Method
Starting the investigation, the working area was first sprayed with disinfectant and then wiped with a cloth, this was done to kill bacteria on the surface that may lead to contamination. The Bunsen burner was then setup and placed on top of a heat proof mat around the working area ensuring a sterile field to prevent contamination when working with petri dishes, antibiotics and culture medium. A bottle of molten agar was then opened and the neck of the bottle flamed, 1ml of bacterial culture was then mixed with bottle of molten agar and was flamed again to prevent contamination, the lid was then placed on top and was gently inverted. The bottle was then opened again with the neck of the bottle flamed, a petri dish was lifted up slightly and the agar was poured into the petri dish as quickly as possible to prevent contamination from microorganisms in the air. The same steps were undertaken to create another petri dish with the bacterium culture mixed in the molten agar and then poured into the petri dish using the same aseptic techniques and the plates were then left to set.
Table 1
Different concentrations of antibiotic were then created for the unknown antibiotic 1 & 2. Each antibiotic it had its own 5ml syringe and water having the 10ml syringe. The correct volumes were then used by looking at table 1 by adding the required volume of water and antibiotic to create the concentration of 25% and 50% for antibiotic 1 and 2 in the 4 medium sized bottles, with each bottle having its own concentration. 0% and 100% concentrations of antibiotics did not have to be created for each antibiotic as pure water and the original solution was used.
A marker was used to mark the location with the percentage of antibiotic concentration on top of the petri dish, indicating the placement of each sterile disc. A tweezer was then sterilised by flaming, killing any bacteria on the tweezer. The tweezer was used to pickup a sterilise disk and dipped into pure water (0% antibiotic concentration) and the lid of the petri dish was slightly raised so I could place the sterilise disc in the correct location and then closing it. The tweezer was then sterilised again by flaming. This technique was used then for each concentration of antibiotic 1 and antibiotic 2 until all the sterile discs were placed in the correct location for each petri dish after dipping it into the correct concentration of antibiotic. The petri dishes were then resealed using sellotape and scissors and placed in incubation for 24 hours with the temperature set to 25%.
Precautions
The work surface area was wiped with disinfectant prior to starting the experiment to kill bacteria on the surface and a Bunsen burner was setup ensuring the field of air was sterile. Opening and closing of the bottle was quickly flamed each time to remove contaminants from those areas, more over the tweezer was also flamed to prevent contaminations from entering the sterile medium when placing the discs within the petri dish. Lab coats and goggles were worn to prevent contamination from our clothing and our hands were washed before and after the experiment ensuring our hands were free our bacteria.
Results
Analysis
The findings show conclusive evidence between the effectiveness of both unknown antibiotics. Antibiotic 2 at 0% antibiotic concentration had a mean clear zone of 5.33mm and antibiotic 1 had a clear zone of 5.33. Antibiotic 2 showed a greater inhibition zone when compared to antibiotic 1 even though there was no antibiotic. This can be seen as not an anomalous result as both discs were dipped in water which could be used to compare, this is further backed by the overlapping of the confidence level, the upper confidence level for antibiotic 1 5.83 is greater than the lower confidence level of 4.996 for antibiotic 2 therefore there is a greater chance than 5% probability the results are by chance, so the null hypothesis is then accepted. Increase in concentration of antibiotic from 0% to 25% showed a greater difference in the clear zones. Antibiotic 1 had a clear zone of 15.36 where was antibiotic 2 had a clear zone of 10.88 with a difference of 4.48mm between antibiotic 1 and antibiotic 2. In-addition, there was no overlapping within the confidence levels therefore there is less than 5% probability the results are by chance therefore the null hypothesis is rejected. The findings at 25% antibiotic concentration indicates antibiotic 1 is more effective than antibiotic 2 at preventing bacterial growth which is evident through the significant difference in the mean diameter zone. The effectiveness of antibiotic 1 compared to antibiotic 2 is further backed up when increasing the antibiotic concentration from 25% to 50%. The mean size of the clear zone for antibiotic 1 was 18.40mm whereas antibiotic 2 the inhibition zone was 11.40mm, showing a 7mm difference between both antibiotics at 50% concentration. Within these concentrations the confidence limits do not overlap as before therefor there is less the 5% probability the results are by chance and the null hypothesis is rejected. At 100% antibiotic concentration antibiotic 1 had a mean clear zone of 19.43 where was as antibiotic 2 the mean clear zone was 12.86mm. Confidence levels for both antibiotics showed no overlapping therefor the null hypothesis is rejected as there is less than 5% probability the results are by chance. Overall, 100% antibiotic was the most effective at preventing bacterial growth, as 1 antibiotic had the greatest mean clear zone and 0% antibiotic concentration was the least effective at preventing bacterial growth. Therefore, it can be concluded antibiotic 1 is more effective at preventing bacterial growth when compared to antibiotic 2 and overall the results suggest there are no anomalous results.
Both antibiotics show evidence for the reduction of bacterial growth however there are 4 mechanisms the way antibiotics to reduce the growth of bacteria. The first way in which antibiotic operate is the disrupt the cell membrane, the cell membrane is disrupted which allows substances to move inside causing the bacteria to burst therefore preventing the growth of bacteria. The second way antibiotics function is inhibiting nucleic acid synthesis, this prevents the bacteria from dividing by binary fission and not able to replicate its DNA, causing the growth level to stay the same. The Third way antibiotics slow down bacterial growth is inhibiting the synthesis of the cell wall. The antibiotic inhibits the enzyme which provides structure to the cell wall, this prevents cross links therefore the bacteria loose its structure and inhibits their ability to function properly. Final way antibiotics way is inhibiting the mRNA translation, this prevents the mRNA from binding to the ribosome therefore the proteins the bacteria requires are not produced leading to the death of the bacteria. The control for both unknown antibiotics was by dipping the paper disc in water, which is classed as 0% antibiotic concentration, this was then placed in both petri dishes. This was a control to show the clear zone was produced due to the effect of antibiotic and not any other factors.
Limitations
One of the limitations of the experiment is human error, human error lead to the results being affected. In the experiment the antibiotic 1 soaked disc for 50% and 100% concentration were placed in close proximity. This error led to the overlapping of the clear zones therefore it made it really difficult to assess the effectiveness of the concentration of the antibiotic and made it difficult it measures the size of the clear zone, therefore this will have affected my conclusion in assessing which concentration for antibiotic 1 was most effective. Furthermore, there can be human error when measuring the size of the clear zone using a ruler, this will lead to different results being recorded.
Another limitation is contamination, slight opening and closing of the petri dishes to place the paper disc on the agar plate. Whilst opening the petri dishes microbes in the air may enter into the nutrient rich petri dish which could affect the effectiveness
of the antibiotic in preventing the growth of the bacteria, which will lead to different clear zones being produced therefore affecting the results which could lead to a different conclusion at which concentration and which antibiotic is most effective at killing the bacteria.
Improvements
The first improvement that should be made is doing repeats, the repeats will allow you to assess the validity of the results and spot anomalous results as the experiment was only carried out once. In-addition, instead of using a ruler to measure the inhibition zone a digital Vernier Caliper should be used which is more accurate than measuring with a ruler, therefore the results will be more precise and reliable, this will give a better indication into the effectiveness of different concentrations of antibiotics on the growth of bacteria.
by
sheeraz321 (student)
