THE EXPERIMENTAL DESIGN
The Fruits of Our Labor
CONTROLS, DEPENDENTS, AND INDEPENDENTS OF THE EXPERIMENT
Independent Variable: Amount and presence of silver nanoparticles.
Dependent Variable: The impact of silver nanoparticles on the growth of E. coli bacteria.
In our experiment, we have many constants. We will be using the same silver nanoparticles and strain of the bacteria, E. coli, throughout our procedure. The number of bacteria that we will be growing for each test will be the same. We will be using the same model of jars when creating the diluted concentration of the silver nanoparticles. Furthermore, we will also be using the same branded paper plates when growing the bacteria.
Our investigation will have a control group. The control group will be a culture of E. coli bacteria that will not contain silver nanoparticles throughout the duration of our experiment. By comparing the control group to other test groups we will be able to understand the precise effects of silver nanoparticles on strains of bacteria.
OUR PROCEDURE
Procedure
Gather all of the materials.
Wear nitrile gloves to prevent spread of germs.
Sterilize five jars using isopropyl alcohol that is 91%.
Label five jars (sizes may vary) with a permanent marker like this:
Jar #1: 500,000 μg/L
Jar #2: 50,000 μg/L
Jar #3: 5,000 μg/L
Jar #4: 500 μg/L
Jar #5: 50 μg/L
Sterilize your workspace with isopropyl alcohol.
Sterilize the tweezers by placing them in isopropyl alcohol and waiting until they dry.
Using a 25 mL graduated cylinder, put 22.5 mL of distilled water into each jar.
Place 25 mL of colloidal silver into Jar #1.
Take 2.5 mL of the solution in Jar #1 and place it in Jar #2. (This is 1:10 dilution)
Take 2.5 mL of the solution in Jar #2 and place it in Jar #3. (This is 1:10 dilution)
Take 2.5 mL of the solution in Jar #3 and place it in Jar #4. (This is 1:10 dilution)
Take 2.5 mL of the solution in Jar #4 and place it in Jar #5. (This is 1:10 dilution)
Using the sterile tweezers, pick up the sterile discs and place three into each jar. Let them sit for 12-24 hours.
Light a candle and place it next to your workspace.
Set out 5 nutrient agar media plates and label them 1-5 with the date that you started experiment on the bottom of the plates (Side without lid). Make sure they are refrigerated before use.
Take out vial of activated E. coli bacteria and insert sterile cotton swab into fluid
Carefully, open the first nutrient agar media plate and streak E. coli bacteria across surface
To streak, touch surface of cotton swab at top of media plate
Move swab right to left across media plate while moving downward
After reaching bottom, turn media plate 60 degrees to the right
Repeat steps a. And b.
Turn 60 degrees once again, and repeat a. And b.
Take the three sterile discs from Jar #1 and place on the bacteria-streaked nutrient agar plate (1) in a triangular shape approximately 3 cm away from one another.
Repeat the streaking process on the next agar plate followed by the placing of sterile discs from the next jar. Continue this process until all agar plates streaked and sterile discs are placed.
Ex. Disks from jar #3 should go on plate labeled 3
Close all the agar plates and carefully flip them over so that the agar is on top. This will allow the bacteria to grow.
Tape the agar plates to secure them from any leakage of bacteria.
Cautiously move the plates from the workplace to an area where they will not receive direct sunlight.
Once again, sterilize the workplace as well as any other things that may have touched the E. coli bacteria with isopropyl alcohol.
Wait 24 hours and measure the width of any zones of inhibition on the agar plate.
Repeat this observation process for the next 5 days.
When finished with the agar plates, sterilize them with 10% bleach for 1-2 hours.
Sterilize the area where the plates were left with isopropyl alcohol.
*If wanted to create another set of media plates as we have done, label 5 more media plates with 1A, 2A, ect. Then, repeat steps 13-22 before continuing with 23-27*
DATA AND DATA ANALYSIS
Our team investigated the effect of the silver nanoparticles on E. coli bacteria by conducting an innovative, measurement-based investigation. In the experiment, we conducted measurements on the zones of inhibition, or the areas in which bacteria have been neutralized in the presence of silver nanoparticles. We compared and tested the E. coli via 5 different concentrations of silver nanoparticles: 500,000 micrograms per liter, 50,000 micrograms per liter, 5,000 micrograms per liter, 500 micrograms per liter, 50 micrograms per liter, and the control. Upon the course of 5 days, our team measured the total diameter of the circular region of bacterial inhibition via a millimeter-measuring ruler. With this information, we were able to express this data in different ways.
First and foremost, we decided to create a line graph expressing the growth of the zones of inhibition for each disk in every plate over the course of the experiment. This representation displays how each of the discs in each plate grew throughout the experiment.
*View graphs above for more information.*
Secondly, we represented the diameters of the zones of inhibition for each of the discs in every plate comparative to the diameters of the zones of inhibition of other discs. We created a bar graph with this information for all 5 days of this experiment. This representation of data clearly shows the progression of each disk in every plate to other discs in other plates on a daily scale.
**View graphs above for more information.**
Last but certainly not least, we conducted testing relating to the surface area of the inhibition. During the same time period, we compared and contrasted the gradually decreasing bacterial population and analyzed the overall growth of zones of inhibition. Using the diameter measurements, we calculated the average surface area of each of the different concentration and the control per day. We did this by finding the average diameter of the 6 discs per concentration per day. Then we took this number and dividing by 2 to get the average radius of the 6 discs per concentration per day. Finally, we squared the average radius and multiplied by pi to gain the average surface area of each concentration per day. With this bar graph, one can clearly view which concentrations averaged in the largest zones of inhibition over the course of the experimentation. This condensation of information gives the simplest overview of the data we have derived from our experimentation.
**View graphs above for more information.**
OUR ULTIMATE CONCLUSION
The results of our experiment conclusively supported our hypothesis. We believe that the presence of silver nanoparticles will be a fast, effective, and reliable method of inhibiting microbial growth due to its ability to diminish permeability of the cell membrane and create pits and gaps in cell structure. Our data indicated this through the presence of inhibition zones on the bacterial media plates. As the concentration of silver nanoparticles increased, the inhibition zone became larger. Lower concentrations of silver had a very minimal impact on the bacteria and in the creation of inhibition zones. Furthermore, our hypothesis was evidently proven by multiple data findings and analysis:
From our “Zones of Inhibition Growth Data, Part 1”, we concluded that higher concentrations of silver nanoparticles had larger inhibition zones. These line graphs presented how each of the inhibition zones on every plate developed over the time period of the experiment. This data proved that higher concentrations of silver nanoparticles developed large inhibition zones while lower levels of concentrations of silver nanoparticles showed little to no growth of inhibition zones.
Further, our hypothesis was supported by our “Zones of Inhibition Growth Data, Part 2”. This clearly showed how the growth between plates compared to one another on a day to day basis. The data on the graphs detailed how, over time, the difference between inhibition zones of the plates slowly developed further apart. These charts also gave us day by day comparison for each of the plates to one another. It explained how plates with higher concentrations of silver showed earlier signs of inhibition.
Finally, our “Zones of Inhibition Growth Data, Part 3” promotes and proves our data in a logical and simplistic manner. This aspect is most essential in the overall representation of our data. This graph not only compares the average surface area(of inhibition zones) of each concentration to one another, but it also displays the overall surface area(of inhibition zones) growth of each of the concentrations over the course of the time period. Taking the average surface area of each of concentrations of silver nanoparticles, it is evidently proven that higher concentrations of silver nanoparticles produce larger zones of inhibition.