Bio IA graded 7 IB score PDF

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this is my bio ia that got a pretty hight score hope its usefull...

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Aim: To determine the effect of different concentration of Tea Tree Oil on bacterial growth of E. Coli (Escherichia Coli), in order for investigation of antibiotic properties of tea three oil. Personal Engagement: Ever since I have moved to my current school, where I am living abroad with many students, I got to meet a close friend of mine and a roommate, who had a passion about essential oils. Through her I have learned the multifunctionality and different uses of essential oils, like food supplements, skin care and even treatment of internal diseases. One of the oils that caught my attention the most was Tea Tree Oil (Melaleuca Alternifolia), because of its wide uses on the human body, especially its antibacterial properties that interested me the most and led me to investigate its antibacterial effects in different concentration on the bacterial growth of E. Coli (Escherichia Coli). Exploration: Research Question: What effects do different concentration of Tea Tree Oil (Melaleuca Alternifolia) have on bacteria growth of E. Coli (Escherichia Coli)? The purpose of this investigation is to determine the antibacterial properties of Tee Tree Oil, and whether in different concentration its effectiveness may differ as tested on the bacteria E. coli. E. Coli (Escherichia Coli): E. coli (Escherichia coli) is a type of bacteria usually associated to be found in human intestines. Most strands of this bacteria are found to be harmless and even healthy to be part of the human digestive system, however some

Figure 1: Microscopic Picture of E. Coli Source: (“E. Coli under the Microscope -Types, Techniques, Gram Stain, Hanging Drop Method,” n.d.)

strands can cause to the development of serious problems like diarrhoea, usually caused by consumption of contaminated food or drink. E. coli is a gram-negative bacterium, which associates with its strong antibiotically resistance, with its optimum temperature of growth at 37 1

° C ( Albrecht , 2015) . A typical characteristic of the E. coli bacteria is its thin cell wall, however exactly because of this phenomenon, E. Coli has a developed doable sided tick cell membrane, called diderm, which is the reason for its hight antibiotic resistance, making it a gram-negative bacterium in terms of its membrane permeability. The bacterial cytoplasmic membrane in the gram-negative bacteria such as E. coli provides a permeability barrier to the passage of small ions. (Cox et al., 2001b) This occurrence is regulated by the structural and chemical composition of the double tick cell membrane itself. Maintaining ion homeostasis is an essential characteristic of preserving the energy status of the cell as well as membrane-coupled, energydependent processes such as solute transport, regulation of metabolism, control of turgor pressure and motility. (Cox et al., 2001b) Therefore, even relatively slight changes to the structural integrity of cell membranes can detrimentally affect cell metabolism and lead to cell death, as the change in the concentration of the substance intake, like in the case of this investigation, the change in Tea Tree Oil concentration in different solutions. (Cox et al., 2001b) Tea Tree Oil (Melaleuca Alternifolia): Tea tree oil also known as Melaleuca oil is an essential oil extracted from a plant called Australian Tea tree. Tea tree oil can be applied in many cases of

Figure 2: Picture of Pure Tea Tree Essential Oil

skin infections such as acne as well as it is believed to be good for inhalation in cases of cough, bronchial infections etc. because of its broad spectrum of inhibitory activity on bacterial spread, proved and discussed by many other studies in the field.

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With consideration to that this is why I chose to examine how can different concentrations of tea tree oil affect the production and spread of the bacteria E. coli, whether or not certain trend of reaction would be observed.

H 0 Hypothesis: With the increase of the concentration of tea tree oil in the oil mixture there would not be an effect of reduction of E. coli growth. Variables: Independent Variable: Concentration of the tea tree oil solution attained by dilution of pure tea tree oil (100%) into Polysorbate 80 since it is a natural vegetable sourced emulsifier that creates solubility between waterbased ingredients and oil-based ingredientsand therefore does not possess any antibacterial properties to interfere with the tea tree oil (“POLYSORBATE 80,” n.d.).

Dependent Variable: The zone of inhibition/ growth of the bacteria in the container, measured by a ruler with accuracy of Table 1: Identification, importance and control of the controlled variables.

± 0.1mm .

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Controlled variable

Importance of the Controlling method controlled variable The total amount of oils In order to keep the In different trials the total mixture each trial. concentration of tea tree oil amount of oil mixture should be kept consistently at 1 ml consistent. each trial. The amount of E. coli In order to control and insure Using a laboratory micro pipette, with exactly 1 ml E. bacteria placed in each Petri equal starting point of all coli solution placed in each dish. solution trials, so to receive Agar Plate. accurate results. The temperature in which the In order to ensure accurate Agar Plates are kept in. results all Agar Plates must be in the optimum temperature for E. coli growth. The concentration of nutrients In order to ensure accurate in each Agar Plate. results each one of the E. coli samples has the same nutritious potential. Purity of the Teas Three Oli.

In order to create accurate concentration levels for the investigation, 100% pure Tea Tree Oil is used so it keeps its potency and properties.

Placing all Agar placed used in the experiment in an incubator at the optimum temperature of 37 ° C . (Albrecht, 2015) Using an already preparade laboratory Agar Plates using the laboratory micro pipette to ensure the same amount of nutrients is obtained. In order to ensure valid results, the investigation would be placed with the use of 100% pure Tea Three Oil.

Materials and tools required:       

Rubber gloves- 1 pair Laboratory Googles1 pair Laboratory Apron- 1 Test tube -5 Test tube rack-1 Tweezers-1 Bunsen burner for sterilization-1

    

Ruler-1 Filter Paper discs-50 Agar Plates-5 Tea tree oil ≈ 1 Bottles (30ml) Polysorbate 80

≈ 30 ml 

Black permanent marker-1

   

Laboratory Film- 5 Mechanical Pipette- 1 Disposable Pipette Tip- 3 Saline Solution

≈ 500 ml   

Cell spreader- 1 Laboratory Mixer-1 Incubator-1

Methodology:

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1. Dilute the tea tree oil into five different mixtures: 0%, 25%, 50%, 75%, 100% of tea tree oil into the total oil solution with the use of laboratory pipette, ensuring total solution of 1 ml each mixture. 2. Name accordingly each test tube of the different concentration. 3. Divide each Agar plate into four sections with a permanent marker on the outside surface for repetition of the experiment (the last one divide into five sectors). 4. Name accordingly each Agar plate for the different solutions. 5. Soak about 10 paper discs in a beaker with the oil solution. 6. Seal the beaker so to control the amount of solution inside and to avoid air contamination. 7. Sterilize the tweezers in order to use them for placing the paper discs on the Agar Plates. 8. Enculturating the E. coli into the Agar Plates with laboratory pipette to ensure exactly 1 ml of E. coli in each Agar plate, under Bunsen Burner for sterilization. 9. Open the test tubes and use 5 filters out of each to place in the centre of the sector of the Agar Plate contaminated with E. Coli. 10. Keep the Agar Plates in incubator for 48 hours at 37 ° C. 11. After 48 hours, remove the Agar Plates out of the incubator and measure the E. coli growth on each Petri dish with a ruler ( ± 0.1cm ).

Table 2: Image representation of the set-up of the experiment and the final results on each Petri Dish. experiment.

of Tea Tree Oil and carrier

the Petri dishes, measured

Polysorbate 80.

with a ruler (accuracy:

± 0.1cm )

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Risk Assessment: 

Safety: In order to assure safe work with the bacteria E. Coli protective measurements have been taken such as wearing laboratorial apron, gloves and glasses as well as sterilizing used instruments with the Bunsen burner and disposing single used instruments in a saline solution for disinfection before throwing away.



Environmental Concerns: Throughout the experiment there was a necessary use of single use plastic for easier safety use, which therefore after sterilized in the saline solutions were disposed as trash and ultimately became harmful for the environment as a non-reusable laboratorial plastic waste. A possibility for future experiment is to replace those parts with reusable once, capable of sterilization.



Ethical Concerns: There were no ethical concerns present due to a non-inclusion of animal factor incorporated in the experiment.

Analysis: Data collection: The data collected is representing the area of each individual Agar Plate where the subculturing of E. coli was unsuccessful, therefore the antibacterial properties of the solution given had killed the bacteria around the edges of the circular paper disc placed inside. The following results were obtained as following: XConcentrati

Trial 1

Trial 2

Trial 3

Trial 4

Trial 5

Averag

(cm) (cm) of the experiment (cm) (cm)of the five concentrations (cm) eand trials, Table 3: Representation of obtained results for each combined with the average of each solution. on (%) 0% 25% 50% 75% 100%

0.2 0.3 0.2 0.6 0.4

0.1 0.3 0.2 0.4 0.6

0.1 0.2 0.4 0.5 0.5

0.1 0.3 0.4 0.4 0.5

0.1 0.2 0.4 0.6 0.4

0.1 0.3 0.3 0.5 0.5 6

Data Analysis: With the obtained data at first sight, it could be noticed that there is a positive corelation between the percentage of Tea Tree Oil in the concentration and the effectiveness of antibacterial properties of the solution. Therefore, in order to sufficiently answer the research question in the scope of the research this quantitate data of diameter of antibacterial activity would be taken to test with ANOVA test method. The results of the carried-out ANOVA test as following: Data calculating: Mean Example: For the concentration of the 25%, with the following results being obtained from previous table of values it could be calculated:

m=

∑ of the data of each trial = 0.3 +0.3 +0.2 + 0.3+ 0.2 = 1.3 =0.26 total number of trials

5

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Standard Deviation Example: For the concentration of 25% with the following results being obtained from previous calculations, the standard deviation could be calculated through One-Way ANOVA Calculator, with

Table 4: Summary Data of the experiment including the mean and standard deviation for each trial (treatment).

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the following results obtained. (In order to accumulate instant calculations One-Way ANOVA Calculator was used and the collected data was placed into a table.) (Stangroom, 2019) From the obtained data it could be clearly observed that there is a positive relationship between the means, with an indicates that with the increased around of Tea Tree Oil in the solutions examined, the wider the radius of the circle around the paper discs becomes, therefore possibly the more antibiotic properties the solution acquires. However, in order to reach to a conclusion on the null hypothesis, the ANOVA test should be examined until reaching the F value, in order to accept it or reject it, with comparison to the F critical value obtained from the following table. Table 5: Critical values of F for the 0.05 significance level. Source: (F ratio table 2005)

In order to obtain the correct F critical value there is a need to compute the numerator and denominator’s degrees of freedom as following: 

Numerator degrees of freedom: d 1=treatement −1 =5−1 =4



Denominator degrees of freedom: d 2=total observation−treatement=25 −5=20

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Therefore, from those values it could be concluded that the critical F value:

4,20 F0.05 =2.87 . However, in

order to conclude with rejection or acceptance of the null hypothesis, the observed F value should be calculated through One-Way ANOVA test for independence, performed by an online ANOVA calculator. Table 6: Results obtained from ANOVA online calculator with the represented degrees of freedom and the F value observed for the dana analyzed.

From the graph it could be concluded that since F observed value is larger than F critical value, there is a rejection of the null hypothesis.

H0

Hypothesis: With the increase of the concentration of tea tree oil in the oil mixture there

would not be an effect of reduction of E. coli growth.  Re-stating the Hypothesis: With the increase of the concentration of tea tree oil in the oil mixture there is an effect of reduction of E. coli growth. In order to re-check the validity of the observation of the calculation an Excel Chart is created with the appropriate data used.

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Gra

.

Radius of no E. clo growth around the paper (cm)

Graphic representation of the avagage radius growth in different concentrations of Tea Tree Oil 0.6 0.5

0.5

0.48

75%

100%

0.4 0.32 0.3

0.26

0.2 0.12 0.1 0

0%

25%

50%

Concentration of Tea Tree Oil Solutions

Conclusion: Tea tree Oil, with its antibacterial properties is widely spread in different concentrations in the cosmetic industry and in the daily use of many people. This investigation aimed to explore whether there is a relationship between concentration of tea tree oil in a solution and the growth inhibition of the bacteria, with the initial prediction, stated as a null hypothesis, that there is no correlation between concentration and inhibition. However, as proved by the investigation, there is a very strong correlation between the concentration of Tea Three oil and the antibacterial properties of the mixture, as when tested onto the gram- negative bacteria E. coli it was observed that with the increase of the percentage of it, the radius around the filter paper was increasing, therefore killing and stopping the growth of the bacteria. When exploring the results of the investigation, it could be concluded that indeed this experiment is proving the null hypothesis, by obtaining results from the zone of inhibition of the bacteria, that indeed as starting with the 0%, where the effects were the least, to the biggest effect of 75%. However, when observing it could be clearly seen that despite the 75% being the highest, it is also with the

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highest standard deviation, meaning the values are differing the most from each other, showing a possibility of an error in this section of the experiment. When using the data and results obtained from this investigation it could be generally concluded that indeed, with highest percentage of Tea Three Oil, there is a lower inhibition growth of bacteria, proving the antibacterial properties of this essential oil. Evaluation: This investigation is strongly performed and therefore concluding on the base of E. coli- a gram negative bacteria. In such cells the Tea Tree Oil because of the specific structure of their membrane, has the ability to inhibit cellular respiration and increase membrane permeability therefore its lethal actions are a result of inhibition of membrane-located metabolic events and a loss of control over the cell’s movement of ions across the semi-permeable membrane of the cell, known as chemiosmotic movement. (Cox et al., 2001b) Therefore, if testing only on gram negative bacteria, where there is a different structure of the membrane allowing chemiosmotic movement of the Tea Tree Oil particles to the inside of the bacteria, it would be inefficient to draw a major conclusion that Tea Tree Oil has effective antibiotic properties for all bacteria, if not investigating the limitations of the research regarding gram negative bacteria. Type of error

Systematic Error

Table 7: Error representation and evaluation. Weakness Significance of the error performed

Instrument As

Imperfect

the

calibration when using performed

investigation on

is

five

originally

Possible improvement

Using

another

different measuring

type

instrument

the mechanic pipette for concentrations, equally away from each avoid

technic

the creation of different other, it might be the case due to the imperfections

in

concentration in solutions imperfect calibration, that two mixtures calibrations. therefore

the

mixtures are closer percentage based of the

11

th

themselves might slightly

solution compared to others.

differ form the original

Random Error

values presented. Delay in closing the Agar

Since E. coli could be very sensitive to Ensure proper preparati

Plates after contamination the outside environment and external of with E. coli, due to

instruments

an

germination, the openings of the Agar solutions beforehand, so

inserting Tea Tree Oil plates, once contaminated with E. coli, minimize the opening concentrations

paper should be minimum and only if

discs into the Agar plates.

each agar plate.

specifically required for the experiment, since it can affect the future growth of

Human Error

Probable

contamination

the bacteria and effect the experiment. Since while carrying out the experiment Creating a better organiz

of the Agar plates in the it was incubator.

done

with other students schedule

of

experiments and therefore kept in the incubator, same

incubator,

a

so

possible experiment

use that

ea

could

b

contamination with other factors might separately carried out, change the growth of the bacteria.

providing

multip

incubators capacity.

Bibliography:



Albrecht, J. (2015, August 13). Escherichia coli O157:H7 (E Coli). Retrieved from UNL Food website: https://food.unl.edu/escherichinia-coli-o157h7-e-coli

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for

mo



Carson, C. F., Hammer, K. A., & Riley, T. V. (2006). Melaleuca alternifolia (Tea Tree) Oil: a Review of Antimicrobial and Other Medicinal Properties. Clinical Microbiology Reviews, 19(1), 50–62. https://doi.org/10.1128/cmr.19.1.50-62.2006



Cox, S., Mann, C., Markham, J., Gustafson, J., Warmington, J., & Wyllie, S. (2001). Determining the Antimicrobial Actions of Tea Tree Oil. Molecules, 6(12), 87–91. https://doi.org/10.3390/60100087



E. Coli under the Microscope -Types, Techniques, Gram Stain, Hanging Drop Method. (n.d.). Retrieved February 18, 2021, from MicroscopeMaster website: https://www.microscopemaster.com/e-coli-under-mi...