It is now possible to add the values of the tea tree oil and the chlorhexidine.
(Σx2/nx) – (x)2 + (Σy2/ny) – (y)2
nx –1 ny – 1
354 - 361 + 240 - 225
7 7
356 – 361 = - 7
240 –225 = 15
-7 + 15 = 8
7 7
Step 3
It is now possible to add both the results for values for Tea tree oil and chlorhexidine into the statistical T –test equation.
T = (x) – (y)
(Σx2/nx) – (x)2 + (Σy/ny) – (y)2
nx – 1 ny-1
-7 + 15 = 8
7
8/7 = 1.1492857143
1.14 (3.s.f)
Step 4
After adding both the values for tea tree oil and chlorhexidine. It is now possible to square root this value.
√1.14 = 1.067707825
1.07 (3.s.f)
T = (x) – (y) as shown above the equation, represents the mean of x minus the mean of y.
As we know (x) is 19 from (y) is 15
Subtracting (x) 19 from (y) 15 will give me 4.00 (3.s.f)
These values 1.07 and 4.00 can be used to find the value of T
T = 4/1.07
T= 3.738317757
T = 3.74 (3 s.f)
A two-tailed test was done on the effectiveness of the two antiseptics. This would indicate two possible outcomes, which was the null hypothesis and the alternate hypothesis.
Step 1 - Degrees of freedom
D.o.f = (nx – 1) + (ny-1) = (8 – 1) + (8 –1) = 14
Step 2 - significance level = 0.05
Step 3- Null hypothesis states there is no significance difference between the two sets of data used to chance
Alternate hypothesis – There is a significant difference between the two sets of results. This is due to an underlying factor, i.e. such as the active properties in tea tree oil (terpinen- 4-ol ) is more effective in causing lysis (cell death) than chlorhexidine glucanoate.
Step 4- critical value of T at 14 degrees of freedom and 0.05 level of signficance
Critical value : 1.761. If the value of T is less than 1.761, this supports the null hypothesis with 95% chance of the means being insignificantly different.
However if the value of T is greater than 1.761, this supports the alternate hypothesis, with 95% chance of the means being significantly different.
Statistical tests allow us to make statements with a degree of precision, but cannot actually prove or disprove anything. A significant result at the 95% probability level tells us that our data are good enough to support a conclusion with 95% confidence (but there is a 1 in 20 chance of being wrong). Through analysing this experiment we can accept this level of significance as being reasonable leading to a justified conclusion.
Looking at my Statistical t test equation, the value for T is above 1.761. This supports the alternate hypothesis, which states that there is a significant difference between the two sets of data due to an underlying factor.
Conclusion-1
Through my mathematical analysis the T value, was worked out to be 3.74, again this approving of the alternate hypothesis, indicating there is a significant difference between the two sets of data. Generally speaking there were large zones of inhibitions on average for the tea tree oil than for the chlorhexidine on the E. coli bacterium (gram negative). This portrays the fact that tea tree oil is a better antiseptic on gram negative bacteria than chlorhexidine, with respect to the above averages.
T-Test for gram positive bacteria (bacillus subtillis)
The effectiveness of the antiseptics were analysed by carrying out a statistical T -test on the Bacillus Substillus (gram positive), from this it would be possible come to justified conclusion.
Statistical T test
T = (x) – (y)
(Σx2/nx) – (x)2 + (Σy/ny) – (y)2
nx – 1 ny-1
Null hypothesis - There is no significant difference between the two sets of results, due to chance.
Alternate hypothesis – There is a significant difference between the two sets of results. This is due to an underlying factor, i.e for example the active properties in tea tree oil (terpinen- 4-ol ) is more effective in causing lysis (cell death) than chlorhexidine glucanoate, or vice versa)
Calculations for the Tea tree oil
X= Tea tree oil (mean of diameter)
Σx = 18 + 20 + 17 + 14 + 21 + 24 + 15 + 18 = 147
Σx2 = 324 + 400 + 289 + 196 + 441 + 576 + 225 + 324 = 2775
nx= 8 number of samples
(x) = Mean of x is 147/8 = 18.375 18.0 (3.sf.)
(x)2= 182 which is 324 (3.s.f)
Calculations for the Chlorhexidine
y = Chlorhexidine (mean of diameter)
Σy = 24 + 23 + 15 + 20 + 23 + 26 + 21 + 22 = 174
Σy2 = 576 + 529 + 225 + 400 + 529 + 676 + 441 + 484 = 3860
ny = 8 samples
(y) = Mean of y, therefore 174/8 = 21.75 22.0 (3.s.f)
(y)2 = 222 which is 484 (3.s.f)
Inserting the values of the Tea tree oil and the chlorhexidine into the statistical T- test equation
T = (x) – (y)
(Σx2/nx) – (x)2 + (Σy/ny) – (y)2
nx – 1 ny-1
Inserting the values for the Tea tree oil
Step 1
2775/ 8 324
347
(Σx2/nx) – (x)2
nx – 1
7
Σx2= 2775/8 =346.875 347 (3.s.f)
nx = no. of samples which is 8
(x) = 18.0
(x)2 = 324
Inserting the values for the Chlorhexidine
Step 2
3860 / 8 484
482
(Σy2/ny) – (y)2
ny – 1
7
Σy2= 1924
ny = no. of samples which is 8
Therefore 3860/8 =482.5
(y) = 22.0
(y)2 = 484
It is now possible to add the values of the tea tree oil and the chlorhexidine.
(Σx2/nx) – (x)2 + (Σy2/ny) – (y)2
nx –1 ny – 1
347 - 324 + 482 - 484
7 7
347 – 324 = 23
482 – 484 = -2
23 + -2 = 21
7 7
Step 3
It is now possible to add both the results for values for Tea tree oil and chlorhexidine into the statistical T –test equation.
T = (x) – (y)
(Σx2/nx) – (x)2 + (Σy/ny) – (y)2
nx – 1 ny-1
23 + -2 = 21
7
21/7 = 3.00 (3.s.f)
Step 4
After adding both the values for tea tree oil and chlorhexidine. It is now possible to square root this value.
√3 = 1.732050808
1.73 (3.s.f)
T = (x) – (y) as shown above the equation, represents the mean of x minus the mean of y.
As we know (x) is 18 from (y) is 22
Subtracting (x) 18 from (y) 22 will give me +/- 4.00 (3.s.f)
These values 1.73 and -4.00 can be used to find the value of T
T = 4/1.73
T= 2.312138728
T = 2.31 (3 s.f)
A two-tailed test was done on the effectiveness of the two antiseptics. This would indicate two possible outcomes, which was the null hypothesis and the alternate hypothesis.
Step 1 - Degrees of freedom
D.o.f = (nx – 1) + (ny-1) = (8 – 1) + (8 –1) = 14
Step 2 - significance level = 0.05
Step 3- Null hypothesis states there is no significance difference between the two sets of data used to chance
Alternate hypothesis – There is a significant difference between the two sets of results. This is due to an underlying factor, i.e. such as the active properties in tea tree oil (terpinen- 4-ol ) is more effective in causing lysis (cell death) than chlorhexidine glucanoate or vise versa.
Step 4- critical value of T at 14 degrees of freedom and 0.05 level of signficance
Critical value : 1.761
If the value of T is less than 1.761, this supports the null hypothesis with 95% chance of the means being insignificantly different.
However if the value of T is greater than 1.761, this supports the alternate hypothesis, with 95% chance of the means being significantly different.
Looking at my Statistical t test equation, the value for T is above 1.761. This supports the alternate hypothesis, which states that there is a significant difference between the two sets of data due to an underlying factor.
Conclusion-2
Through my mathematical analysis the T value, was worked out to be 2.31, again this approving of the alternate hypothesis, indicating there is a significant difference between the two sets of data. Generally speaking there were large zones of inhibitions on average for the chlorhexidine than for the tea tree oil on the Bacillus subtilius bacterium (gram positive).
Bacteria belong to the prokaryote class because they do not have a true nucleus, as the main chromosome is not surrounded by a nuclear membrane., have few organelles except for small ribosome’s and flagella, which lack microtubules. They have circular pieces of DNA called plasmids. Most bacteria may be placed into one of three groups based on their response to gaseous oxygen. Aerobic bacteria thrive in the presence of oxygen and require it for their continued growth and existence. Other bacteria are anaerobic, and cannot tolerate gaseous oxygen. The third group is the facultative anaerobes, which prefer growing in the presence of oxygen, but can grow without it. Bacteria are rod-shaped (bacilli), spherical (cocci) or spiral (spirilla) in form. Bacilli or cocci may adhere in small groups or chains.
The bacteria that were used in this experiment were Bacillus subtillus and Escherichia coli, both of which have many common features, but however have key differences in their metabolism and gene regulatory mechanisms.
Bacillus substilis is a gram-positive bacterium. It is rod shaped and is an endospore forming aerobic bacterium. E.coil on the other hand is a gram-negative bacterium, cylindrical in shape/i.e known as cocci.
The average diameters for Tea tree oil and chlorhexidine for E.coli (gram negative)
Tea tree oil had a mean value of 19.0mm and Chlorhexidine, which had, mean value of 15.0mm
Whereas the averages for Bacillus substilis (gram positive)
Tea tree oil had a mean value of 18.0mm and Chlorhexidine had a mean value of 22.0mm
Looking at these averages tea tree oil seems to work best on gram negative bacteria than on gram positive bacteria although there doesn’t seem to be a significant difference between the two means. ”Treatment with Tea tree oil on gram negative bacteria (E.coli) had a mean diameter of 19.0mm, whereas gram positive bacteria had a mean diameter of 18.0mm. These four active properties of tea tree oil play an important role of its effectiveness as an antiseptic on gram positive as compared to the chlorhexidine antiseptic.
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Anti-parasitic contains chemicals destructive to parasites
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Anti-fungal inhibits the growth of fungi.
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Anti-microbial which means its is capable of destroying or inhibiting the growth of disease causing micro organisms.
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Anti-biotic properties, a drug used to treat bacteria infections, however it is also termed as a chemical substance produced by micro-organisms (mainly bacteria/fungi) at low concentrations, which have that selectively inhibit the growth or destroy other microorganisms.
Treatment of tea tree oil on E.coli (gram negative), suspensions resulted in the larger zones of inhibition and approves of the fact that tea tree oil works better on gram negative than in gram postive, due to the nature of their cell surface membranes. Gram negative have a thin cell surface membrane size around 2nm layer of peptidoglycan which is allows a better diffusion pathway as compared to gram positive which has a thick outside cell surface membrane (around 8nm) with a ridig layer of peptidoglycan The active ingredient in Tea tree oil Terpinen-4-ol caused damage to the cell membrane and the peptidogcylan. The function of the peptidogyclyan is to prevent osmotic lysis. Once this has been destroyed, the bacteria loses its genetic material was also lost from bacterial cells through the damage of the cell wall (lysis). This experiment has established the fact that tea tree oil (terpinen-4-ol) has a significant anti -microbial activity and that the complex properties of tea tree oil make it difficult for the microorganism to develop resistance to the antiseptic The antibiotic properties in tea tree oil also seem to have an effect and interfere with 70S ribosome in the bacteria and this affects the binary fission of bacteria
Binary fission is when the bacterium divides into two daughter cells, with the same genetic composition.
The picture on the left simply illustrates how the bacteria such as E.coli divide.
1. DNA replicates and the DNA attaches to the mesosomes, which help to separate the DNA.
2. Septum cross wall develops in the cell and finally divide into to daughter cells of the same genetic makeup. The terpine-4-ol in tea tree oil also seems to interfere with the synthesis of the cell wall and its constituents, thus causing disorientation of protein synthesis such as the, metabolic reactions and transcription needed for cell growth.
Chlorhexidine on the other hand seems to work better on the gram-positive bacteria rather than on the gram-negative bacteria.
“ Treatment with Chlorhexidine on gram positive bacteria (Bacillus substilis) had a mean diameter of 22.0mm, whereas on the gram negative bacteria (E.coli) the mean diameter value of 15.0mm”
This resistance arises because E.coli are able to produce specific enzymes for destroying the active properties of chlorhxidine glucanoate and due to the plasmids that may be carried. These plasmids are extra chromosomal pieces of DNA, which code for the resistance to a number of antibiotics. Resistance can be developed within the bacterium’s chromosome.
This happens because gram-negative bacteria are generally more resistant to the antiseptic properties of chlorhexidine. The main active property in chlorhexidine, ‘chlorhexidine glucanoate’ is slow acting antiseptic and its chemistry is not as complex as terpine-4-ol, resulting in the development of resistant strains of an organism in otherwords enables development of resistant enzymes in E.coli. The plasmid DNA carries the resistance and can developed within the bacterium’s chromosome. The antimicrobial agents such as the chlorhexidine glucanoate naturally act selectively on specific types of organisms or on specific strain of organism. This action of selectivity favours the survival of organisms that either escape full-strength assault, or those capable of resisting the assault. It seems from the practical investigation I have carried that the E.coil (gram negative) was able to resist full assault of the chlorhexidine, while the Bacillus subtillus (gram positive) endured the full effect of the active ingredient in chlorhexidine and produced less resistance to the antiseptic. Using scientific knowledge, It could be that the outer membrane of gram-negative bacteria (E.coli) however acted as a barrier (layer of peptidoglycan is protected by a complex membrane including lipopolysaccarides, lipids and phospholipids, which limits the entry of many chemically unrelated types of antibacterial agents such as the chlorhexidine gluconate. This could also be a partial reason why the chlorhexidine worked better in gram-positive bacteria, than that of the gram negative.
