PRELIMINARY WORK
Before doing this experiment, I carried out some preliminary experiments, to give me better idea of the experiment I am to do.
- Our first experiment was growing bacteria on agar plates. I did this by rubbing my fingers on the agar, and left this for 1 week to grow. Next I identified the different cultures that had grown on the plate.
- In my second experiment I made some agar plates aseptically. I did this by mixing boiling water and agar powder together. Using aseptic techniques I poured this as evenly as possible into the agar plate. It was important that this was carried out aseptically, so that no bacteria is present before the agar plate will be used.
- The last preliminary experiment I did was that I put milk onto the agar plate and left that for 1 week. The result was that bacteria found naturally in milk was grown on the agar.
These preliminary experiments have helped me use aseptic techniques and given me a better idea of how to successfully do the experiment.
Bacillus Subtilus
For the experiment I have decided to use Bacillus Subtilus which is cultured at school. This type of bacteria is harmless and found naturally in the body. I am not going to use milk because I found that it was difficult to spread and did not produce a lot of bacteria growth.
Method:
- Get 4 clean petri dishes. Run them under hot water to kill bacteria that may be on the dish already. As disinfectant will be put on them that should kill any bacteria on there, but as the control will contain distilled water, this will not kill any bacteria so run all the petri dishes under hot water to keep the experiment a fair test.
- In one petri dish, place 50ml of ‘Dettol’ and place 4 hole punched pieces of filter in the petri dish. In another petri dish do the same with Liquid Hand Soap, Clearasil and distilled water (the control).
- Leave them to soak for 20 minutes.
- Whilst the discs are soaking, the agar plates need to be prepared.
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Get 4 autoclaved agar plates. The agar plates are autoclaved by putting them in a large pressure cooker where the temperature rises to 100oC to kill any bacteria or spores. This is called an ‘Aseptic’ technique. These techniques are used to eliminate microbiological contaminants . The lid of the agar plate should be lifted only a few centimetres, so other micro organisms from the air have minimal chance of entering. At this stage the agar plates should contain no organisms.
- Each agar plate should be labelled with the date and the disinfectant that will be put in it.
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2.5ml of the bacteria Bacillus Subtilus, which is in a suspension form, will have to be poured onto 10ml of autoclaved water in the agar plate. This will be done using an aseptic technique. One hand will hold the bottle, the other needs to open the agar plate. The lid of this bottle with have to be opened with the little finger of one hand, whilst the other hand opens the agar plate slightly and the bacteria is poured in with one swift action.
- Make sure the bacteria is spread over the agar plate evenly by using a glass spreader or inoculation loop. All equipment used should be flamed in a Bunsen Burner to eradicate any micro-organisms.
- Make sure this is done for all 4 agar plates.
- After the discs have been soaked for 20 minutes they need to be transferred to the agar plates.
- Transfer 4 discs from each disinfectant and distilled water to the agar plates by using tweezers. Before transferring each disc, the tweezers should be heated in a bunsen burner.
- Selotape each agar plate in four corners. Do not selotape all around the agar plate as anaerobic respiration will take place and harmful toxins may be produced.
- Leave the agar plates for 1 week and check them everyday recording any observations.
Diagram:
PREDICTION:
I predict that the Dettol will kill the most bacteria and will therefore produce the largest diameter of a clear zone, next will be liquid hand soap and then Clearasil. I predict because:-
Dettol- This is an antiseptic and a disinfectant. It is very strong as it needs to kill all known bacteria. Its impregnated with a bleach solution that cleans and kills bacteria and reduces the spread of harmful bacteria.
Liquid Hand Soap - This anti bacterial hand wash claims to remove germs and bacteria but moisturise the skin as well. It cannot be very strong, or it may harm the skin.
Clearasil- This cleansing lotion removes dirt and bacteria from the skin which cause spots. This must also be quite gentle as it is being put on the most sensitive skin on the body.
The reason why I believe Dettol will produced a larger clear zone is because it actually kills the bacteria so they cannot reproduce by mitotic division. With Liquid Hand soap and Clearasil, these do not kill bacteria but actually just remove it, so any that have not been removed can still carry on multiplying.
Analysis
From my results and graphs it is obvious that Dettol produced the largest clear zone followed by Liquid Hand Soap and then Clearasil. I noticed a trend in all three antibacterial substances that the clear zone got large and then started to decrease in size.
At first Dettol killed the bacteria so the reproductive procedure had been stopped so they were unable to reproduce by mitotic division. But after the first day the clear zone decreased in size. This was because all the of the chemicals in the Dettol that killed bacteria got used up so bacteria was not being killed and therefore able to reproduce. Also some of the Dettol had evaporated, so chemicals were lost that way. Dettol produced the largest clear zone because it contained the strongest chemicals, killing the most bacteria .
The liquid hand soap and the Clearasil did not have as large clear zones as Dettol because the chemicals in them were not as strong as that of Dettol. With liquid hand soap and Clearasil, they are applied to the skin and therefore have to be quite gentle so they do not harm the skin. Dettol on the other hand is used on work surfaces where sometimes food is kept and therefore needs to be tough to kill all known bacteria. It is also often used diluted so the strength of the anti bacterial substance is reduced.
This supports my prediction very well, as what I thought would happen did. This experiment does not undermine it in any way.
Evaluation
I believe that this experiment went satisfactorily. When I did this experiment for the first time, it was unsuccessful and did not work because I had used too used too much bacteria in the agar plates so it had not settled evenly on the agar plates and the results obtained were inaccurate. When I did the experiment a second time using less bacteria I found that the results I had obtained were much more reliable and accurate. However I did get a few anomalous results. For example when looking at Clearasil, at the fourth disc on days 4 and 5, the clear zone was 3mm on day 4 and then went up to 4mm on day 5. This was an inaccurate result and could have been due to human error of not measuring accurately or it could have been due to too much Clearasil being in one place.
If I were to redo this experiment and make it more accurate I would:
- Repeat the experiment more times to obtain more results. I would do 12 discs for each antibacterial substance instead of 4 on 3 agar plates.
- I would test more antibacterial substances to obtain a better analysis of why some substances kill or remove bacteria better then others.
- I could test the rate the bacteria reproduces to see what conditions bacteria work best in.
- I could leave the antibacterial substances in for longer than 5 days to see how time has an effect on bacteria.
- I could put the agar place in different temperatures or light intensities to see where this has an effect on the bacteria or antibacterial substances.
The evidence is quite reliable but I believe that with better aseptic techniques and the spreading of bacteria on the agar plate more evenly the experiment could have gone better.
By
Serina Ramkhelawon
