Equipment:
- 24 agar plates- of standard size and volume. To spread the bacteria onto, and for ideal growing surface.
- Glass spreader- choice of glass as it can withstand high heats when undergoing sterilisation. I has a smooth surface so won’t damage the agar when spreading the bacteria.
- Sterile pipette- 1mls calibrated in 0.01 intervals for high levels of accuracy. Transfers the bacteria to the agar plate, and needs to be sterile to eliminate the chance of spreading other bacteria.
- Bunsen burner- used for sterilisation purposes and to help reduce contamination in the air in a 10-metre radius.
- Disinfectant- to remove possible contaminants from the working area.
- Forceps- to avoid using fingers to transfer the antibiotics and herbal products into the agar plate.
- Alcohol (ethanol) - to sterilise glass spreader, pipette and forceps.
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Incubator- set at 25°C to reduce the growth of pathogens.
- Filter paper discs- 6mm area to soak in herbal oils.
- 2 sterile bottles- to empty the herbal products into.
- Cotton wool- to wipe down the area with.
- Matches- to light the Bunsen burner.
- Sellotape- two strips per agar plate to hold them shut- Don’t seal the whole way around as harmful pathogens could be produced.
- Ruler- to measure the inhibition zone and the effectiveness after 48 hours in the incubator.
- Calculator- to work out the area of each inhibition zone.
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Bacteria- E.coli and M.luteus, mixed with a broth to make them easily spread able.
- Antibiotics- Penicillin 10units and Streptomycin 25micro grams, absorbed onto 6mm filter discs.
- Herbal products- tea tree oil 100% pure, and peppermint oil 100% pure, absorbed onto filter discs.
Safety:
Due to this investigation looking at microbiology and using bacterial microorganisms, it could be potentially dangerous if the safety precautions are not followed correctly.
When performing this investigation:
- Make sure the surface you are working on has been disinfected.
- No running occurs in the lab to restrict possible accidents.
- Make sure there is no food or drink consumption in the lab, especially near your experiment.
- Take care when using matches.
- Keep the ethanol away from the naked flame.
- Make sure the bacteria broth is handled with care to prevent contamination.
- Don’t remove the lids fully off the agar plates to reduce unwanted bacteria.
- If you spill of break anything make sure it is cleaned up immediately and reported to a teacher.
- Make sure you wash your hands prior to and following the experiment, and whenever else you feel is necessary.
Following up to this safety list is a risk assessment, which you will discover at the end or this coursework, discussing the main safety precautions in a more serious depth and with more detail.
Ethics:
Taking into consideration the ethics of this investigation and the fact that living organisms are involved, it can be established that both E.coli and M.luteus’ nucleus’ are so minute that they have no recollection of pain. When in contact with the herbal products and the standard antibiotics, although they are unable to reproduce they do not become damaged and after contact they are able to grow naturally.
The agar plates need to be disposed off in biohazard bags to ensure no damage is placed upon the environment, as not many of the products used are environmentally friendly, yet they are used in small amounts to reduce unnatural causes.
Pilot study:
Prior to performing the main investigation it is important that a preliminary study is hosted to ensure, that the idea of how to carry it out correctly, the practice of technique and the equipment and method are all convenient. Faulty results that may occur need to be watched out for, and these are to be used at the best advantage possible. Considerable practice of the spreading technique is needed as the success of results depends on the quality of this. Additionally the amount of time to leave the bacteria growing needs to be determined; too long and the bacteria will re-grow over the inhibition zone, too short and the results will not be clearly visible to measure.
Method for pilot:
- Wash your hands prior to the experiment and using cotton wool and disinfectant wipe down the surface area you are using.
- Set up you equipment in an organised manner.
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Flame the neck of the E.coli bacteria bottle, keeping the lid in your hand at all times.
- Using the pipette place 0.2mls of E.coli bacteria into 12 of the agar plates.
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Flame the glass spreader holding it at a 45° angle.
- Spread the bacteria evenly across the agar plates.
- Using the forceps place the penicillin disc’s into the centre of 3 agar plates.
- Place the forceps in ethanol and flame them.
- Using the forceps again place the streptomycin disc’s into the centre of 3 agar plates.
- With the empty sterile bottles, empty the contents of the tea tree oil into one and the peppermint oil in the other.
- Then place 6 paper filter discs in each bottle and allow to soak for a few minutes.
- Using the forceps place the 3 tea tree discs in the centre of 3 of the agar plates.
- Place the forceps back into the ethanol and flame them off.
- Then using the forceps place the 3 peppermint oil discs into the last 3 agar plates.
- Use 2 strips of Sellotape to secure the lid to each agar plate- do not seal the whole way round, as most dangerous bacteria are anaerobic.
- Label each plate with the bacteria name, the herbal oil or the antibiotic name, the date and your initials.
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Repeat the above steps but use M.luteus bacteria instead.
- Clean up all your mess.
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Once finished, place all the agar plates in the incubator at 25°.
- Leave for 48 hours.
- Measure the inhibition zone of each agar plate, by measuring 3 points and adding them together and dividing the answer by 3. Then divide this answer by 2 and square that answer and then times it by pi (the calculation for measuring circles) this is the area of your inhibition zone.
NEVER OPEN THE PLATES ONCE SEALED AND PLACED IN THE INCUBATOR!
Pilot results:
From looking at the pilot results, it can be established that tea tree oil is the most effective against bacteria, both on Ecoli and M.luteus. It can also be said that penicillin is the least effective.
These results have been rounded up to the nearest mm, but the main results will be to 1 decimal place to ensure maximum accuracy.
Pilot study debrief:
Pilot studies help to certify that in the investigation E.coli and M.luteus will be used as the two bacteria’s, this is due to them producing prominent inhibition zones that are fairly easy to measure. Both herbal products seemed to play some affect on the growth rate, therefore these will be used, the tea tree and peppermint oils in the final investigation.
The agar plates were incubated for 48 hours and this proved to show a relevant growth rate of bacteria to be measured, but two rings occurred where the bacteria started to re-grow because the product became weak. Therefore the plates will only be left for 24hours to prevent this from happening. Following this preliminary, the closest form of measurement received was by measuring to the nearest mm, to measure to a greater degree of specificity would be extremely difficult.
In the main investigation care is needed when placing the discs into the centre of each agar plate as some of them failed to be in the correct place. This could explain why some of the results were irregular and this could also be due to the fact that technique lacked when spreading the bacteria and at least twice the agar jelly was damaged.
Taken into account is that it is obvious the results are going to vary slightly as the concentrations are different. An attempt to make the concentrations equal was made but this was pretty impossible as the amount needed was so small and the equipment the school could provide wasn’t good enough.
It can be established that the herbal products, tea tree and peppermint work brilliantly in small amounts in vitro conditions; however in vivo conditions it wouldn’t be necessary to treat a patient with pure herbal products as it could cause harm.
Taking into consideration the concentrations of all the products, should two and a half penicillin discs been used to match that of the streptomycin, as the penicillin results showed a weaker effect than the streptomycin? Due to the larger the concentration of the streptomycin this shows that the rate of reaction was faster and it proves this within the results table. This is something that should have thought about before carrying out the pilot study and may have therefore helped to obtain more accurate results.
In the final method the order of when the discs are soaked in the herbal oils will be altered, this is because waiting a while to ensure the filter discs had fully absorbed the products just wastes time. Therefore this will be made one of the first things to do in the investigation and not leave it till the middle. Although the concentrations were different, you can see from the results that the herbal products worked the best. This is due to the fact that the antibiotic discs were dry and the herbal discs were wet and when placed into the agar plate the concentration diffused out of the disc and onto the agar jelly, causing a larger affect against bacteria growth. So in the final method the herbal discs need to be dried out first to make the test fairer and the results are more accurate and easier to compare.
Final experiment additions:
The Null hypothesis will remain the same but with the hypothesis has had some amendments made as with the prediction:
Final Hypothesis:
The herbal products will prove to be more effective against the growth of bacteria than the standard antibiotics.
Prediction:
The herbal products will be more effective against the growth of bacteria compared with the standard antibiotics.
Equipment additions:
The equipment list will stay the same but the amount of agar plates used will be doubled; two pieces of equipment will be added.
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Oven- to dry the herbal soaked filter discs. Set at 180°, leave the discs in for 15minutes.
- Baking tray- to place the herbal discs on.
Method:
(The highlighted steps are the alterations)
- Wash your hands prior to the experiment and using cotton wool and disinfectant wipe down the surface area you are using.
- Set up you equipment in an organised manner.
- With the empty sterile bottles, empty the contents of the tea tree oil into one and the peppermint oil in the other.
- Then place 12 paper filter discs in each bottle and allow to soak for a few minutes.
- Place herbal soaked filter discs on the baking tray.
- Place in the oven and leave for 15minutes.
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Flame the neck of the E.coli bacteria bottle, keeping the lid in your hand at all times.
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Using the pipette place 0.2mls of E.coli bacteria into 24 (half) of the agar plates.
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Flame the glass spreader holding it at a 45° angle.
- Spread the bacteria evenly across the agar plates.
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Using the forceps place the penicillin disc’s into the centre of 6 agar plates.
- Place the forceps in ethanol and flame them.
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Using the forceps again place the streptomycin disc’s into the centre of 6 agar plates.
- Take the tray out of the oven and allow to cool.
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Using the forceps place the 6 tea tree discs in the centre of 6 of the agar plates.
- Place the forceps back into the ethanol and flame them off.
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Then using the forceps place the 6 peppermint oil discs into the last 6 agar plates.
- Use 2 strips of Sellotape to secure the lid to each agar plate- do not seal the whole way round, as most dangerous bacteria are anaerobic.
- Label each plate with the bacteria name, the herbal oil or the antibiotic name, the date and your initials.
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Repeat the above steps (1-19) but use M.Luteus bacteria instead.
- Clean up all your mess.
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Once finished, place all the agar plates in the incubator at 25°.
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Leave for 24 hours.
- Measure the inhibition zone of each agar plate, by measuring 3 points and adding them together and dividing the answer by 3. Then divide this answer by 2 and square that answer and then times it by pi (the calculation for measuring circles) this is the area of your inhibition zone.
NEVER OPEN THE PLATES ONCE SEALED AND PLACED IN THE INCUBATOR!
This experiment overall will be repeated 48 times- 12 times for each products, where 6 times are for E.coli and 6 for M.luteus.
Final results:
As stated in the pilot the closest degree of accuracy is mm which is what the final results have been measured in. It was necessary to measure to 1 decimal place to ensure the greatest degree of accuracy. Another method which helped to contain accuracy was to collect three radii readings for each agar plate to ensure the average area and the average radii could be calculated. Each radii was taken at 120˚. It should also be mentioned that each condition was repeated 6 times on 6 different agar plates to give a greater range of readings. Following the collection of the data the agar plates were disposed of via autoclaving, this was for environmental purposes.
By looking at the final results table you can see that the most effective product used against E.coli bacteria is tea tree with an average area of inhibition of 333.5 mm2. However with M.luteus, streptomycin has proved to show the most effect with an average area of inhibition of 390.1 mm2 .The least effective on both the bacteria’s is penicillin, and if you look at the pilot results table on page 6 you will see this was also the case there. It is in major interest that such an established and mass produced antibiotic had such minimal effects.
Most of the results appeared to be consistent, however if you look at the final results table for E.coli, streptomycin’s radii averages vary from 14.6 mm to 23.6 mm. By looking at M.luteus, penicillin has one anomalous result, with an average radius of 6.3 mm. This isn’t extremely out of line, but the other 5 results have average radiuses of either 7.3 mm or 7.6 mm.
From studying both graphs it is able to establish that the final results have changed from the pilot results. In the pilot the order of effectiveness for E.coli went:
- Tea tree
- Peppermint
- Streptomycin
- Penicillin
For M.luteus:
- Tea tree
- Streptomycin
- Peppermint
- Penicillin
And in the main investigation the results are as follows:
E.coli:
- Tea tree
- Streptomycin
- Peppermint
- Penicillin
M.luteus:
- Streptomycin
- Tea tree
- Peppermint
- Penicillin
Penicillin stayed the least effective, the main change was streptomycin.
Further data:
Further research has shown that there are two different types of bacteria, gram positive and gram negative, both behave very differently and so are likely to be more susceptible to inhibition from varying antibiotic and herbal products. Research into gram staining has shown that E.coli is that of a gram negative variation and M.luteus is gram positive, therefore providing reason for the results to vary on the different bacteria’s, although they didn’t vary hugely. The final results table shows that penicillin near enough failed to inhibit the E.coli bacteria, and so the growth rate ceased to be restricted. Thus meaning penicillin has minimal effect against gram negative bacteria, but more successful when in contact with gram positive, and by looking at the M.luteus results you can see this. The gram staining procedure involves the use of crystal violet and gram’s iodine solution to identify the difference in cell structure.
In gram positive bacteria the cell wall consists mainly of polymer layers composed of peptidoglycan, which are all connected via amino acid bridges. In gram negative bacteria the walls are very thin and contain very little peptidoglycan. However, they have two unique regions surrounding the outer plasma membrane: The periplasmic space and the lipopollysachharide layer. The periplasmic space separates the outer plasma membrane from the plasma membrane.
These appear as follows:
Analysis:
From looking at both the final results table and graph it is clearly acknowledgeable that with regards to the effect against bacteria growth, there is no significant statistical difference is evident in the performance between standard antibiotics and herbal products. It is evident that tea tree and streptomycin have the highest inhibition rate, as shown by the fact that the inhibition zones on the agar plates were the largest.
Referring back to the hypothesis ‘The herbal products will prove to be more effective against the growth of bacteria than the standard antibiotics,’ this hasn’t exactly proved to be correct. The hypothesis was changed to this after the pilot study was conducted, as this is what the pilot results showed. You can see this by looking at the table on page 6. So, neither did the hypothesis prove to be correct nor false, it seemed to lie somewhere in between. As stated on page 11 in the further data section, E.coli is gram negative bacteria and by looking at the results table it can be established that the results for penicillin against E.coli are a lot lower than the results for penicillin against M.luteus. This therefore supports the fact why penicillin is impartial toward s it as it couldn’t stop synthesis in the cell wall. In gram positive bacteria there is a mesosome and the chemicals released by penicillin affect this leading to cell division not occurring and the cell dying. There is some doubt whether gram negative bacteria’s cell walls contain a mesosome, and even if they do the mesosome is too small to react. Therefore the enzyme released by penicillin cannot affect them to stop bacteria growth so the cells continue to divide.
Tea tree oil is widely known for its effectiveness against bacteria and this clearly seemed to be the case in this experiment and this can be seen by looking at the results table on page 9. Although these results seemed to prove it is highly effective compared to some of the other used products, the results aren’t that consistent. By looking at the graph you can establish that the percentage errors for tea tree against E.coli are relatively high showing that this set of results are fairly inaccurate. This could be due to the placing of the discs, as some were slightly out of the centre. Another reason that may have affected these results could be the fact that the standard antibiotic discs were readily produced, and with the herbal products being soaked then dried the concentrations couldn’t be tested and therefore it is more than likely that they varied. Although tea tree shows it is very effective, as stated in the background it is not possible to use in-vivo conditions. Tea tree has proved to be extremely effective against E.coli but not so effective against M.luteus, whereas most of the other products stayed closely similar in affect with both bacteria’s. This leads me to question whether tea tree oil fails to work at it’s best against bacteria that is gram negative.
Both of the herbal products on M.luteus showed two rings of where the bacteria had first grown to, and a second ring of where it had over-gone this ring due to the product becoming less effective towards the bacteria. This shows that the effects of herbal products are strong against bacteria but are not long lasting.
Most of the results seemed to respond consistently with closely similar averages; however there are a few anomalous readings which have affected the percentage errors on the graph, making them larger and therefore less accurate. If you look at page 13 another set of the results table has been produced where the anomalous results are highlighted in pink. There are 7 out of 48 anomalous results, most of these are not major but are still making the results less consistent. These anomalous results could have occurred for a number of reasons. As already mentioned the concentration of the product on each disc could have been different, therefore either causing the inhibition zone to be larger if the concentration was strong or smaller if it was a low concentration. From knowledge it can be established that the higher the concentration the faster the reaction occurs, this can be related to the herbal products only as both penicillin and streptomycin were used as dry discs mass produced with the same concentration per disc. Where penicillin consisted of 10 units per disc and streptomycin 25ug. So due to the concentrations in the herbal products varying the reaction between the product and the bacteria could be faster in some agar dishes than others. Another reason could be due to my lack of technique where the bacterium wasn’t spread as evenly on some dishes as others.
One interesting anomalous result found was in penicillin against E.coli, it wasn’t massively irregular, however the other 5 results either had an area of around 41 or 45 and this one was at 31, affecting the percentage error quite badly.
Evaluation:
The final practical session was preformed with a great level of accuracy, and the method followed out step by step with every safety precaution taken into account. On most of the agar plates the bacteria were spread evenly across the agar jelly, with no foreign pathogens affecting the results. This is complemented by the fact that there doesn’t seem to be any huge extreme dissenters, just slight mishaps. Obviously there are going to be some differences within results, thus the reason for repeating the experiment six times and why percentage errors were produced, which can be found on graph 2, the final graph. It could have possible that inadequately the whole batch of results could have been chancy and affected the whole lot and equally faults could have occurred with the incubator being either too hot or too cold. Although these problems may have occurred the results still prove to be reliable and show fairly consistent readings throughout the whole table.
As already stated the results do not prove the hypothesis to be correct but nor incorrect so they therefore weren’t what expected and followed a different trend, in which supported the null hypothesis, where no significant statistical difference occurred.
Some problems were encountered with trying to sterilise the bacteria bottle tops and keep the lids tightly held. Although the bacteria was spread evenly in the end it was tough to do so and at least twice the agar jelly became damaged, either due to the glass spreader being too hot when brought into contact with the agar jelly or by not being tender enough. It was difficult to specifically ensure 2.0ml of bacteria was placed onto each plate, so at this point accuracy was a problem but not immensely as no major reading was affected. The yield of accuracy became a problem yet again with the concentrations, although for the main the herbal discs were dried before, the concentration of them was still unaware. Yet this way proved to be more accurate than just soaking them and placing them straight onto the agar dishes.
If this experiment was to be performed again it could be improved by the incubation time, changed from 24hours to either 12 or 18, just something a little less so that the double ring did not occur. Another improvement would involve pictures being taken. If pictures of the agar dishes from both the pilot study and the main experiment were taken then more understanding would be gained and it would help to show the double ring effect. Practice of the spreading technique would be a good improvement to make, as difficulties occurred in this area that has already been mentioned above.
There were two limitations encountered during this coursework production and within the investigations. The first being the time given. If more time was allowed the subject would have been looked into with a lot more detail and precision, and time to just play about with different ways of conducting the study could have been carried out. Also accuracy could be improved with more time by taking greater measures of sterilisation and methods against contamination. Also with the time to produce more beneficial and interesting results the variables could be altered to see their effect on the inhibition zones. Theses alterations would be-
- Looking at varying concentrations of bacteria that the products come into contact with, and used different bacteria’s to see if penicillin could produce inhibition zones equal to or higher than those of streptomycin.
- In conjunction with the above testing could be done to see how different concentrations of bacteria affect the inhibition of antibiotics.
Equipment was another limitation which affected the accuracy of the results. As it has already been said the concentration was a big issue, if the correct equipment was readily available then although it would have been very challenging the concentrations of the products could be made near enough the same.
After conducting this experiment it is conclusive that there is no significant statistical difference in the performance between standard antibiotics and herbal products.
Due to the percentage errors on the graph being large, the highest at 62%, this shows the results aren’t very consistent enough. The results are valid and true but not entirely accurate as if other people conducted the same experiment they would very likely obtain different results. However the tests were repeated as this was an important procedure, but by looking at the results you can see that they lacked value.
As an extension to this coursework further studies could be carried out. For example different bacterial cultures could be used or the doses of the antibiotics could be changed. These extensions would give more supporting evidence and help with creating more detailed and knowledgeable explanations.
Bibliography:
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