Which antibiotic is most effective?

Hypothesis:

There will be a zone of inhibition around the antibiotic on the agar plate due to the antibiotic inhibiting the growth of microorganisms (such as bacteria).

In this case it can be seen that the antibiotic A is far more successful in killing the bacteria than B. This is evident as the diameter of the inhibition zone of A, shown by the arrow, is far larger than that of the inhibition zone of B.

Increasing the concentration of a bactericidal antibiotic such as penicillin should increase the number of bacterial colonies it kills.

Apparatus:

Agar plate seeded with known Bacteria
Sterile Pasteur pipette
Bunsen burner
Beaker of disinfectant, 1% Virkon or equivalent
Bench spray of disinfectant, 1% Virkon or equivalent
Bactericidal soap
Paper towels
Marker pen
Forceps
Mast ring or antibiotic impregnated paper discs
Adhesive tape
Incubator set at 30 °C

Method:

1. Wash your hands with the bactericidal soap. Spray the working area thoroughly with the disinfectant spray and wipe with a paper towel after waiting for the disinfectant to act.

2. Prepare an agar plate seeded with bacteria. This may have already been done for you. If not, follow the instructions on the Student sheet ‘Pouring agar plates’ which can be found with Activity 4.19. Label the Petri dish on the base at the edge with your name, the date, and the type of bacterium it is inoculated with.

3. If not already autoclaved, sterilise the forceps by flaming them and allow cooling. Use them to pick up an antibiotic disc or mast ring. Raise the lid of the Petri dish and place the mast ring firmly in the centre of the agar; If individual discs are used they will need to be spaced evenly around the dish.

4. Tape the dish securely with two pieces of adhesive tape (but do not seal it completely), then incubate it upside down for 48 hours at 30 °C.

5. Wash your hands with bactericidal soap and clean the bench again using ...

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4. Tape the dish securely with two pieces of adhesive tape (but do not seal it completely), then incubate it upside down for 48 hours at 30 °C.

5. Wash your hands with bactericidal soap and clean the bench again using the Virkon spray. 6. After incubation, look carefully at the plate but do not open it. Where bacteria have grown, the plate will look opaque, but where the antibiotics have inhibited growth, clear areas called inhibition zones will be seen. Measure the diameter of the inhibition zones in millimetres and use this information to decide which antibiotic is most effective at inhibiting the growth of the bacterium.

Dependant Variable:

Diameter of the inhibition zone-This will be measured after incubation of 48 hours and should be fairly standardized as the antibiotic and concentration used in each technique is undeviating.

Controlled Variable:

Temperature of the incubator-It is fundamental that the temperature of the incubator is controlled as this can affect the rate of bacterial growth. For any given bacterium there are maximum and minimum temperatures beyond which growth will not occur. Penicillin (one of the antibiotics to be used in the pilot study) is a time dependent bactericidal antibiotic that interferes with bacterial growth-hence the rate of bacterial growth is influential to the final result. Therefore to ensure that the rate of growth is uniform in all samples; the agar plates will be incubated in an oven at a constant temperature of 35ºC.
Volume of Bacteria used-The volume of bacteria pipetted into a Petri dish will remain consistent regardless of the culture technique it is introduced to.

Results

Conclusion

Collectively observing the results of my investigation they demonstrate some evidence of the effectiveness of the antibiotic, penicillin, used and how my hypothesis was correct and as the concentration of the antibiotic increased so did the diameter of inhibition zone. There appeared to be no anomalous results for this experiment.

Evaluation

I believe that my experiment was fairly accurate and reliable; as I didn’t have any outliers this can increase the chance that there was no or very little human error. I used the apparatus and measuring equipment carefully and accurately to reduce the chances of systematic error and increasing the accuracy of my results and as I took 2 sets of data for each concentration and both were the same/similar results this proves that my results were quite reliable.

After performing the experiment I feel there is still room for improvement. In the event of a future investigation, I would;

Make more replicates of both the spread and pour plating technique, as well as other culturing techniques (e.g. streak plating) to produce a clear picture of the overall results.

Alternate the chemical class and concentration of the antibiotic to determine whether the effect of culture technique is universal for various antibiotics.