Purpose
The purpose of this lab is to discover which native plants contain active ingredients that will be able to inhibit the growth of bacteri
Materials
Plant specimen
10 mL syringe
test tubes
Methanol
1 mL pipet
Ampicillin
Inoculating Loop
60x15 Petri dish
E. Coli JM109 (stock plate)
10 mL pipet
100 mL beakers
5mm filter paper
250 mL media bottle
LB Agar
LB broth base
Plastic funnels
Procedure
Part I
1. Prepare an LB broth for the E. coli. Wait 24 hours and then add a colony of the E. coli to the broth.
2. Get six petri dishes. Draw a "+" symbol on each plate and then divide the dish into 4 quadrants. Label the quadrants 1-4 and put your initials and the date on the plate as well.
3. Liquefy LB agar by heating it in the microwave. Pour approximately 20 mL of the agar into each petri dish. Let the agar solidify for 24 hours.
Part II
1. Grind up your plant specimen in deionized water. Filter it through filter paper. Then filter the mixture again through a syringe filter. Collect 1 mL of this mixture and put it in a labeled test tube.
2. Grind up your plant specimen in methanol. Filter it through filter paper. Then filter the mixture again through a syringe filter. Collect 1 mL of this mixture and put it in a labeled test tube. Then place the test tube in a 65 degree Celsius heat block with the cap open for 24 hours. After, put 10 mL of deionized water into the test tube.
3. Repeat steps 2 and 3 until you have 6 total samples. You will have 3 of each sample.
4. Drop 3 filter paper disks into each test tube.
5. Prepare 6 negative control disks of methanol and distilled water. (3 each)
6. Prepare 6 positive control disks of ampicillin.
7. Close tubes and store at 4 degrees Celsuis.
Part III
1. Put 1mL of E. Coli onto your petri dish and spread it around using a cleansed spreading loop. Wait for 15 minutes.
2. Place one disk in its appropriate quadrant using sterilized forceps. Make sure you have a positive and negative control disk and both sample disks.
3. Incubate the petri dishes at 37 degrees Celsius for 24 hours.
4. After 24 hours, examine the petri dishes for zones of inhibition. Photograph and record your results.
The purpose of this lab is to discover which native plants contain active ingredients that will be able to inhibit the growth of bacteri
Materials
Plant specimen
10 mL syringe
test tubes
Methanol
1 mL pipet
Ampicillin
Inoculating Loop
60x15 Petri dish
E. Coli JM109 (stock plate)
10 mL pipet
100 mL beakers
5mm filter paper
250 mL media bottle
LB Agar
LB broth base
Plastic funnels
Procedure
Part I
1. Prepare an LB broth for the E. coli. Wait 24 hours and then add a colony of the E. coli to the broth.
2. Get six petri dishes. Draw a "+" symbol on each plate and then divide the dish into 4 quadrants. Label the quadrants 1-4 and put your initials and the date on the plate as well.
3. Liquefy LB agar by heating it in the microwave. Pour approximately 20 mL of the agar into each petri dish. Let the agar solidify for 24 hours.
Part II
1. Grind up your plant specimen in deionized water. Filter it through filter paper. Then filter the mixture again through a syringe filter. Collect 1 mL of this mixture and put it in a labeled test tube.
2. Grind up your plant specimen in methanol. Filter it through filter paper. Then filter the mixture again through a syringe filter. Collect 1 mL of this mixture and put it in a labeled test tube. Then place the test tube in a 65 degree Celsius heat block with the cap open for 24 hours. After, put 10 mL of deionized water into the test tube.
3. Repeat steps 2 and 3 until you have 6 total samples. You will have 3 of each sample.
4. Drop 3 filter paper disks into each test tube.
5. Prepare 6 negative control disks of methanol and distilled water. (3 each)
6. Prepare 6 positive control disks of ampicillin.
7. Close tubes and store at 4 degrees Celsuis.
Part III
1. Put 1mL of E. Coli onto your petri dish and spread it around using a cleansed spreading loop. Wait for 15 minutes.
2. Place one disk in its appropriate quadrant using sterilized forceps. Make sure you have a positive and negative control disk and both sample disks.
3. Incubate the petri dishes at 37 degrees Celsius for 24 hours.
4. After 24 hours, examine the petri dishes for zones of inhibition. Photograph and record your results.
Conclusion
1. Did any extract give you positive results?
Yes 2. Did your controls work as expected? Yes 3. Discuss errors that could give you false results. Human Error 4. Discuss further experimentation. Try the experiment on different Ecoli strains 5. Discuss next steps. Repeat these experiments in a more sterile environment. |
Think Like a Bio Technician1. If an extract gives a negative result in the antimicrobial assay, does that mean that the extract is not an antimicrobial agent? Most of the time, it does mean that the extract is not an antimicrobial agent. But sometimes, the extract does not diffuse properly due to differences in molar molecules. 2. In preparing the sample disk, some of the methanol extractions smell like alcohol. Why is that a problem? Alcohol kills bacteria and this is a huge problem because it would destroy the experiment. You would not know if the extract had been altered and this would cause your results to considered false. 3. Each extract may have one or more compounds in it. What should be done to begin to identify the exact compound in an extract that is causing the antimicrobial action? Chromatography is one method that is used to separate and identify different compounds within the extract. This separates the different compounds by their polarity. Once separted, this would allow you to test for antimicrobial action. |
What did you like/dislike about the lab?
I found it interesting that our plant was able to repeal the strand of ecoli in our petri dish. I disliked using the filters they were just to to tedious to pry apart. How did you and your partner collaborate? My partner was Logan Gleeson and we worked very well together and if one of us would mess up we help each other throughout the lab. What would you do differently next time? I would try a different plant in this experiment. |