the same clip cut down the chance of superbugs developing. Therefore, there is a really high possibility of increasing the use of honey for intervention of lesions alternatively of utilizing conventional antibiotics and the consequence on bacterial suppression is comparable to that of antibiotics but with a really great advantage of non bring forthing more superbugs.
a-?Results demoing how bacterial suppression alterations with concentration of manuka honey [ 12 ]
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There is no correlativity between the concentration of manuka honey and the growing of bacteriums.
The higher the concentration of manuka honey used, the lower the turbidness ( indicant of bacterial growing ) .
Manipulated variable: Concentrations of honey used
Reacting variable: Turbidity ( indicates growing of bacteriums )
Fixed variable: Temperature of the solutions, temperature of incubation, volume of broth solution used, type and sum of bacteriums used, type and volume of honey used.
Specimen tubings, label spines, 1000Aµl micropipette, electronic balance, Bunsen burner, unfertile swabs, spectrophotometer, unfertile beakers.
Sterile food stock, Staphylococcus aureus suspension, sterilized distilled H2O, manuka honey, antiseptic solution, cuvettes and unfertile cotton wool.
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A test experiment was conducted to find which method and besides which stuffs were most suited to be used in this experiment. All these experiments were carried out utilizing Staphylococcus aureus as it is a common bacteria nowadays on tegument and when there is a lesion, it is about ever present. [ 3 ] This species of bacterium is besides used in research utilizing manuka honey. [ 4 ] [ 9 ]
Trial 1: Type of honey
This test was done utilizing two types of manuka honey which were obtained from a hypermarket and a pharmaceutics severally. The manuka honey obtained from the market was intended for day-to-day ingestion so it was obtained at a comparatively inexpensive monetary value. The manuka honey obtained at the pharmaceutics was lab tested for its antibacterial belongingss and it was referred to as medical class manuka honey. The antibacterial belongingss of these 2 honeys were tested by blending the honey with broth so that the antibacterial belongingss of these 2 honeys will be more outstanding as it is in direct contact with bacteriums.
Broth assorted with manuka honey from hypermarket and bacteriums
The broth solution was non clear
Broth assorted with medical class manuka honey and bacteriums
The broth solution was clear
a-?Table 1: The consequences obtained with regard to the different types of honey used
Harmonizing to the consequences above, medical class manuka honey showed seeable antibacterial activity. The medical class manuka honey was chosen.
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Trial 2: Methods of proving the antibacterial activity
This test was carried out utilizing a assortment of methods to prove for the antibacterial belongingss of the manuka honey. These methods included the phonograph record diffusion method, the well diffusion method, blending the honey solution straight with the stock and incubating the agar seeded with bacteriums prior to adding the honey. Disc diffusion method involves utilizing a unfertile paper phonograph record soaked in the honey and so placed on coagulated alimentary agar seeded with bacteriums. Well diffusion besides involves utilizing alimentary agar, but alternatively of utilizing a paper phonograph record to present the honey onto the medium, a well is made utilizing a unfertile cork bore bit and the honey is placed in the well. A control utilizing sterilized distilled H2O was used as comparing in this test.
Disc diffusion method
No important difference from control
Well diffusion method
No important difference from control
Blending honey straight with bacteriums and stock
The stock assorted with honey showed a clear solution whereas the stock assorted with sterilized distilled H2O showed a solution that was non clear
Introducing the honey onto the alimentary agar seeded with bacteriums
No important difference from control
a-?Table 2: The consequences obtained with regard to the different methods used
Blending honey straight with the broth solution showed consequences that were most important. This method was chosen to prove for the antibacterial activity of manuka honey against wound infecting bacteriums.
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Trial 3: Using different concentrations of honey
The concentrations used were set in a broad scope so that patterns or tendencies could be identified. The concentrations used were 100 % , 50 % , 25 % , 12.5 % and 6.25 % . The honey solutions were prepared as follows.
Mass of honey ( g )
Volume of distilled H2O ( cm3 )
Concentration of solution ( % )
a-?Table 3: The different concentrations of honey solutions used and how they were prepared
The undermentioned solutions of honey were assorted with alimentary broth solution and a fixed volume of bacteriums. The clarity of the solution was determined utilizing a spectrophotometer. The 100 % honey solution was non used in obtaining the consequences as the honey itself contained coloring material and would impact the reading. The other more dilute solutions would non impact the readings so much as it has a light coloring material. The spectrophotometer was calibrated utilizing the alimentary stock. The undermentioned consequences were obtained.
Concentration of honey solution ( % )
Reading of spectrophotometer
a-?Table 4: The consequences obtained with regard to the different concentrations of honey used
The higher readings indicate a solution that is less clear. The 25 % solution is an anomalousness, but the other readings show a seeable tendency.
From this test, I learnt that more concentrations need to be used to obtain a more accurate image of tendencies shown. Alternatively of merely 5 concentrations, I increased the figure of concentrations in the chief experiment so that the immense spreads in concentrations could be filled and clearer tendencies could be seen. I besides learnt from this test that repetitions need to be done to obtain an norm and besides to increase the sum of informations available to execute a important statistical analysis. Repeats carried out can besides increase the truth of the consequences obtained. I besides learnt that graduating the spectrophotometer with the alimentary stock together with the different concentrations of honey would bring forth a more accurate reading of optical density.
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Real experimental process
Preparation of honey solutions
1. A mass of 2 g of honey was weighed utilizing an electronic balance in a unfertile beaker.
2. Different volumes of H2O was added as shown in tabular array to fix the different concentrations of honey required in the experiment. The concentration of honey was calculated utilizing the undermentioned equation:
*1 milliliter of H2O was assumed to be 1 g
Concentration of honey/ %
Mass of honey/ g
Volume of water/ milliliter
a-?Table 5: Mass of honey and volume of H2O needed to fix the different concentrations of honey
1. 35 unfertile specimen tubings were prepared.
2. Each of these tubings was labeled by saying the concentrations of honey solutions that would be put into each specimen tube. The tubings were divided into 6 sets, so that 5 repetitions could be performed for each concentration of honey. The staying 5 tubings were used as controls.
3. Each tubing was filled with 4ml of unfertile alimentary stock.
4. 400 Aµl of bacterium was pipetted into the tubing followed by 600 Aµl of honey solution.
5. Each of the specimen tubings were vortexed somewhat to blend the contents of the tubing.
6. The tubings were placed in a rack and placed in the brooder at a temperature of 37A°C for 24 hours.
7. At the terminal of the incubation period, the tubings were removed from the brooder.
8. The tubings were vortexed once more so that all the contents were assorted equally.
9. A spectrophotometer was calibrated utilizing alimentary stock together with each concentration of honey which was already prepared earlier.
10. The solution in the tubing was removed and inserted into a cuvette and placed in the spectrophotometer after guaranting that the cuvette is clean.
11. The reading was recorded.
12. This was repeated for all the other solutions in the other specimen tubings.
13. All the consequences were recorded in a tabular array.
14. A graph of optical density was plotted against concentration of honey.
15. A Pearson product-moment correlativity coefficient statistical trial was carried out to analyze the information.
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Throughout the whole experiment, sterile technique was used and given the highest precedence to guarantee accurate consequences and to forestall taint. The tabular array or bench used was cleaned with 70 % ethanol solution to kill all bacteriums present on the surface.
Handss were washed utilizing antibacterial handwash to take the hazard of bacteriums from custodies polluting the experiment.
Baseball gloves were worn at all times to forestall bacteriums from custodies polluting equipment and the experiment and besides to forestall bacteriums from the experiment acquiring on custodies as Staphylococcus aureus can do serious infections if it gets into the organic structure.
The specimen tubings were besides kept near at all times and opened really somewhat when solutions were to be inserted into them to forestall taint from milieus.
Great attention was taken so that none of the contents of the specimen tubings were spilled. Care was besides taken that none of the solution was spilled on the spectrophotometer when consequences were obtained.
The flasks incorporating alimentary stock and the bottles incorporating bacteriums civilization were flamed before and after usage.
All the solutions and specimen tubes from the experiment were disposed of in a biohazard waste bag so that it can be autoclaved before disposal.
Concentration of honey
a-?Table 6: The optical density by solution when different concentrations of honey was used
a-?Graph 1: Graph of optical density against concentration
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I chose Pearson product-moment correlativity coefficient ( PMCC ) to mensurate the strength of the correlativity between the 2 variables tested in this experiment which is the optical density which indicates bacterial growing and concentration of honey. The correlativity coefficient, R has a scope between -1 and 1. 1 indicates a absolutely positive correlativity where an addition of one variable is accompanied by an addition in another variable. A value of -1 indicates a absolutely negative correlativity where an addition in a variable is accompanied by a lessening in the other. A value of 0 indicates that there is no correlativity between the 2 variables. [ 6 ]
a-?Table 7: Calculations required for Pearson product-moment correlativity analysis
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Using expression to happen the correlativity coefficient, R
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Degree of freedom= N-2
N peers figure of informations observed which in this instance is 30. Degree of freedom is 28.
The critical value for grade of freedom 4 and a one tailed trial at 0.05 significance has a value of 0.3172. [ 6 ]
R = 0.7706 & gt ; 0.3172
An analysis utilizing PMCC showed a statistically important negative linear relationship between concentration of honey and growing of bacteriums since the deliberate R value was greater than the critical value at 0.05 significance. The deliberate R value was really much larger than the critical value and this signifies that there is merely a 5 % opportunity that the negative correlativity obtained is by happenstance.
The void hypothesis is rejected.
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Table 5 shows the optical density obtained utilizing a spectrophotometer from the solutions which contained a fixed volume of alimentary stock, fixed volume of bacteriums and a fixed volume of the 6 concentrations of honey. The optical density obtained from the 5 repetitions carried out indicates the extent of growing of bacteriums in these solutions. The higher the reading of optical density obtained, the more extended the growing of bacteriums. The statistical analysis utilizing PMCC verified the negative correlativity between the 2 variables. The tabular array showed a general tendency of the values bespeaking that the lower the concentration of honey the lower the suppression towards bacterial growing.
Graph 1 shows clearly the tendency of a negative correlativity between these two variables, farther exemplifying what is shown by the informations in the tabular array. A tendency line of the information was plotted to demo the general tendency and relationship between the variables. The least growing of bacterium was observed when 50 % of honey solution was used, it gave the lowest reading on the spectrophotometer.
Unfortunately, there were a few anomalousnesss in the information obtained as they did non demo a additive negative correlativity. There were fluctuations particularly with the lower concentrations. At 3.125 % and 12.5 % concentration of honey solution, the growing of bacteriums increased when it was supposed to diminish harmonizing to the expected tendency.
A control experiment was carried out for each set of concentrations so that a valid comparing can be made between the tubings with honey and those tubings without. The absence of honey in the control experiments allowed bacteriums to turn freely as they would in a normal environment which contained no antibacterial agents. This clearly demonstrates the effectivity of the different concentrations of honey against the growing of bacteriums.
The information indicates that merely the 50 % honey solution shows suppression of growing compared to the control. The concentrations of honey below 50 % all showed a growing of bacteriums higher than the control experiments without the honey. This indicates that honey at high concentrations can suppress or curtail the growing of bacteriums but at lower concentrations this does non go on.
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From the information, it is safe to state that the minimal repressive concentration of manuka honey is about 50 % . This is because as stated above, the concentrations of honey solution lower that 50 % did non suppress bacterial growing as they showed more growing compared to the control experiments. The minimal repressive concentration obtained here was compared with an probe carried out by the Honey Research Unit at University of Waikato in New Zealand. There is a important difference between the consequences obtained by them and my consequences. The grounds for this difference will be farther discussed in the rating.
a-?Results obtained by the Honey Research Unit at University of Waikato, New Zealand [ 8 ]
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Broth was used is this experiment to enable the honey to be assorted straight with the bacterium. This direct contact of the honey with the bacterium gives significantly more seeable consequences. The tendency of growing of bacteriums can be seen clearly as the concentration alterations. The stock was allowed to chill down before being poured into the tubings so that the high temperature of the stock does non kill the bacteriums and therefore impacting the consequences. The volumes of the stock, bacterium, honey and besides incubation temperature was kept changeless so that the lone factor impacting the growing of bacteriums would be the concentration of honey solution.
There is some fluctuation in the consequences obtained in this experiment. First of all, for one peculiar concentration of honey, the readings from the spectrophotometer show rather a broad scope. For illustration, the readings obtained for 12.5 % of honey. The maximal reading obtained was 1.074 and the lower limit was 0.570. One ground that might hold caused this was that the contents of the tubing which was the stock, bacteriums and honey were non assorted equally, in this instance ; vortexing the mixture. Problems may besides originate when the bacteriums sinks to the underside and signifiers deposits which stick to the underside of the tubing and do n’t blend with the solution. This could give an inaccurate reading. The highest reading on the spectrophotometer of 1.074 was chiefly caused by the froth nowadays in the solution due to vigorous commixture of the solution. The same could be said for the concentration of 6.25 % . The inconsistent information was chiefly caused by the same grounds as mentioned above.
To minimise the mistakes caused by the jobs above, repetitions were carried out. Even though, there was considerable fluctuation in the repetitions carried out, this was overcome by the mean that was calculated. The average values were the values used in plotting the graph. The average values are a better representation of the consequences as they are the norms of the consequences obtained. The anomalousnesss mentioned above were included in the computations of the mean for each concentration to demo consistence in the informations used, in this instance, 5 informations for each concentration.
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The control experiments carried out above were for comparing with those incorporating honey. Harmonizing to the consequences obtained, the control experiments had a lower bacterial growing compared to the lower concentrations of honey. This indicates that even with the presence of honey, the bacterial growing is more extended than the bacterial growing in the control experiments. The lone concentration that showed bacterial suppression was 50 % concentration of honey. There is more bacterial growing in the lower concentrations of honey because it promotes the growing of bacteriums at lower concentrations alternatively of suppressing it. [ 10 ] At low concentrations of honey, the typical belongingss of honey has been minimized. The high sugar concentration that is frequently associated with the osmotic consequence is minimized in low concentrations of honey solution. This provides an ideal environment for bacterial growing since both wet and nutrient is present. Bacterias like to multiply on alimentary nutrient [ 5 ] , like rice which is a signifier of saccharide. These saccharides when broken down are sugars and these sugars are readily present in the diluted solution of honey, this favours bacterial growing, hence the lower concentrations of honey have more bacteriums than the control experiments.
The bacteriums used have to be from the same bottle. This is due to the fact that different batches of bacteriums which have been cultured at different times may incorporate different concentrations of bacteriums. This may do incompatibilities in consequences if a different batch of bacterium is used in the same experiment. This can be avoided by utilizing bacteriums from the same bottle to guarantee that each tubing has the same get downing concentration of bacteriums, and the lone factor that will be impacting the bacterium is the concentration of honey.
Another restriction is that during the experiment, the stocks used may be unfertile, but while transporting out the experiment, it is exposed to air. This gives an chance of bugs in the air to pollute the stock. This can be avoided by covering the stock every clip it is non being used. The surfaces of the bench and the equipment are besides exposed to the same hazard, so baseball mitts are used to minimise the sum of taint to equipment and hence obtaining more accurate consequences.
One more restriction is that when the honey solution is assorted with the stock, the honey is farther diluted by the stock. This means that the honey concentration in the tubing is non precisely the concentration of honey prepared from the pure honey. This will impact the consequences obtained and besides the minimal repressive concentration. This could hold been overcome by taking into history this and fixing a honey solution of higher concentration so that when it is diluted by the stock, the wanted concentration of honey is obtained. This was ignored in the experiment because the mistake is cancelled out when the same mistake is present in all the solutions, and it still showed the expected negative correlativity. The honey solutions used were still in diminishing concentrations, so the bacterial growing increased.
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All these restrictions and anomalousnesss mentioned above caused the mistake in finding the minimal repressive concentration but still managed to demo a important negative correlativity between concentration of honey and bacterial growing. This indicates that honey has a immense potency to replace conventional antibiotics in lesion attention and therefore cut down the production of “ superbugs ” .
The higher the concentration of manuka honey, the lower the bacterial growing. The minimal repressive concentration organize the experiment is 50 % manuka honey.
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Beginning 1 and 2 is about the antibacterial belongingss of manuka honey and besides about how it is effectual on lesions. The information from these beginnings tally and since one of the beginnings is from the consequences and research done by the Honey Research Unit at Waikato University and information from here is considered believable. Beginning 3, 4 and 5 are all about Staphylococcus aureus, how it infects and besides the conditions required for its growing. The information obtained are from medical web sites and this can function as reassurance that the beginning is dependable. Beginning 6 is a published book about the statistical analysis in research. It was written by a Professor at Jacksonville University and hence it is a really dependable beginning. Sources 7,8, 9 10 and 11 are on-line diaries about research done on manuka honey, the type of bacteriums it can be used against and the active ingredient in manuka honey. These diaries were published on web sites like Science direct and Springerlink and besides some are published by research units of universities. The diaries obtained from these beginnings are all equal reviewed and this means that other scientists agree with the work done in these diaries and the information is dependable.
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