• Join over 1.2 million students every month
  • Accelerate your learning by 29%
  • Unlimited access from just £6.99 per month

An investigation into the effect of ampicillin on the growth of two different species of bacteria

Free essay example:

An investigation into the effect of ampicillin on the growth of two different species of bacteria

        I will be investigating the effect of an antibiotic on two different species of bacteria.  The two species I will be using are Escherichia Coli and Bacillius Subtilis.  I am using these two species is because Escherichia Coli is gram negative and Bacillius Subtilis is gram positive.  These two are safe species in the sense that being incubated at 25°C will not create any pathogens.  Since they are two different species of bacteria, I can see if the antibiotic will affect one species more than the other.  The difference between gram positive and gram negative bacteria is that gram positive bacteria has a thicker peptidgolycan wall around the outside of it's cell whereas gram negative bacteria has an outer wall composed of [other compound] and a thin peptidoglycan wall further into the cell.  

        The antibiotic I will be using is ampicillin.  Ampicillin is a synthetic derivative of penicillin.  Penicillin works by affecting the formation of peptidoglycan cross links inside the bacterial cell wall.  With a weakened wall, the bacteria is more susceptible to cell lysis.  The outer wall of gram negative bacteria is not composed of peptidoglycan, however, ampicillin has an amino group in its structure which helps in penetrating the outer wall.  

        Ampicillin works by inhibiting the enzyme which is responsible for making the cell walls, so as a hypothesis, I'd predict that ampicillin will affect gram negative and gram positive equally.  I have predicted this because ampicillin still retains the ability to disrupt cell wall synthesis, this makes gram positive species susceptible to cell lysis.  Also, gram negative bacteria are affected due to the presence of the amino group which is not present in penicillin, so gram negative bacteria will be killed at the same rate as gram positive, hence, an equal affect is achieved.  

H0 – The antibiotic will have no effect on either of the two species

H1 – The antibiotic will affect both species equally

        For the experiment, I will be using the bacterial lawn method.  The reasons for choosing this method is because with a poured plate, there is a possibility for the bacteria to disperse as I pour the bacteria into the dish, showing a clear area with a halo effect even though there has been no antibiotic applied.  I will carry out the experiment using the following equipment and measurements:

  • Glass rod for spreading bacteria
  • Bunsen Burner
  • Six bottles of 25cm3 agar
  • Bottle of Escherichia Coli
  • Bottle of Bacillius Subtilis
  • Varying concentrations of ampicillin: 0 μl, 0.2μl, 0.4 μl, 0.6 μl, 1μl and 2μl
  • Six petri dishes

        I chose these different concentrations of antibiotic to see if there is a direct, proportional relationship between the strength of antibiotic and bacteria.  If I chose higher concentrations it is likely that that concentration would have killed all the bacteria on the plate, making it impossible to have any relevance in my results.  The zero concentration is to show that it is the ampicillin causing the clear area around the  disc and not anything else.  I will apply the ampicillin via micropipetting a set volume of antibiotic onto the disc because dipping the disc into ampicillin and flooding it with antibiotic is not practical.  

I will use sterile technique in order to prevent foreign microbes contaminating the plate before incubation as they could affect my results:

  1. Sterilise bench with disinfectant.
  2. Start a bunsen flame to start an updraft and prevent microbes from falling into the work area
  3. Flame end of glass rod used to spread bacteria
  4. Shake bottle to lift bacteria settled at the bottom
  5. Twist the lid off the bottle containing bacteria, ensuring not to turn the lid upside down
  6. Flame neck of the bottle
  7. Insert glass rod, ensuring only the flat end comes into contact with the bacteria
  8. Flame neck and reapply lid
  9. Lift lid of the petri dish slightly, ensuring the lid is not fully removed so other microbes do not contaminate plate
  10. Spread bacteria all over the agar using glass rod and close lid
  11. 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.                  

Mean diameter of zone of clearance (mm)

Bacillus subtilis

Concentration of Ampicillin (µg / µL)

0

0.25

0.50

1.00

2.00

4.00

0

37

43

50

46

58

0

40

36

37

44

43

0

0

29

38

40

47

48

0

28

31

37

40

41

0

24

36

31

39

50

0

27

33

32

40

50

0

41

43

44

50

49

0

40

43

40

45

45

0

38

40

46

48

51

0

34

26

33

40

61

0

38

35

43

43

48

0

35

36

46

45

45

0

36

43

41

45

46

0

37

39

44

48

63

Mean:

0

35

37

40

44

50

Standard Deviation:

0.00

5.48

5.08

5.71

3.46

6.56

Mean diameter of zone of clearance (mm)

Escherichia coli

Concentration of Ampicillin (µg / µL)

0

0.25

0.50

1.00

2.00

4.00

0

23

26

31

33

42

0

25

27

33

27

37

12

25

27

30

31

36

0

8

18

23

27

31

0

11

19

21

23

26

0

20

20

26

33

34

0

16

19

22

27

30

0

18

26

27

28

29

0

18

22

27

28

28

0

20

24

28

30

30

0

17

21

21

44

50

0

21

20

24

32

25

0

15

16

26

26

26

0

16

20

24

26

28

0

24

27

31

30

29

Mean:

1

18

22

26

30

32

Standard Deviation:

3.09

4.95

3.71

3.80

4.93

6.77

        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.         image00.pngimage01.png

        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.

Concentration of ampicillin

0

0.25

0.5

1

2

4

Student T Test:

0.312225379

6.06837E-09

6.14661E-09

1.64508E-08

9.41077E-10

1.05899E-07

Null hypothesis accepted

Null hypothesis rejected

Null hypothesis rejected

Null hypothesis rejected

Null hypothesis rejected

Null hypothesis rejected

        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.  

This student written piece of work is one of many that can be found in our AS and A Level Molecules & Cells section.

(?)
Not the one? Search for your essay title...
  • Join over 1.2 million students every month
  • Accelerate your learning by 29%
  • Unlimited access from just £6.99 per month

Related AS and A Level Science Skills and Knowledge Essays

See our best essays

Related AS and A Level Molecules & Cells essays

  1. Marked by a teacher

    An investigation into the inhibiting effect of tomato juice on the germination of cress ...

    3 star(s)

    This shows that tomato juice does indeed have a property that significantly affects the amount of germination. For instance, if tomato juice did not have an effect on germination, then the results after tomato juice had been added would

  2. Marked by a teacher

    Beetroot Practical Write up

    3 star(s)

    To conclude the betalain pigment in beetroot diffuses out of the cell when the membrane proteins are damaged and the lipids are dissolved. This is because of the change in pH to the cell membrane which causes the proteins to denature and no longer hold a complete structure.

  1. Investigating how different concentrations of a antibiotic effects the growth of a bacterium.

    Penicillin kills bacteria by interfering with the ability to synthesize cell wall. The diagram to the right shows the chemical structure of Penicillin. THE GROWTH OF BACTERIA-BINARY FISSION The next point to consider on the scientific explanation is how the bacteria grow.

  2. Catalyse Investigation

    Let me write specifically about the enzyme, catalase and the substrate, hydrogen peroxide. In organisms, hydrogen peroxide is a toxic by-product of metabolism, of certain cell oxidations to be more specific. Hydrogen peroxide on its own is relatively stable and each molecule can stay in this state for a good few years.

  1. Investigating the effect of sodium chloride on the action of antibiotics on bacteria growth

    They primarily live in the mammalian gut. They are part of a group of bacteria known as enteric bacteria" The non-pathogenic strains are the ones to be used in this investigation. This is because they are safe to handle and are easy to grow under optimum conditions compared to other microbes.

  2. Cost effectiveness of mainstream bench cleaners against generic supermarket cleaners on the number of ...

    a cleaner on an agar plate that has already been inoculated with bacteria. This will be done after serial dilutions and a viable count. I will then repeat the same process with the other cleaners and incubate them all along with a control agar, which has been inoculated with bacteria

  1. Rate of diffusion investigation

    If two solutions containing different concentrations of a solute are separated by a selectively permeable membrane (permeable to water but not to the solute), water will move from the solution with low solute concentration to the solution of high solute concentration.

  2. Catalase investigation

    structure, meaning the folded structure that forms the active site. Aim In this experiment I hope to determine the effect that increasing concentration has on rate of reaction. The enzyme in question will be catalase. I will use liver as a source of catalase for this investigation.

  • Over 160,000 pieces
    of student written work
  • Annotated by
    experienced teachers
  • Ideas and feedback to
    improve your own work