3. Methodology

Variable: 
Quantity:
Amount of bacteria
Number of Colonies
Amount of bacteria sample spread
100 µL
Amount of cleaning agent spread
10 µL



Materials Needed:
Amount:
Petri Dishes with LB Agar
25 Dishes
L Shaped Glass Rods
10 Packs
Inoculation Loops
10 Packs
Pipette (100µL+1000µL)
2 Pipettes
Pipette Tips (100µL+1000µL)
1 Box/Pipette
Centrifuge Tubes, Plastic
1 Pack 
Distilled Water
2 x 500mL
E. coli B. subtilis Samples
-
Preparing the bacteria for experiment

  1. Prepare 10 agar filled petri dishes, and label them 1-10 
  2. Prepare 10 centrifuge tubes of 9mL distilled water each, and label them 1-10
  3. From the sample of E.coli, using the 1mL pipette, transfer 1mL of the sample into the distilled water in tube 1, and cap the tube and invert tube to make sure bacteria is mixed evenly
  4. From the sample in tube 1, take out 1mL of the mixture into 9mL of distilled water in tube 2, cap the tube and invert tube to make sure bacteria is mixed evenly
  5. Repeat step 4 with different tubes until all 10 tubes are filled
  6. Pipette 0.1mL of the mixture from centrifuge tubes 1-10 into petri dish 1-10 respectfully, and spread evenly with the L shaped glass rod
  7. Incubate the 10 petri dishes overnight at 37 degrees Celcius
  8. After the incubation, check each petri dish for the number of colonies. Use the petri dish that has 30 - 300 colonies.
  9. Check the petri dish number of the dish that is chosen, and use the corresponding dilution for future incubation (in experiment)

* In any case that 10 dilutions still produce too much bacteria, continue to dilute further.


Conducting the actual experiment
  1. Label 4 agar filled petri dishes according to the antibacterial agent to be spread on it and leave one as a control
  2. Using the tube that was chosen previously, spread 0.1mL into each petri dish
  3. Using the L shaped glass rod, spread the bacteria evenly across the petri dish
  4. Pipette 0.01mL of each anti-bacterial agent onto its respective petri dish
  5. Using the L shaped glass rod, spread the anti-bacterial agent evenly across the petri dish
  6. Incubate all the dishes overnight at 37 Degrees Celcius overnight
  7. Repeat steps 1-6 two more times.
  8. Gather results by counting the number of visible bacteria colonies after step 7 using a permanent marker and a black coloured paper (place the paper at the bottom of the petri dish), and fill up the table as below.

*In any case that the agent used is too thick, using a pair of flamed scissors, cut the tip before pipetting the agent.


Result Planning

* Sample table


The values in the table represent the amount of remaining bacteria (E.coli) colonies left "unkilled" by the antibacterial agents. These values are obtainable by counting the amount of visible bacteria colonies in the petri dish.
With our results after filling in the table, we will plot two different bar graphs. 
The two types of bar graphs we will be plotting will be the percentage of colonies each petri dish has against the number of colonies in the control, and the amount of colonies every petri dish has against the number of colonies in the control.
The reason why we chose to use a bar graph to represent the data was because we wanted to show the contrast of the number of colonies from each petri dish against another, and a bar graph would be able to show the obvious differentiation. If other graphs were used, the graphs such as pie charts, line chart etc. would not be able to show the contrast between the number of colonies in each dish.

The graphs we would plot would look like these below:

Sample Graph 1 (Average amount of colonies)                                          

Sample Graph 2 (Average percentage of colonies) 

This graphs shows the percentage of colonies in each petri dish compared to the control (100%), using the average number of colonies through the 3 trials conducted.


*The above graphs are sample graphs, and do not have any actual results taken from the experiment.



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