To test plant extracts to see if they possess antibacterial activity. PROCEDURE First create a list of plant extracts being tested and choose 2 extracts per group. The plants being tested are Oregano, Tea tree, Eucalyptus and Colloidal Silver. Obtain two nutrient agar plates and using a permanent marker draw four quadrants on the bottom of each agar plate. Using a sterile pipet transfer 250 ml of E. coli broth to the middle of each petri dish and evenly spread bacterial culture around the agar plate. Cover and allow the culture to soak into agar for at east 15 minutes.
Using sterile forceps, carefully place one filter disk from designated sample into the middle of each quadrant. The samples should read as follows: Quadrant #1: water, Quadrant #2: Ampicillin, Quadrant #3: Plant extract 1, Quadrant #4: Plant extract 2 (also see figure 1) Make sure the disks are adhering well to the surface of the agar. Label your plates with group name and place them in the 37°C dry heat incubator for 24-48 hours. Once incubation period was over measure the zone of inhibition for each quadrant.
Figure 1: Quadrant sample plant # 13 4 plant #2 water 1 2 Ampicillin RESULTS For this lab, the class was split up into several groups with each group using 2 different plant extracts. As a group, we chose oregano and colloidal silver as our samples. In our agar plate #1, the zone of inhibition for oregano and colloidal silver measured at 7mm. For agar plate #2, the zone of inhibition for oregano measured at 7mm, and for colloidal silver at 6.75mm. We were not the only group who used oregano as a sample, their results measured at 7.5mm. The results for the groups that tested tea tree for antibacterial activity, varied greatly. For one group, tea tree’s zone of inhibition measured at 9mm and for another group it measured at 12mm.
Eucalyptus was measured by only one group and the z.o.i measurement for eucalyptus was 9.75mm. The negative control (H20) and positive control (Ampicillin) were tested by all groups. For water the z.o.i. measured at 6.5mm, 7mm, and 7mm. Ampicillin’s z.o.i. measured at 11.5mm, 11.25mm and 9.5mm. Figure 2 and table 1 depicts our results as a class. The results show that tea tree and eucalyptus have about the same antibacterial activity as the positive control (ampicillin) used in this experiment. Oregano and colloidal silver’s zone of inhibition were closer to h20, which concludes that at the amount used for both there is no antibacterial activity. FIGURE 2
Assaying antibacterial activity in pant extracts. Class averages for Zones of Inhibition. TABLE 1: Zone of inhibition measurements for each sample DISCUSSION There were changes made to the experiment which may or may not have affected the results. The amount of E.coli used changed from 1ml to 250ul. This change could’ve affected the results of tea tree and eucalyptus especial because they demonstrated antibacterial properties. A larger amount of E.coli might have proved too much for both samples and maybe the z.o.i. would’ve measured differently, smaller in size.
On the other hand, it might not have had any affect on the results. The concentration of the sample disks is unknown therefore it is hard to say how much tea tree and eucalyptus you would need to use as potential antibiotics. One way to change the experiment would be to have known concentrations of each substance. By knowing the concentration of each sample and testing them, would determine at what level antibacterial activity occurs. The disk themselves were heavily saturated with substances which caused disk to move around on agar plate.
In order to make sure that disk would not continue moving on plate, the plates were set aside to allow for disks to settle in their spot before incubating. This could have caused some results to vary since the zone of inhibition could’ve have been compromised. As you can see in Table 1, some of the substances were not tested by all the groups. Oregano and tea tree were tested by two of the groups. Colloidal silver and eucalyptus were tested by only one of the groups. The results could’ve been more conclusive had all samples been tested by all the groups. The measurement of zones of inhibition were made with different instruments which may have led to varying results of the same substance.
Also, not all z.o.i. were a perfect circle, some were oddly shaped which meant you had to use you best judgement on where to start your measurements. This may have led to some inconstancies of results. In future experiments when measurements are involved, the use of the same instrument could minimize errors. When comparing the results of tea tree and eucalyptus to that of ampicillin, it is clear that they both have antibacterial properties similar to ampicillin. Changing the antibiotic or positive control and retesting both tea tree and eucalyptus would be one way to demonstrate their effectiveness.
Oregano has been used for medicinal purposes, in this experiment it failed to prove that it has antibacterial properties. I wonder if at a higher concentration or using a different bacterial culture would change the results of oregano. This experiment called for the use of 37°C dry heat incubator with an incubation time of 24-48 hours. E.coli grows any where from 10°C – 45°C so changing the temperature to a slightly higher temperature could’ve caused the samples to activate antibacterial properties otherwise not activated by a lower temp. Also, the amount of incubation could be lengthen to see if that changes the results.