I will use sterile technique in order to prevent foreign microbes contaminating the plate before incubation as they could affect my results:
- Sterilise bench with disinfectant.
- Start a bunsen flame to start an updraft and prevent microbes from falling into the work area
- Flame end of glass rod used to spread bacteria
- Shake bottle to lift bacteria settled at the bottom
- Twist the lid off the bottle containing bacteria, ensuring not to turn the lid upside down
- Flame neck of the bottle
- Insert glass rod, ensuring only the flat end comes into contact with the bacteria
- Flame neck and reapply lid
- Lift lid of the petri dish slightly, ensuring the lid is not fully removed so other microbes do not contaminate plate
- Spread bacteria all over the agar using glass rod and close lid
- Apply antibiotic disc and incubate at 25°C for 48 hours, allowing bacteria to fully grow.
After the bacteria have been incubated, I expect to see a clear area with a halo effect around the antibiotic disc where the antibiotic has diffused out via disc diffusion. The clear area shows the absence of bacterial growth and how far the antibiotic has diffused through the agar.
I will measure the diameter of the area three times; once along the longest distance, one across the shortest distance and another random distance and take an average of the three measurements in order to calculate a final result for that concentration of antibiotic.
After gather results I plan on using the student t test. I am not choosing to use the chi squared test because the chi squared test counts individuals whereas the student t test considers measure values. Seeing as we're collecting paired data, it is more practical to use the t test.
The student t test is designed to show whether the experiment was statistically valid and did not occur by chance. If the experiment has less than a 5% chance of error, then the null hypothesis is rejected in favour of another hypothesis.
Analysis
The investigation was carried out as I planned except for the following changes. Instead of repeating the experiement, I used pooled data in order to get repeat values because there was not enough time to carry out repeats of the experiement. Applying the ampicillin onto the disc using wicking instead of a micropipette; does not guarantee an even distribution of antibiotic, making each disc have a contain a different volume of ampicillin and affect the overall results. The concentration I used in the investigation were also too high; if lower concentions were used, it would have decreased the amount of overlapping between zones of clearance and increased accuracy when measuring diameters.
The table of results shows pooled data I have collected, the highlighted cells being my own data. Obvious anomalous results have been omitted seeing as they have they could negatively influence my final result and increase the percentage of error in the investigation.
My own results showed a 0 for all my Bacillus Subtilis plates and an positive correlaton between concentration and mean diameter of zones of clearance in the Escherichia Coli samples, therefore we can say that higher concentrations of ampicillin has a greater effect on bacterial replication. The reasons for the all of the results being 0 could have been due to several possibilities. No bacterial growth could have occurred from the glass rod used to spread the bacteria being too high a temperature, killing the bacteria on contact. There is also the possibility that the concentration of bacteria was so thin and dilute that even the weak concentrations of ampicillin inhibited the growth of bacteria on the entire plate. A more dilute concentration of bacteria would have probably occurred from the bottle of bacteria not being well shaken beforehand, leaving much of the bacteria at the bottom of the bottle and resulting in none or very little being transferred onto the glass rod.
Bacterial growth decreased as ampicillin concentration increased because ampicillin acts as a competitive inhibtior against the enzyme transpeptidase which forms cross links and finalises bacterial cell wall synthesis. A higher concentration has more ampicillin present, allowing more enzyme inhibition to occur. Water moves into the cytoplasm via the semi permeable cell wall using osmosis, diffusing from a less negative water potential to a more negative water potential. Normally, with the presence of a cell wall, the cell would become turgid and not rupture due to osmotic pressure, however, after ampicillin has inhibited cell wall formation, the bacteria can no longer withstand the pressure being exerted from the water and dies due to cell lysis. When looking at the two different species, gram positive bacteria have a much thicker peptidoglycan outer wall so the results agree with my initial hypothesis that gram positive would be more sensitive to ampicillin than gram negative bacteria. But, gram negative bactera were also affected by ampicillin so we can assume gram negative bacteria do not fully utilise the transpeptidase enzyme but have a cell wall composed of another compound and therefore is still affected by the mechanism of ampicillin.
However, the two variables are not directly proportional even though we would expect them to be. For example, if we look at 2µg / µL and 4µg / µL in both experiments, we see that the mean diamater increases from 44mm to 50mm in the Bacillus Subtilis and in the Escherichia Coli the mean diameter increases from 30mm to 32mm. Also, when looking at 0.5µg / µL and 1µg / µL, the mean increase in diameter is from 37mm to 40mm and 22mm to 26mm. Since these two variable are not directly proprotional, it would prove innaccurate for me to extrapolate the points from my graph to predict zones of clearance at higher concentrations. If the two variables were directly proportional, we would expect a straight line, making it straightforward to extrapolate a line of best fit for higher, unknown concentrations.
The table also shows that the gram negative species (Escherichia Coli) was less affected by the antibiotic than the gram positive species (Bacillus Subtilis); the gram negative bacteria had lower means at all concentrations than the gram positive. Since ampicillin has a larger effect on gram positive bactera, this shows that the mechanism of ampicillin mainly affects peptidoglycan walls because gram positive species have a much larger amount of peptidoglycan present in their outer walls than gram negative bacteria.
Above are the results for the Student T Test which test the null hypothesis of the experiement. If the p value is less than 0.05, the experiment is proven to be statistically valid and the outcome has not occurred due to chance, allowing the null hypothesis to be rejected in favour of another hypothesis. The control sample which only had sterile water on it had a p value higher than 0.05, showing that the null hypothesis can be accepted and there will be no effect on the different species. This agrees with the results collected because no affect would be expected due to no antibiotic being present on the disc so there would be no difference in effect. All the other T Tests values are less than 0.05; meaning that the null hypothesis can be rejected in favour of another hypothesis. The other hypothesis states that there will be a difference in sensitivity to ampicillin on different bacterial species.
Limitations
Limitations and errors were expected in the experiment due to so many techniques and apparatus used. There was a large amount of error in the filter paper discs used to hold the antibiotic. The first issue was the discs not being a uniform diameter and thickness, because the discs varied in these dimensions, different amounts of antibiotic could be held in the discs, causing some discs to have a larger volume of ampicillin than others and creating a larger zone of clearance than there should be. The zone of clearance is larger because there is more antibiotic available to diffuse through the agar using disc diffusion.
There were also a zone of clearance produced from some control discs due to the bacteria solution not fully drying and the disc being saturated with water. As the disc is placed and pressed upon the agar, the bacterial solution is pushed away from the filter paper disc, creating a zone of clearance when there should not be one, creating a clear zone which is not from the antibiotic.
Sterile technique was also quite difficult to carry out strictly in a school laboratory. Even though there was no visible evidence of any foreign microbes growing on the agar, microbes resistant to ampicillin could have contaminated the antibiotic disc whilst being transferred onto the agar and affected transpeptidase inhibition. Another problem was the time period in which the plates were checked for bacterial growth; checking the plates after incubation for 24 hours could have showed if the plates showing 0 for all concentrations was due to no bacteria being present on the plate or the weaker concentration diffusing completely through the plate.
Only using two species of bacteria, one gram positive and one gram negative, limited the investigation by implying that the effect applies to all bacteria from the two different groups. Using more species of bacteria could have shown whether ampicillin has the same or a different effect on different species.
There was also a limitation with antibiotics; there were too few concentrations used. More concentrations used could have increased accuracy and also shown whether there is a maximum effect on the inhibition of bacterial growth because ampicillin acts as a competitive inhibitor on the enzyme transpeptidase, preventing cross link formation, leading to cell lysis. If ampicillin acts as a competitive inhibitor, then there must be a point of maximum effect where no more active sites can be occupied.
Despite having results which agreed with my hypothesis, the average values were not directly proportional. This means that extrapolating any line of best fit would not produce an accurate prediction for the area of effect for higher concentrations of ampicillin. Even with mean values and a line of best fit, the only way to improve the results would be to use more concentrations of antibiotic. More points plotted would results in a great level of accuracy and therefore a more accurate prediction for higher, unknown concentrations of antibiotic. The investigation could have also been extended to include other similar antibiotics, such as penicillin or amoxycillin, to see whether or not they are more effective in inhibiting the growth of gram positive and gram negative bacteria.
Conclusion
As a result of the investigation of the effect on ampicillin on different species of bacteria, I can conclude: the higher the concentration of ampicillin, the greater the effect it has on bacterial growth and that gram negative bacteria are less sensitive to ampicillin than gram positive bacteria.
