Natural Products and their Antimicrobial Properties

2013-2016

Natural products and their antimicrobial properties Alexander Robinson – CREST Award submission

11AlRobi

[COMPANY NAME] [Company address]

Introduction to the Project Message to the reader, This project was first begun in April 2013 and through a number of setbacks within testing and periods of time where other commitments took priority, it has taken over three years and in excess of 100 hours to complete. It is the culmination of countless ideas and trials, in an attempt to find the best way to carry out my project.

This project has been organised into a somewhat chronological list, where new information is explained in the order it was discovered. By presenting my discoveries and understandings as a set of changing methods, I hope to demonstrate my attempt to find the perfect scientific method.

This project could never have been completed without the support and guidance of my CREST leader, Mrs Lebreuilly.

I hope that you find this project not only relevant but also interesting and accurate.

Alex

Table of Contents Project Context and Initial Research...............................................................................................................1 Project Planning ..............................................................................................................................................2 Research (continued) ......................................................................................................................................3 Selection of approach .....................................................................................................................................5 Aseptic Technique...........................................................................................................................................6 Risk Assessment ..............................................................................................................................................7 Method 1 ........................................................................................................................................................9 Development of Approach............................................................................................................................11 Optimal method........................................................................................................................................18 Final iteration of method selection. Method 8.........................................................................................18 Measuring Results.....................................................................................................................................22 My Results.....................................................................................................................................................23 Conclusion.....................................................................................................................................................27 Impact of my project on the wider world .....................................................................................................28 Evaluation .....................................................................................................................................................29 Personal Reflection .......................................................................................................................................30 Works Cited...................................................................................................................................................31 Acknowledgements ......................................................................................................................................31

Project Context and Initial Research For thousands of years, humans have had infections, illnesses and ailments – but no pills or pharmaceutical drugs to treat them with. Whilst death was far more common, some people were ‘cured’ after consuming potions of countless substances; Sage, willow, Mandrake and cinnamon could be mixed together to cure a far-fetched and exuberantly named disease - and in some cases it would have appeared to have worked. Whether or not placebos were involved or if it was simply a strike of luck, something must have taken effect for an illness to be cured.

Potions like these had been used to treat infection for vast periods of time. Looking at some treatments in the past we can cringe at the miserable understanding of the human body, but some of the treatments seem to have some logic behind them. Whilst modern medicine is deemed to be far superior to any of these natural products, perhaps some are still extremely useful.

For years, natural products have been used as remedies for ailments of all kinds – and whilst many of these remedies have been proven to be useless, there must be some substance behind their claims or there would be no reason to believe in them. After scientists discovered an ancient recipe known as “Bald’s eyesalve” – A nineteenth century anglo-saxon remedy made from a whole host of ingredients including “onion, garlic and part of a cows stomach” – which has successfully treated MRSA, what reason would there be to believe that other herbal and ‘spiritual’ remedies used for centuries by traditional people wouldn’t bring about the same effect, (Sugg, 2015) (McKenna, 2015).

If many diseases, infections and illnesses are rumoured to be treatable through herbal medicine, and a selection of these diseases are caused by bacteria, would common household herbs and spices be able to achieve the same effect and have visible effect on inhibiting the growth of or killing certain bacteria?

Natural products that we know have medicinal effects are plants such as Aloe Vera and tea tree oil, both of which are used and prescribed to many people to treat illnesses. But what about other plant products such as spices and herbs usually used in cooking. Many cultures incorporate herbs and spices into their cuisine as a daily essential. Is it just the taste of these plant materials that make them so attractive, or do they have hidden properties which could explain why some are more prone to disease than others?

When Fleming first discovered Penicillin in 1928 he revolutionised modern medicine and led to millions of lives being saved through the avoidance and resistance of preventable diseases.

Antibiotics were seen by manufactures as a ‘miracle drug’ back in the 1940’s and were prescribed far too liberally for the effect to be sustained for many centuries to come. Through Darwin's Theory of Evolution

by Natural Selection, the antibiotic acts as a selection pressure and forces the few bacteria which are resistant to select antibiotics to multiply whilst killing off the ones non-resistant. This has resulted into us entering into a period of time in which every antibiotic known to medical science cannot stop the toughest bacteria. Therefore it is essential that we develop new possibilities to treat infection to make sure we don’t end up in a world where people are dying from the common cold. Finding natural products with antibacterial or antimicrobial properties has the potential to change the way we fight infection and hopefully provide doctors with a new way to cure patients.

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Project Planning Hypothesis: Natural Products such as many herbs and spices are commonly thought to be able to cure minor illnesses.

As many of these ailments are caused by pathogenic microbes or bacteria, it is not unreasonable to suggest that some will have antimicrobial or antibiotic properties. I think that natural products will cause an inhibition of growth in some microorganisms.

Aims: In this project I aim to test the antibacterial and antimicrobial properties of natural products, specifically herbs, spices or other natural items.

I aim to observe how certain bacteria are prevented from growing and measure this inhibition to plot data onto a graph. After testing how successful the plant materials are at preventing or inhibiting the growth of bacteria, I aim to find the most effective natural antibacterial /microbial substance.

Objectives: -

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Test different herbs or spices to see how well they work against one type of bacteria.

See how efficient the herbs and spices are compared to two different bacteria, whether one is more inhibited than others.

Come to a conclusion in which I can identify whether a natural product has or does not have an impact on surrounding bacteria.

Research (continued) Through research using the internet I have developed a list of possible natural products to test against different bacteria.

Many vegetables and spices have antibiotic properties that can be used and utilised to help us fight bacterial infections naturally. “Some of these food items are vegetables like the cabbage and carrot, fruits like lemons and limes, spices like clove, cumin seeds, garlic, ginger, onion and turmeric, and honey and yoghurt”. (Livestrong) Tea tree oil and Aloe Vera are two examples of where natural products are used in their pure form to help cure minor ailments; regularly prescribed by doctors they reminded me that just because something is not synthetically produced does not mean it is any less effective at treating us.

An enormous range of the fresh produce we eat helps to fight bacteria (Suite101), Spices and herbs such as Clove, Cinnamon, Cumin, Ginger, Garlic and Turmeric all help our body to resist harmful bacteria. It is the concept of eating a balanced diet that really helps us thrive.

Many of our traditional herbs such as Mint, Basil and Thyme have amazing health benefits and many have been shown to have Antibacterial properties. I researched into the properties of Basil, and found that it had been “shown to provide protection against unwanted bacterial growth” (The George Mateljan Foundation). The essential oils within its leaves help to combat bacterial growth and so it may prove invaluable within my project – as it is evidence of this inhibition of growth which I am trying to prove.

It is not a new idea that traditional herbs and spices have antimicrobial properties, a scientific article titled “Plant Products as Antimicrobial Agents” (Cowan, 1999), noted different antimicrobial compounds found in plants and suggested how they might be used to help treat bacterial infections.

I have decided that the following plant substances might be useful in my research: Dried Cinnamon Garlic Turmeric Clove Cumin

Fresh Basil Mint Lavender Coriander Parsley Thyme Ginger I intend to use dried substances over the fresh variety as these seem to be far easier to use and manage, and I can accurately control the source and variety of each plant material.

Choice of Bacteria At my school, I had the choice of a number different bacteria: Staphylococcus albus Micrococcus luteus Bacillus subtilis Pseudomonas fluorescens Escherichia coli

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After researching into the properties and features of different bacteria, I decided that I would use two in my experiment, Escherichia coli and Staphylococcus albus. I have chosen to use these two because they are similar strains to bacteria which cause sickness among humans. By choosing strains of bacteria which are similar to those which can cause illness, I will be able to observe the effects of natural products, without the risks associated with dangerous bacteria.

Escherichia coli is a gram-negative bacteria and Staphylococcus albus is a gram-positive bacteria. ‘Gram’ refers to a Gram stain test which denotes whether or not bacteria have a thick peptidoglycan layer within its cell wall.

This layer retains a stain placed onto the bacteria, and so can help us to classify differences between bacteria and show how they may differ. Bacteria which respond to the Gram stain in particular ways tend to have similar properties to each other. As the two bacteria I have chosen have different Gram stain results, it could be thought that they may react differently to each sample and may show me different, important results.

E. coli is most often known for causing food poisoning in humans and some strains are considered to be rather dangerous, especially to the risk of consumers from poorly prepared food. It is one of the most commonly known bacteria, so I thought it was fitting to use it within my experiment as the results could give a real-life indication of whether or not the herbs or spices could be used to fight common disease.

Staphylococcus albus is commonly found on the human skin and is usually completely harmless, yet patients in hospital with compromised immune systems are at risk of developing an infection.

“Staphylococci cause abscesses, boils, and other infections of the skin, such as impetigo… The most common form of food poisoning is brought on by staphylococcus-contaminated food,” (The Columbia Encyclopedia, 6th ed., 2015). I chose to use this bacteria because I felt that it was a good contrast to the E. coli. It was gram-positive as opposed to gram-negative and it was commonly found on the human body, whereas E. coli is not. Both bacteria can cause infection due to food related processes, such as improper hygiene, so this meant that both can easily get into food.

Growing bacteria.

The bacteria that I’ve selected will be grown in a nutrient broth by my school, but I will need to find a way to culture them for my own experiments.

I need to source a growth medium, a material which will allow the bacteria to grow within or atop, whilst providing them with nutrients to allow them to thrive. The best medium to use is Agar jelly. Whilst there are many different types, for safety reasons I will use “Nutrient agar”. This contains various different substances all intended to promote life, such as beef/yeast extract for nutrients, sodium chloride to minimise diffusion of compounds out of the bacteria and also agar for the main solidity and texture.

(Wikipeida, 2015) I began to wonder if certain cuisines contained particular herbs or spices which gave their consumers an advantage over other populations. If so, could these substances be included into meals to help us live longer and live better. Hopefully my project will be able to point us into the right direction.

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Selection of approach Project Strategy During the early planning stages, I became aware that this project was going to be slightly more complicated than I had first thought. It was therefore important that I set about creating a plan which I could follow to achieve my end goal.

I understood that I would need to complete a few preliminary tests before I could begin my proper experiments, so I decided that I would begin creating methods for the experiment, test it and see how effective it was and where I needed to change concepts and procedures.

Producing results I realised that there were two possible ways of carrying out the project. The first was to spread the bacteria and test material equally over each agar plate and count the number or individual bacterial colonies by eye, dividing up the plate by an arbitrary number and counting colonies within each area, to see how many bacteria were permitted to grow.

Alternatively, I could spread the bacteria evenly throughout the plate and apply the test material to a specific location. This would allow me to measure the area surrounding the material where inhibition of the bacteria had occurred. The former would be difficult to measure effectively, for if the bacteria was spread improperly in areas or the application of the natural product was imperfect then certain parts of each plate could look drastically different to others- making it difficult to come to any notable conclusion.

The latter seems to be a better way of tackling the issue as I could ensure that both the bacteria and the test material were placed in specific locations and measuring would be able to take place by eye, using a simple ruler – much more easily than counting individual colonies.

I decided to use the latter method as I felt it was far more accurate and would reduce the chances of human error.

Suitable ranges of the data set When I came to collecting data, I needed to have a specific resolution to which I was measuring. As I was using a ruler graduated in millimetres, I decided that 1 millimetre would be the resolution of my experiment. This made good sense as less than a millimetre is difficult to measure by eye and larger than a millimetre would make the results of the tests virtually indistinguishable.

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Aseptic Technique Aseptic Technique is the means through which we ensure that the samples we are producing are uncontaminated by unwanted microbes or any substance which could affect our results.

As my project involves the study of microorganisms, it is imperative that I understand the importance of good technique as it can seriously affect the validity of a scientific study. Agar plates are infected and contaminated incredibly easily, so this meant that I will have to try hard to keep my plates clean.

This will be achieved through… 

Minimising the agar jelly’s exposure to the air. o When the lid of each Petri dish is lifted off to either add bacteria, a sample, or to pour agar, it will be held beneath a flame (a Bunsen burner lit to a moderate temperature).

This will prevent airborne particles from falling into the dish. As the flame heats the surrounding air, it creates an area of less dense air which rises, preventing it from falling onto the plate. o The lid of the dish will only be removed from the plate for very limited times, and when it is removed it will be held no more than a few centimetres above the plate to prevent contamination.

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Using sterile equipment o For each time I use a certain bacteria, I will make sure to use a new sterilised pipette to avoid any risk of contamination from previous bacterial cultures. o All non-disposable equipment will be sterilised within an autoclave machine before use.

This machine reaches high temperatures where almost all conventional bacteria cannot withstand the heat. o Any equipment which I need to use multiple times during an experiment will be dipped into a high concentration of ethanol, left to air dry for a moment then passed through a hot flame.

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Ensuring surfaces are wiped down Washing hands and avoiding any face contact during preparation of plates as bacteria from the skin could be placed onto the plate and allowed to grow Keeping windows and doors closed to prevent airflow from outside which could allow pollen and other particles to contaminate the experiment.

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Learning to keep all sterile equipment from touching the table if it is still to be used. For instance, stopping bacterial contamination by preventing each test tube from touching the table.

Risk Assessment

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Description/ details of steps in activity

Hazards

Possible accident / ill health

Usage of glassware during experiment

Broken glassware

Use of bacterial cultures during experiments

Potentially dangerous bacteria could contaminate areas

Fetching additional resources to proceed to the next step of the experiment

Trip hazards such as chairs or bags.

Holding items in arms may make it difficult to see the floor

Injury sustained from falling over a chair and bruising a body part.

A Bunsen burner is used to sterilise equipment and prevent particulates from falling onto the agar plates.

The flame becomes very hot and can easily set things alight

1) Clothes become inflamed and are set alight.

2) Residual alcohol on the sterilised item catches alight and spreads fire across workbench.

3) Particulates could enter the flame and fly off into eyes or onto clothing

Injury sustained from contact with broken glassware involving skin or possibly eyes.

Bacteria could develop into a dangerous strain and become pathogenic, potentially causing illness if handler became infected.

Severity (1-5)

Likelihood

(1-5)

Risk level

Action to take in case of accident

Handle all glassware with care and set it carefully onto any surfaces. Ensure that goggles are worn.

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Avoid all contact with the bacteria as much as possible, avoid cross-contamination.

Ensure all utensils are disposed of or sterilised immediately after use. Ensure that any wounds or cuts are plastered and sealed, to minimise chance of bacterial entry to bloodstream.

Ensure that all chairs are tucked underneath desks and all bags are left at the front or outside the room. Place all items needed for the experiment in one single area to minimise movement throughout the room.

Non-flammable lab coats must be worn to protect from fire.

Goggles worn at all times to stop flying particulates from causing damage.

Ensure utensils are completely dry before sterilising, and ensure that ethanol is kept far away from the flame.

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Remove glass from the site of wound (as long as safe to do so), disinfect and bandage.

Seek medical advice immediately.

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Use emergency first aid and tent to the person.

Clear up any broken glassware and remove any equipment from the floor.

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Fire: Reach for a Dry powder fire extinguisher (or at the very least a Foam extinguisher [Class A&B]) to extinguish flames if set alight.

Eyes: Wash with eyewash and contact professional help if pain persists.

Existing risk control (Mitigation)

The risk assessment constructed on the previous page uses the following chart to evaluate the severity and relative probability of an accident taking place. From this we can see how dangerous the situation is and whether or not it is safe to proceed with the intended plan.

TABLE 1 - EVALUATING THE RISKS OF THE EXPERIMENT For my experiment, I needed to develop a range of Safety Procedures, which I have listed below.

Using a Bunsen burner Using a Bunsen burner is very important to maximise the cleanliness and sterile environment in which we work, but they can be very dangerous if not handled properly.

When using the Bunsen burner, one must exercise a great deal of care to remove any chance of accidental burns or causing damage to any of the equipment. When not in use, the flame must be changed to a yellow ‘safety flame’ to minimise risk.

When sterilising equipment, allow ethanol to dry after dipping tools, to prevent it from being set aflame.

Heating tools is important to kill bacteria, but it is equally important to remember that tools become very hot and can cause injury if left in the flame too long as they can burn skin.

Goggles and Lab coats Goggles protect the eyes from a number of possible hazards. From chemicals such as ethanol, to the flame from the Bunsen burner, the goggles provide a layer of protection, preventing the loss of eyesight.

Lab coats also provide a layer of protection as they prevent spillages of bacteria onto clothes, which could lead to them growing and developing. Instead, the lab coat can be removed and washed. It also protects from the risk of fire from the Bunsen as it can be instantly removed before allowing any other clothes to set alight.

Specifics regarding agar plates The incubator within the laboratory is always set to a maximum of 30°C, so as to minimise the risk of developing potential pathogens. This temperature is useful to the bacteria we wish to incubate but does not encourage potentially dangerous microbes as much as 37°C would.

Plates are to be sealed in four places using sellotape. Care must be taken to ensure that the Petri dish is not sealed shut as this could encourage the growth of harmful anaerobic bacteria, which we are not looking to produce.

By following these procedures, I hope to make sure that my experiment is as successful as it can be – and minimises errors.

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Method 1 My first experiment consists of using samples of dried spices purchased from a supermarket as I believe that these will have the greatest effect on my bacteria. I think that as they are dried, their oils and chemicals could be intensified.

The samples I used were: Clove, Cinnamon, Cumin, Ginger, Garlic and Turmeric.

Apparatus and Materials Item

Number required

Needed …

Pre-poured Agar plates Sterile pipettes Escherichia coli culture Staphylococcus albus culture Sterile pestle and mortars Jar containing disinfectant Tweezers Small pot of ethanol Samples of dried Clove, Cinnamon, Cumin, Ginger, Garlic and Turmeric Sterile 20ml measuring cylinders Sterile Water Weighing boats Pot of paper disks A balance Sellotape Bunsen burner Heatproof mat Matches Safety goggles Labcoat Black marker pen

22 2

20 to culture bacteria within, 2 for the control tests

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To grind up the fresh plant substances.

For used pipettes and other equipment To transfer paper disks to the plate For sterilisation before being flamed

2 1 1g 1

For use as solvent To weigh out the dried substances 1 1 1

To accurately measure the amount of solute.

To partially seal the plates for incubation

To protect eyes from any harmful chemicals or flame.

To ensure safety around the flame and chemicals.

To label the plates

Variables Independent Variable: The solute used: Clove, Cinnamon, Cumin, Ginger, Garlic or Turmeric.

Dependant Variable: The amount of inhibition that the bacterial growth has experienced around the circumference of the bored hole.

Controlled Variables: 

The ratio of herb/spice solute to sterile water.

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Application of the solution to the Petri dish: o Method of Application (Use of paper disk) o The volume of plant solution added to the quantity of agar jelly.

Application of the bacteria to the Petri dish: o Method of application (Spreading) o The volume of bacteria to use in each application.



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Methodology 1. Sterilise all surfaces using a disinfectant spray, ensure lab coats and goggles are worn. Wash hands thoroughly and avoid hand-to-face contact.

2. Set up a Bunsen burner on the desk, with a heatproof mat beneath it. Light the flame and set to safety until it is ready to be used.

3. Decide what plant solution will be used for this experiment, repeat the experiment for all 6 solutes.

4. Retrieve the pre-filled petri dishes from the box and name them according to the bacteria and solute they are going to contain. Leave a control plate for the bacteria and a plate for the plant solute.

5. With the bacterial culture in one hand, lift the lid off and insert a sterile pipette. Place a single drop onto each plate (except the solute controls) labelled with that bacteria and then apply evenly using a glass spreader. Between each spread of the plate, sterilise the spreader to eliminate the risk of cross-contamination.

6. Take 1g of plant substance and place into a pestle and mortar.

7. Add 5ml of sterile water to the substance and grind together to form a thin paste.

8. Using a pair of sterile tweezers, take a paper disk and dip into the solution of solute and water, before allowing to dry for a brief moment, then placing atop an agar plate, directly in the centre.

9. Repeat this for all of the plates (except the control, which should have a paper disk dipped in the sterile water). Ensure that the tweezers are sterilised after each step.

10. Place any dirty equipment into the disinfectant jar.

11. Take four pieces of sellotape and tape down 4 edges of the plate, so as to stop the lid from being lifted off. Ensure that the sellotape does not cut off the oxygen supply as the bacteria are aerobic and need oxygen to survive.

12. Leave the bacteria for between 3 and 4 days to divide and multiply.

13. Place a thick sheet of black cartridge paper beneath the plates, to make the contrast between bacteria and agar easy to see.

Evaluation of the method: When producing solutions of the plant materials, I first attempted to use only water, yet I had problems with getting the dried solute to dissolve properly, namely cinnamon and garlic powder. The method was written rather vaguely written, leaving out key details such as the exact number of plates needed, or exactly what needed to be written on them and this led to time delays and some slight issues.

At this early stage of the project, I was only testing one substance per session. This meant that I could not be sure of the consistency of the bacterial samples I was receiving and was unaware if they were always from the same strain on each occasion, as this responsibility was left with the laboratory technicians. This resulted in different experiments which were very difficult to compare and overall this made the tests invalid.

To develop this experiment, I have to:

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Find a way of getting the solute to properly dissolve into solution.

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Test all substances in one sitting, to give a set of results which are directly comparable

Development of Approach After completing my experiment for the first time, I realised that there were going to have to be many, many changes and tweaks made for it to be as efficient as possible. From my first idea of how to complete the project, to my optimal solution in completing it; I went through a long development process in order to find the most effective method which produced visible and substantial results.

I identified 3 areas within which the method which could be altered. These variables can be broken down into the following areas: 1. The solution of plant solute and solvent a. The quantity of plant solute b. Volume of Solvent c. Concentration of water and any other additional chemicals, 2. The application of the plant solution to the petri dish a. Using a paper disk or another method b. Volume of the plant solution to use in each application 3. The application of the bacteria to the petri dish. a. The method of application (Spreading or Inoculation) b. Volume of bacteria to be used in each application It is important that I address each subsection of each area as even a tiny change in the method that I take could make results look wildly different. My aim is to find the optimal value for each so that the method I chose works as effectively as possible, showing a clear result. These were variables which needed to be controlled but I did not yet know in which way I wanted to control them. Over the following pages, I have documented my journey to find the optimal method in which I believe each criterion is met correctly.

Controlling Variables.

Throughout the experiment, I knew that it would be important to understand to the fullest extent what effect the samples were having on the microorganisms. By creating a sample which used only the solvent and bacteria (not the plant solute) I was able to test the degree to which the solvent was having an effect on the bacteria.

Variables which I had no control over: Type and strain of chosen bacteria, I was unable to ensure that it was the exact same for every test that I undertook as this job was left with the technicians to sort out. I wanted to control the strength/intensity of the plant solutes, but I found no definitive way in which to do this. I considered the use of a colorimeter to measure intensity, but eventually found this unsuccessful.

N.B. I would consider methods 1 to 7 as being “Preliminary tests�. Each one provided me with a great deal of information and helped to form constructive observations about the way that each experiment was carried out, but their results have not contributed to the final readings which are displayed at the end of this project. This ensures that all variables are appropriately controlled and minimises the room for error in my experiments.

Figure 1 shows a basic overview of each experiment. Each is explained more over the next few pages but does not go into a high level of detail to avoid unnecessary information.

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FIGURE 1 - AN OVERVIEW OF THE APPROACH TAKEN TO FIND THE OPTIMAL METHOD

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•Solution: (Dried natural product) and water •Application of solution: Paper disk •Application of Bacteria: Spread over the surface of sterile agar growth medium

•Solution: (Dried natural product), ethanol and water (1:9) •Application of solution: Paper disk •Application of Bacteria: Spread over the surface of sterile agar growth medium

•Solution: (Dried natural product), ethanol and water (1:19) •Application of solution: Paper disk •Application of Bacteria: Spread over the surface of sterile agar growth medium

•Solution: (Dried natural product), ethanol and water (1:39) •Application of solution: Paper disk •Application of Bacteria: Spread over the surface of the sterile agar growth medium

•Solution: (Dried natural product), ethanol and water (1:19) •Application of solution: Paper disk •Application of Bacteria: Innoculated within the agar growth medium

•Solution: (Dried / Fresh natural product), ethanol and water (1:19) •Application of solution: 1ml of solution poured into the centre of a hole bored into the agar •Application of Bacteria: Innoculated within the agar growth medium

•Solution: (Fresh natural product), ethanol and water (1:19) •Application of solution: 1ml of solution poured into the centre of a hole bored into the agar •Application of Bacteria: Spread atop the agar growth medium

•Solution: (Fresh natural product), ethanol and water (1:19) •Application of solution: Paper disk •Application of Bacteria: Innoculated within the agar growth medium

Method 2 After finding it difficult to get the dried solute to dissolve, I decided to create a solvent using some ethanol. I was aware that ethanol is very useful in dissolving substances, so I believed that adding a small amount might enable the solute to dissolve.

From previous knowledge, I already knew that ethanol was a powerful antimicrobial agent, so if I were to use it as a solvent I would have to heavily dilute it. This presented a challenge to me as I needed to find the perfect balance between having no effect on the antimicrobial properties of the solute (And thereby not altering the results), and also enabling the solute to dissolve. I had to use a control plate to test if the alcohol solution had any effect on the bacteria, in the case of which I would have to adjust my results to fit.

I came to the decision to use a 10% solution of Ethanol. This comprised of 9 parts water and 1 part Ethanol. This was easy to produce as I used a measuring cylinder to do so.

I also developed from my first method by spending a longer period of time within the laboratory for each experiment and tested every solute right after one another.

Evaluation of this method: The decision to test each solute in one sitting was a good one. It allowed me to ensure that certain variables were controlled, such as the precise strain of each bacteria and the amount of bacteria within the nutrient broth. The only negative I encountered was that I had only requested a single sterilised pestle and mortar, which may have meant that there was some cross-contamination in the preparation of individual samples.

The 10% solution of ethanol was very effective in working as a solvent, as there was noticeable distribution of the solute across the agar jelly. There was a slight zone of inhibition surrounding a few of the samples, but a smaller zone was also visible on the control plate – therefore the ethanol was interfering with the readings.

To develop this experiment, I have to:  

Attempt to find a better concentration of ethanol-water solution, to minimise the impact that the ethanol has on the zone of inhibition.

Request 6 pestle and mortars to be autoclaved – every single piece of equipment I used has to be completely sterile to avoid any chance of cross contamination.

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Method 3 This method was very similar to method 2, but two adaptations.

Firstly, the rather excessive 10% alcohol solution was lowered to a 5% ethanol solution comprised of 19ml of sterile water and 1ml of M ethanol. This was made to reduce the risk of the ethanol affecting the results and altering the data The second adaptation was to use separate pestle and mortars for every solute

Evaluation of this method: This method was a massive improvement on the previous.

The zone of inhibition on the control was far smaller, barely 1mm wide, which meant that there was far less interference with the actual results.

The use of separate pestle and mortars was an improvement because it removed any chance of crosscontamination. Again, only a few plates showed a noticeable zone of inhibition, which was rather frustrating as it meant I was unable to draw any conclusions.

To develop this experiment, I have to: 

Consider altering the ethanol solution again, in case it can be reduced further.



Change an essential part of the method to produce a positive result

Method 4 Due to the success of my last test, I decided to try a lower percentage of alcohol. This was a 2.5% solution with the other 97.5% being distilled water. This would follow all of the other procedures I had used previously.

My aim in this test was to discover if the solution would allow the solute to dissolve just as easily into solution but with a lower alcohol content. As the alcohol itself affects microbial growth, I wanted to minimise the effect as much as possible, to maximise the visibility of the natural products I was testing.

Evaluation of this method: This turned out to largely be a failure. Whilst my aim had been for the products to create a solution just as they had before, this was clearly not the case.

I had used cinnamon in the first test of this method and it refused to dissolve in the same quantities as it had with the slightly higher 5% solution. After testing this I decided to remain with the 5% solution as although it has an effect on the final result, it allows the solute to dissolve, which is the only way that I had found to test each of the products.

To develop this experiment, I have to:

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Return to the 1:19 ethanol: water solution

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Change an essential part of the method to produce a positive result

Method 5 As Method 4 was no better than 3, my approach needs to change in order to continue progressing. I returned back to the 5% ethanol solution because I knew that this worked.

During the planning stages, I realised that there were two methods of bacterial application: Spreading and inoculation. This was the first instance where I inoculated the bacteria within the growth medium and so the process was, at first, a little difficult to perfect.

To inoculate the bacteria within the agar I first took liquid agar from a water bath at 90°C and left it to cool on the side atop a heatproof mat. Then, I measured out 6ml of bacterial broth into 200ml of molten agar and poured this into empty agar plates. After waiting for the agar to set, I applied the paper disk as previous.

Inoculation is a great method of bacterial application because it distributes the bacteria evenly throughout the agar, as opposed to a thin film upon the top.

Evaluation of this method: Staph. A: Not grown or inconsistent.

I believe that when pouring the bacteria into the agar, I had not yet allowed it to cool sufficiently. This led to a majority of the bacteria dying due to the high temperatures which caused me to be unable to see any relevant results.

E. coli: No effect was seen on inhibition of growth.

There was almost a promotion of growth, which led to me feeling very confused about the situation. As this test had been such a failure, I decided that I would try a different a approach. It was due to the excess amount of bacteria that I had added that the E. coli bacteria had performed so well, this was clearly far too much so in my next test I will lower the amount drastically.

Inoculating the bacteria within the agar created many new problems and issues that I needed to perfect, yet the potential for use in my experiment is so promising that I will try to work through these issues.

Another item to note is that some solute refused to diffuse into the agar, and this led to it having no effect at all. Without it diffusing properly, it cannot work.

To develop this experiment, I have to: 

Adjust the bacteria to agar ratio to a suitable level



Find a suitable temperature at which I can add the bacteria to the agar



Consider how I can get the solute to diffuse better into the agar

15

Method 6 With the issues found previously, I decide to change my approach yet again.

As I had identified problems previously about the solute not properly diffusing into the growth medium, I decided to apply much more of the solution by the means of a well. Instead of using a paper disk, I bored a hole into the agar plate and dropped the solute into it, using a sterile pipette. I believed that this might allow more solute the chance to diffuse across into the agar.

In the inoculated agar, I reduced the quantity of bacteria to 1ml within 300ml (1:300) rather than the (1:33) that it was previously.

Evaluation of this method: This amount of bacteria was a far better choice. The bacteria seemed to grow naturally in both the E. coli and Staph. albus cultures.

Despite the bacteria growing well I still had issues with the solute. The solute seemed to be staying in the well and not diffusing throughout the agar plate. This meant that either the solute was never really in solution, only suspended within the fluid or that the water was evaporating far too quickly. This was even worse than the paper disk.

FIGURE 2 - ONE OF THE BACTERIAL TEST PLATES

At this point in the project, I realised that my current approach wasn’t working. What I needed was a drastic change to my original idea.

During the project, I had wondered at multiple times if the issue was not with my execution, but instead the samples with which I was working. During my research (page 3), I identified two different forms of sample I could use. The dried variety, purchased in packaged containers from a shop, or fresh herbs, which I would pick myself and source locally. Up until this point I had chosen dried herbs for ease of use, yet I wondered if whether switching to fresh herbs would change my project and produce some results which were clear and had a meaning. With this in mind, I decided to change my focus away from dried products to fresh herbs instead, with the hope of seeing a noticeable change.

Method 6.1 Now that I had decided to use natural products, I had to change the ratio of solvent to solute as the plants had a far higher water percentage and were therefore less concentrated.

I collected 5g of each fresh herb from the school gardens and potted plants from supermarkets, then chose 1g when in the laboratory. By eye, around 80% leaf or flower and 20% stem. The herbs used were: Basil, Thyme, Parsley, Mint and Lavender. To the 1g I added 4ml of solution.

Evaluation of this method: Largely a success, the plant products that managed to diffuse into the agar showed distinct zones of inhibition, but on some tests the result was not very clear, as once again there were issues with the diffusion of the solute – although thankfully this was far less than previously. Due to the small successes, I will proceed through my project using fresh instead of dried herbs from this point.

16

Method 7 For this method, I decided to try something a little different.

The concept of the well in the centre of the petri dish really resonated within me as it seemed to be the right way to move forward, yet I was still having a few issues when it came to the solute diffusing into the agar efficiently.

As the solution was placed into the bored hole, the water would evaporate within the incubator before the solute had diffused across. My solution to this problem was to place the solute into the bored hole already suspended within another piece of agar jelly. This would mean that the sample would not dry out as it would have an almost identical osmotic potential as the surrounding agar. As there would be no reason for the agar to dehydrate, the solute would find it far easier to diffuse across as it is.

One issue with this method was that the hole borers provided by the school were different sizes to each other, one creating an 8mm hole and the other creating a 7mm hole, but this was soon fixed by simply requesting more of the same size.

To do this required extra equipment: 5 extra petri dishes for producing the samples of each substance; 2 sterile Tweezers to remove the substance and place into the bored hole and 5 conical flasks, within which to mix agar and plant substances.

When it came to measuring my results, I also introduced a lightbox in this method instead of black cartridge paper underneath the samples. This gave a much better contrast between the areas of bacterial growth and the zone of inhibition.

Evaluation of Method: It added another layer of difficulty and complication to my project, which arguably wasn’t needed. I needed to ensure consistency across each test and this proved difficult, attempting to ensure the same amount of solute was poured to the same height which was bored with the same size hole. Eventually I came to realise that whilst the idea was sound, the additional work required complicated matters too much.

To develop this experiment, I have to: 

Return to the use of paper disks



Ensure each step of the method is clear to follow and precise – including the measurement stage

FIGURE 3 - USING A LIGHTBOX FOR CLEAR VIEWING OF THE SAMPLES 17

Optimal method Final iteration of method selection. Method 8 The production of this method was due to the culmination of all the information I had gained in the past experiments. As far as I am aware, this is the most efficient and effective way to test my hypothesis.

Apparatus and Materials Item

Number required

Needed ‌

Sterile Petri dishes Large containers of liquid Agar nutrient jelly Large conical flasks Escherichia coli culture Staphylococcus albus culture Sterile pestle and mortars Vials/Small jars.

Small Bottle (~50ml) Jar containing disinfectant Tweezers Small pot of ethanol Samples of fresh Basil, Thyme, Parsley, Mint and Lavender.

Sterile 20ml measuring cylinders Sterile Water Weighing boats Ethanol Pot of paper disks A balance Sellotape Bunsen burner Heatproof mat Matches Safety goggles Labcoat Black marker pen Sterile pipettes

22 2 2

20 to culture bacteria within, 2 for the control tests To pour into Petri dishes To mix the agar and bacteria in

5 5 1

To grind up the fresh plant substances.

To temporarily store plant substances within For storing the solvent in For used pipettes and other equipment To transfer paper disks to the plate For sterilisation before being flamed

3 5g each 2 38ml 2ml 1 1 1 >15

For use as solvent To weigh out the plant substances For use as solvent To accurately measure the amount of solute.

To partially seal the plates for incubation

To protect eyes from any harmful chemicals or flame.

To ensure safety around the flame and chemicals.

To label the plates

Variables Independent Variable: The plant material, i.e. Basil, Thyme, Parsley, Mint or Lavender.

Dependant Variable: The radius of the zone of inhibition that the bacterial growth has experienced around the circumference of the bored hole.

Controlled Variables: 

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Solution of plant solute and solvent: o The quantity of the solute (plant material) o The volume of the solvent o The concentration of the water and ethanol that make up the solvent.



Application of the solution to the Petri dish: o Method of Application o The volume of plant solution added to the quantity of agar jelly.



Application of the bacteria to the Petri dish: o Method of application o The volume of bacteria to use in each application.





Temperature… o …of incubator o …of agar when bacteria is added Type of agar nutrient jelly



The strains of the two types of bacteria

Method Preparation 1. Sterilise all surfaces using a disinfectant spray, ensure lab coats and goggles are worn. Wash hands thoroughly and avoid hand-to-face contact.

2. Set up a Bunsen burner on the desk, with a heatproof mat beneath it. Light the flame and set to safety until it is ready to be used.

Preparing Petri dishes 3. Remove the molten agar from the water baths and lave to sit on a heatproof mat to cool until comfortable to touch, but not yet beginning to solidify.

4. Meanwhile, line up 22 petri dishes on the counter and label 11 of them with “E. coli” and the other 11 with “Staph. A”. Then, label two of each group with “Basil”, “Thyme”, Parsley”, “Mint” and “Lavender” so that each dish is ready for pouring. Label the remaining two dishes as “Control”. Ensure that the labels are written on the bases of the dishes as the lids can easily become misplaced and this would cause the results to become invalid. Separate the dishes into two separate locations on the workbench. One for E. coli and one for Staph. A. 5. Once the agar has cooled sufficiently so as not to kill the bacteria, pour 300ml of agar into a sterile conical flask and add to it 1ml of the E. coli culture. Using the Aseptic technique researched earlier, ensure the mouth of the flask is shielded from the air above at all times. Label this using a marker pen as E. coli on the side of the flask, covering the flask with a lid once all bacteria has been added.

6. Repeat step 6 for Staph. albus.

7. Take the flask of bacteria and agar and bring it to the workbench by the Bunsen burner – which you will now need to switch to a hotter, blue flame. Placing the empty petri dishes onto heatproof mats for easy transport, begin to pour the E. coli – agar mixture evenly into the 11 dishes labelled “E. coli”. Be sure not to overfill and try to allow a decent depth of agar, maintaining consistency as far as possible.

8. Repeat step 7 for the Staph. albus – agar mixture before allowing the plates to cool and set.

Preparing the plant solution 9. Whilst waiting for the plates to cool, begin to prepare the plant solution. First, make the solvent by using a measuring cylinder to pour 38ml of sterile water into a small bottle. Then use the same equipment to pour 2ml of Ethanol into the bottle. We will use this when making our solutions.

19

10. Remove the basil from any packaging surrounding it and place onto a clean sheet of plastic towel. Remove the leaves from their stalks and place onto a weighing boat atop a balance. Place around 80% leaves and 20% stalk/stems onto the balance until the value reads or rounds to 1.0g.

Please try to avoid using hands to touch the leaves as this could infect them with bacteria.

Instead, use tweezers or wear a pair of clean sterile gloves if needed.

11. Ground up the basil leaves and stalks within a sterile pestle and mortar, gradually adding the solution created a drop at a time until it is fully ground and 8ml of solution have been added.

12. At this point, transfer into a glass vial, label the vial with a marker pen as “Basil� and set aside to allow chemicals within the basil to diffuse through into the solution.

13. Repeat steps 10 to 12 using all 5 plant substances.

Placing the sample onto the plate 14. After the plates have fully set, place a set of 11 of them close to you at your workbench.

15. Take a sterile set of tweezers and remove a single paper disk from the pot.

16. Dip the paper disk into the basil solution, before lightly tapping the tweezers on the side of the jar to remove any large clumps of solute. Place in a position of one half of the basil-E.coli plate, giving it a firm push to settle it into position.

When positioning onto the plate, do it beneath a hot Bunsen burner, whilst the lid of the plate is used to cover it partially.

17. Ideally, use another pair of sterilised tweezers for the next step, but instead of this, ensure to resterilise the tweezers by dipping into alcohol then running through a flame. Place the second paper disk onto the plate and again sterilise. It is important to sterilise between every step because any transfer of bacteria into the basil solution could contaminate the entire basil test for both bacteria.

18. Repeat steps 15 to 17 for all 5 plant products for E. coli.

19. Repeat steps 15 to 18 for Staph. albus instead of E. coli, using a different pair of tweezers to remove any chance of cross-contamination of bacteria.

20. For the control samples, simply dip the paper disk into the solvent without using any solute and place firmly onto the control plates.

21. Place any dirty equipment into the disinfectant jar.

FIGURE 4 - PREPARING THE SOLUTIONS OF PLANT MATERIAL 20

Sealing the samples 22. Take four pieces of sellotape and tape down 4 edges of the plate, so as to stop the lid from being lifted off. Ensure that the sellotape does not cut off the oxygen supply as the bacteria are aerobic and need oxygen to survive.

23. Place the plates into the incubator inverted (upside down) carefully so as not to allow the paper disks to move about. The plates are inverted to stop any condensation that has developed on the lid falling onto the plate and interfering with the developing microbes.

24. Clear away everything. Anything that has had contact with bacteria needs to be sterilised but all other items only need to be cleaned. Turn off the Bunsen burner and wipe down benches with a disinfectant spray. Thoroughly wash hands before removing lab coat and goggles.

25. Leave the samples in the incubator for a period of between 72 and 96 hours before checking on them and taking readings.

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Measuring Results Once I had completed the method, placed the plates into the incubator and then waited for an appropriate period of time, I had to take readings from each plate.

Apparatus required Item

Number required

Needed …

Large lightbox (~200mmx800mm) 22 Petri dish samples created previously Clear 15cm rule, graduated in mm Black marker pen

1 1 1

To see contrast between bacteria and inhibition

To mark each sample as measured

Method 1. Draw a blank table as shown in “Results” section.

2. Take the petri dishes and place them atop a lightbox, to increase the contrast between the areas of growth and areas of inhibition.

3. Measure from the edge of the paper disk to the area in which the inhibition stops and the bacteria is able to grow. Measure another value at the same point on the opposite side of the circumference of the paper disk.

4. After taking a measurement in millimetres, write an arbitrary test number next to the disk so that you remember it has been measured already. (As shown in Figure 3).

5. Repeat until all samples have been measured.

In the tables on the following pages I have given the data which I collected from the dishes which I prepared. To ensure that each result was as accurate as possible, I inverted the plate and measured it using a clear rule, so as not to obscure the view.

This provides me with 8 pieces of information with which I can build a graph.

N.B. Despite carrying out my project to a resolution of 1mm, I have calculated the mean to an accuracy of 0.1mm.

FIGURE 5 - MEASURING THE AREA OF INHIBITION

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Whilst this is not strictly accurate or professional, I needed to be able to see differences between each figure on such a small scale if I were to be able to draw any kind of a conclusion.

Therefore, it was needed for any useful results to be seen.

My Results Experiment 1: Escherichia coli

Substance Basil Thyme Parsley Mint Lavender

Distance of visible inhibition (mm) 2 3

1 3 3 Error with Tests 1 1 Error with tests

2

2

Mean 4

2

1

3

2

1

1

1 2 2

0 1 2

2 1 1

2 1 1

1 1 2

0 1 1

2.0 1.0 1.2 1.7

Staphylococcus albus

Substance Basil Thyme Parsley Mint Lavender

Distance of visible inhibition (mm) 2 3

1 1 1 Error with tests 1 1 Error with tests 1 1

Mean 4

2

2

2

3

2

3

0

0

0

0

0

0

2

2

1

2

1

1

2 0.3 1.4

Control tests 1 Escherichia coli Staphylococcus albus

1 0

2 0 1

0 0

0 1

0.3 0.5

Unfortunately, this first test contained quite a few errors. Some of the plates became contaminated, or refused to grow at all. For instance, the thyme plates became contaminated with a foreign body growing atop the agar, seemingly promoting rather than inhibiting microorganism growth.

This is likely due to the fact that both tests used the same sample of thyme solution for each plate, therefore carrying the fungus across to both petri dishes and allowing it to thrive in the incubators conditions.

FIGURE 6 - ONE OF THE CONTAMINATED PLATES

23

FIGURE 7 – EXPERIMENT 1

Results from Experiment 1 Bacterial inhibition (mm)

2.5 2

1.5 1 0.5 0 Basil

Thyme

Parsley E. coli

Mint

Lavender

Control

Staph. albus

The results from experiment 1 were rather poor. This bar graph shows how it is difficult to draw any conclusions, other than that Basil and Lavender were consistent in providing antimicrobial properties.

Both of these two worked well, but as the other tests were unsuccessful, I will label this experiment as inconclusive.

Thyme, Parsley and Mint were unsuccessful in providing any valid or consistent results and so this experiment can be regarded as a failure.

The control plates showed a slightly higher than desired zone of inhibition than I would have liked, but even if this was lower, this wouldn’t have changed the faults with the results.

Notes for the next experiment: 

Ensure every single piece of equipment is sterile, and that there is no chance of crosscontamination.



Reduce contact with air and surrounding particles as much as possible

FIGURE 8 - USING A LIGHTBOX TO CLEARLY SEE THE AREAS OF BACTERIAL INHIBITION 24

Experiment 2: Experiment 2 went far more smoothly than experiment 1, with accurate results being produced for every single test. Perhaps due to an improved aseptic technique or perhaps due to using slightly different bacterial colonies, the results were far more conclusive and exact.

Escherichia coli

Substance Basil Thyme Parsley Mint Lavender

Distance of visible inhibition (mm) 2 3 2 3 3 2 1 2 4 4 3 2 2 2 1 1 2 1 3 2 4 4

1 1 6 4 4 5

2 3 2 1 3

Mean 4 3 2 3 2 2

3 1 1 1 1

2.4 2.9 2.4 1.6 3

Staphylococcus albus

Substance Basil Thyme Parsley Mint Lavender

Distance of visible inhibition (mm) 2 3

1 6 4 2 5 3

3 2 2 3 5

5 1 3 2 2

3 3 2 1 2

4 2 4 5 4

3 3 1 5 4

Mean 4 2 2 2 5 2

2 2 2 2 1

3.5 2.4 2.3 3.5 2.9

Control tests 1 Escherichia coli Staphylococcus albus

1 0

2 0 1

0 0

0 0

Mean 0.25 0.25

Due to the extremely small size of the alcohol controls, and their similarity, I will disregard the effect that they have had on the experiment. Their miniscule effect means that any visible inhibition was caused not by the alcohol, but by chemicals within the substances.

I have highlighted the result for mint, in the E. coli test, on the first plate. Whilst some might consider this to be an anomaly as it does not fit with the trend, I believe this to be a very valid piece of data as I measured it correctly at the time and it was physically there – not an error in measuring. Due to its physical presence, rather than being as a result of an error I will not omit it from the calculated mean.

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FIGURE 9 – EXPERIMENT 2

Results from Experiment 2 4 3.5

3.5

3.5 2.95

Bacterial Inhibition (mm)

3

3

2.9 2.65

2.5

2.4

2.4

2.4

2.9 2.95

2.55 2.3 2.35

2 1.6 1.5 1 0.5

0.250.250.25

0 Basil

Thyme

Parsley E. coli

Staph. albus

Mint

Lavender

Control

Average

This graph shows the effect that the natural substances have had on the zone of inhibition of bacterial growth. This experiment has gone remarkably well in contrast to experiment 1.

In all of the test samples, bacteria has been inhibited and a zone of inhibition larger than that of the control group has been reported.

The control group’s zone of inhibition is rather low at an average of 0.25mm wide, helping to validate the other results and show that it is not the presence of alcohol, but rather the chemicals from the plant samples themselves.

The grey bar represents the average zone of inhibition, and from this we can tell that Basil and Lavender had the largest zone of inhibition, encompassing an average of 2.95mm each. The most successful individual result was a tie between Basil and Mint, both inhibiting Staphylococcus albus.

In this experiment, the least successful substance was the Mint when attempting to inhibit the growth of Escherichia coli. It only managed to produce a zone of inhibition of 1.6mm which was by far the smallest.

26

Conclusion The aims of my project were to test the antibacterial and antimicrobial properties of natural products and to observe how certain bacteria are prevented from growing due to these properties.

Before starting the experiments, I wrote in my hypothesis that I thought the substances were going to act as antimicrobials purely based on the notion that they have been used for many years in ‘potions’ and herbal cures. Now that I have completed my experiment I can say that I still believe strongly in this hypothesis, but instead of speculating I can refer to the evidence collected throughout this project to prove that some plants act as a natural antimicrobial. Regardless of the magnitude of their effect, I can definitively say that all of my samples produced a defined zone of inhibition in which bacteria were prevented from growing.

When viewed graphically, it is clear that the natural substances really have had an effect on the bacterial substances. This is visible proof of my hypothesis as the natural products have inhibited growth and shown that they could be used as a possible antimicrobial in the future.

Perhaps the successes were due to the strength of their oils – for instance basil and lavender both have a very strong and noticeable aroma – maybe it is the strength of these smells (or rather the chemicals that cause them) which has led to the bacterial inhibition. For example, Basil contains the chemical Linalool which is a “naturally occurring terpene alcohol chemical…used by pest professionals as a flea, fruit fly and cockroach insecticide,” (Wikipedia, 2016) suggesting that Basil’s antimicrobial effect may be due to this alcohol. Lavender also contains Terpinen-4-ol which is used to treat multiple skin related diseases and has an “anti-inflammatory, antibacterial and antifungal effect” (Wikipedia, 2016) (US National Library of Medicine, 2015). This information about the chemicals contained within the herbs gives scientific backing to the reasoning behind why an antimicrobial inhibition was observed.

When the data from both the E. coli and the Staph. Albus plates are combined, we can clearly see the direct result of plant substances inhibiting the growth of bacteria. Despite the chart having a higher resolution than the results I collected, the data represented here is a great way to see just how effective the natural products were compared to one another.

The basil and lavender samples created a zone of inhibition 2.95mm around their paper disk. This is an increase of 11.8 times the inhibition of the alcohol control.

The thyme samples with an average inhibition of 2.65mm is 10.6 times more effective, with the mint at 2.55mm and Parsley at 2.35mm, following closely behind with 10.2 and 9.4 times the control’s zone of inhibition respectively.

Combined Results from Experiment 2 3.5 3

2.95

2.95

Bacterial Inhibition (mm)

2.65

2.55 2.35

2.5 2 1.5 1 0.5

0.25

0 Basil

Thyme

Parsley

Mint

Average effect

FIGURE 10 - SUCCESSFUL RESULTS

When we look at it from the perspective of each different bacteria, Basil was 1.46 times more effective at inhibiting the growth of Staphylococcus albus than it was for Escherichia coli.

Lavender Control

27

On average across all natural products Escherichia coli was inhibited by 2.46 mm whereas Staphylococcus albus was inhibited for an average of 2.92mm.

If we were to look at this a different way – instead of the distance from the sample to the zone of inhibition we analysed the surface area of the inhibited zone, the two bacteria behave more differently than one might expect.

E. coli has an average inhibited area of 23.09mm2 and Staph. albus has an average inhibition area of 32.05mm2. Viewing the data in this way shows a clear trend where the E. coli was inhibited far less than the Staph. albus, making it better at resisting antimicrobial agents.1 Perhaps this is due to the key difference between the two, with E. coli being gram negative and Staph. albus being gram positive. Some Gram-positive bacteria are more susceptible to antibiotics and antimicrobials than Gram-negative. Perhaps it is due to the design of their cell walls and membranes that makes them have different properties. Nevertheless, there is a clear difference in volume of inhibition.

My results allow me to conclude that the medicinal properties of natural products such as garden herbs and flowers are real, and not simply a placebo. By creating a solution of these products and recording their inhibition of bacteria, I have shown that there are many antimicrobial properties of foods that we eat every day and that these may impact our daily health.

Impact of my project on the wider world In our current climate of growing antibiotic resistance, it is becoming more and more important to find new ways of killing bacteria or stopping their growth. My project provides a new direction through which scientists could look at everyday plant materials and find different ways of fighting bacterial related diseases.

Methicillin-resistant Staphylococcus aureus is responsible for several difficult to treat infections in humans. One of the bacteria which I studied is related very closely to aureus. Staphylococcus albus shares many of the same properties, so by looking at my research, scientists could find a basis on how to treat a very deadly super-bug.

But it is not just MRSA that we need to worry about. The over-prescription of antibiotics has led to so many common bacteria developing resistant strains. By researching into as many possible natural products and herbs, we can try to avoid total resistance for as long as possible. In my project, I have tested a large number of different herbs and spices, which scientists could use to see what is and isn’t effective.

Basil was 11.8 times more effective at inhibiting the growth of both bacteria than the water-ethanol solution was on the control plate. If we are able to research more and more into herbal products such as Basil and Lavender, we could develop drugs based on them which may form part of the attack on antimicrobial resistance, but we must act quickly if we are to have any noticeable effect.

Hopefully my project can ensure that one day we aren’t at risk of dying of what were once preventable diseases.

1

To produce these figures, I had to do some calculations. For each I took the size of each paper disk to be 0.7mm and therefore I added 0.35 to the ‘inhibition value’ to produce the radius of the zone. After calculating the area, I subtracted 1.54 from it to exclude the area of the paper disk.

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Evaluation This project has taken an inordinate amount of time to complete. Over the course of three years I have manged to bring it to fruition, but it should not have taken this long. Perhaps if I had made the project a higher priority or simply made it far less complex, it may have been completed quicker. In future projects, I will attempt to reduce the complexity and ensure that my time is well spent. Nevertheless, I am proud that I have managed to complete it and come to a firm conclusion about my hypothesis.

My research was constructed to a high standard, with the aim to find as much information and relevant facts about the topic as I could. I think that it creates a great start to the project and helps to engage the reader. The use of Harvard-style citations helps to add to the validity and quality of the research, proving that everything is accurate and not made up.

To get to my optimal method, I went through a process of development and iteration. This stage of the project was very time and resource consuming, but it did prove valuable in helping me find the best method possible. In my opinion this was the most important stage of the entire project, as it allowed me to test every idea that I had and find the most effective way to execute them. For each controlled variable, I found the best value for which it should be maintained i.e. the ratio of ethanol to water should be 1:19. Without this stage, my project likely would not have succeeded as there were far too many variables which – at the beginning of the project – had not yet been properly considered. I think that it was a large success, although if I were to do it again, I would improve the developmental stage by conducting isolated preliminary tests for each variable. I would study them independently before the experiment could start as a whole – as this might cut down on the amount of time and resources wasted from unsuccessful experiments.

The protocol for measuring results (page 22) was written before I had started experiment 8. This page provided guidance and ensured consistency across all of the data that I collected. By writing out a set of clear instructions and detailing exactly how to collect results, I removed any chance of ambiguity in data collection that could have occurred.

If I had more time I would complete many more tests using my optimal iteration of the method to increase the validity of the experiment, although budget constraints and other commitments stopped me from being able to do this at the time. I could also have looked into concentrating the plant substances in an attempt to increase their potency.

By the time that I reached method 8, my aseptic technique was well thought-out and developed to a high standard, although this did take time to get to an acceptable standard. In Method 8, experiment 1, the Thyme plates for both bacteria were contaminated. It is likely that at some point, fungus or spores were able to get into the thyme mixture and this was spread onto both plates. As a result the test was invalid.

Setbacks like this were very frustrating and this meant that I had a much smaller data set upon which to draw my conclusions.

Overall I believe that my project was very well considered and carried out to a high standard. My dedication to it has led to the proof of my hypothesis and data which can form a very clear conclusion.

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Personal Reflection This task has helped me greatly to develop my project management skills. Throughout this I’ve learnt about proper lab techniques, project writeups and how to display my results effectively. I have gained so many skills which no doubt will help me in the future with my science write-ups.

I’ve learnt that most scientific investigations don’t always yield results that you were expecting. It took me a very long time from first setting out on this project to actually achieving an accurate and valid set of results. Once I had come to results I could use, I found there was less difference between them than I was expecting, but I have come to learn that I cannot expect anything from a project as everything can change.

There were points in this task where I felt as though the project had no direction at all – specifically the long approach taken to find the method, but after persevering and trying every different technique I could think of, I found that gradually the method began to work and finally produced a decent set of data.

I believe that my project was a massive success, and therefore I hope that it can be used by others in the future as a way to look into different possibilities and may one day it will be partly responsible for the lifesaving drugs that we may develop.

This project has taken over a large period of my life, but I am very happy I have completed it. I’ve learnt so much about the field of microbiology and it has inspired me to look at going into a science related field as a career.

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Works Cited American Public Health Association, 1920. In: Standard Methods for the Examination of Water and Sewage. s.l.:s.n., p. 95.

Cowan, M. M., 1999. Plant Products as Antimicrobial Agents, Miami: s.n.

Livestrong, Fighting Bacterial Naturally with Food. [Online] Available at: http://suite101.com/article/fighting-bacteria-naturally-with-food-a275165 [Accessed 23 05 2013].

McKenna, M., 2015. National Geographic, Phenomena. [Online] Available at: http://phenomena.nationalgeographic.com/2015/04/07/anglo-saxon-mrsa/ Nuffield Foundation, 2011. Incubating and Viewing plates. [Online] Available at: http://www.nuffieldfoundation.org/practical-biology/incubating-and-viewing-plates Sugg, R., 2015. The Guardian. [Online] Available at: http://www.theguardian.com/commentisfree/2015/apr/06/ninth-century-remedy-mrsapowdered-poo Suite101, Fighting bacteria naturally with food. [Online] Available at: www.suite101.com/article/fighting-bacteria-naturally-food-a275165 [Accessed 2013].

The Columbia Encyclopedia, 6th ed., 2015. Staphylococcus. [Online] Available at: http://www.encyclopedia.com/doc/1E1-staphylo.html [Accessed 2015].

The George Mateljan Foundation, n.d. World's Healthiest foods. [Online] Available at: http://whfoods.org/genpage.php?tname=foodspice&dbid=85 US National Library of Medicine, 2015. HSDB: 4-TERPINEOL. [Online] Available at: https://toxnet.nlm.nih.gov/cgi-bin/sis/search/r?dbs+hsdb:@term+@rn+@rel+562-74-3 Wikipedia, 2016. Linalool. [Online] Available at: https://en.wikipedia.org/wiki/Linalool#Uses Wikipedia, 2016. Terpinen-4-ol. [Online] Available at: https://en.wikipedia.org/wiki/Terpinen-4-ol Wikipeida, 2015. Nutrient agar. [Online] Available at: https://en.wikipedia.org/wiki/Nutrient_agar#cite_note-1 Wikipedia was used for information on Gram stains, E. coli, Staph Albus

Acknowledgements https://www.hsdirect.co.uk/free-info/risk-assessment.html was used to source information about constructing a risk assessment

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