Lab 6d
Purpose:
Which plants in my area contain anti-bacterial ingredients?
Materials:
Procedure:
Part II
4. Grind 2 g of plant tissue (leaves) with 10 mL of deionized water in mortar and pestle, and let sit for 3 minutes. Filter in 11 cm funnel, sterilize extract with syringe filter, and collect 1 mL of extract in labelled 1.7 mL micro tube.
5. Repeat Step 4, except replace deionized water with methanol. Place 1.7 mL tube with 1 mL of methanol extract in 65*C heat block (caps open) for 24 hours to evaporate methanol. Reconstitute dry matter in micro tube with 1 mL of deionized water.
6. Repeat Step 4 and 5 for six samples and label them.
7. Drop filter paper disks in each filtered extract tube using sterile forceps (sterilized by being flamed in alcohol).
8. Prepare three negative control disks of only methanol and sterile and distilled water.
9. Prepare six positive control disks of ampicillin solution.
10. Allow disks to be saturated with the extract (perhaps overnight).
11. Close tubes. Store all samples at 4*C until ready to use.
Part III
12. Transfer 1 mL of the E. Coli broth to middle of Petri dish with sterile pipet. Sterilize spreading loop with alcohol and flame and spread bacteria culture in Petri dish. Cover and let culture soak in agar for at least 15 minutes.
13. Separating the methanol-extracted samples and the water-extracted samples in different dishes, place one disk on each quadrant at least 2 cm away from edge of dish. Block excess liquid before placing disks in.
14. Repeat step 13 twice. (3 methanol extraction replicates and 3 deionized water extraction replicates)
15. Place one of negative control disks in center of the appropriate plate. Place positive control disk with ampicillin in another quadrant of each plate.
16. Finish with 6 Petri plates with a negative control in the center, a positive control, and 3 sample disks. Record which solvents and plant extracts are in each quadrant. Let soak for a few minutes.
17. Ensure disks adhere to surface of agar. Invert the plates and incubate at 37*C for 24 to 48 hours.
18. After incubation, look for at the plates with plant extract disks for zones of inhibition, clear area formed by inhibitory (decrease in action) action of a substance in the plant material around the disk. Photograph the plates, labeling any inhibition of bacterial growth.
19. Create a data table for the replicates and averages. Include descriptions of the bacterial lawn around each disk. Record the diameter and clarity of any cleared areas around the disks in quantitative measurements.
Which plants in my area contain anti-bacterial ingredients?
Materials:
- Balance, weigh boat, lab scoops
- LB broth base
- Media bottles, 250 mL
- Sterilizer/autoclave
- Water bath, 37 degrees Celsius, shaking
- Sterile LB agar
- Laminar flow hood and disinfectant
- Plastic safety goggles
- Bunsen burner and gas lighter
- Inoculating loop, Ni/Cr wire
- Petri dishes, 60x15 mm, sterile
- E. coli JM109 (stock plate)
- Plant specimen
- Mortar and pestle
- Pipet, 10 mL and pump
- Short-stemmed plastic funnels
- Filter paper disks, 5 mm diameter
- 100 mL beakers
- Syringe, 10 mL and filter, 0.2 micrometers
- Reaction tubes and rack, 1.7 mL
- Methanol, absolute
- Pipet, 1 mL and pump
- Dry block heater/heat block
- Forceps, fine tipped
- Ampicillin
- Glass spreader
- Incubator oven, 37* C
Procedure:
Part II
4. Grind 2 g of plant tissue (leaves) with 10 mL of deionized water in mortar and pestle, and let sit for 3 minutes. Filter in 11 cm funnel, sterilize extract with syringe filter, and collect 1 mL of extract in labelled 1.7 mL micro tube.
5. Repeat Step 4, except replace deionized water with methanol. Place 1.7 mL tube with 1 mL of methanol extract in 65*C heat block (caps open) for 24 hours to evaporate methanol. Reconstitute dry matter in micro tube with 1 mL of deionized water.
6. Repeat Step 4 and 5 for six samples and label them.
7. Drop filter paper disks in each filtered extract tube using sterile forceps (sterilized by being flamed in alcohol).
8. Prepare three negative control disks of only methanol and sterile and distilled water.
9. Prepare six positive control disks of ampicillin solution.
10. Allow disks to be saturated with the extract (perhaps overnight).
11. Close tubes. Store all samples at 4*C until ready to use.
Part III
12. Transfer 1 mL of the E. Coli broth to middle of Petri dish with sterile pipet. Sterilize spreading loop with alcohol and flame and spread bacteria culture in Petri dish. Cover and let culture soak in agar for at least 15 minutes.
13. Separating the methanol-extracted samples and the water-extracted samples in different dishes, place one disk on each quadrant at least 2 cm away from edge of dish. Block excess liquid before placing disks in.
14. Repeat step 13 twice. (3 methanol extraction replicates and 3 deionized water extraction replicates)
15. Place one of negative control disks in center of the appropriate plate. Place positive control disk with ampicillin in another quadrant of each plate.
16. Finish with 6 Petri plates with a negative control in the center, a positive control, and 3 sample disks. Record which solvents and plant extracts are in each quadrant. Let soak for a few minutes.
17. Ensure disks adhere to surface of agar. Invert the plates and incubate at 37*C for 24 to 48 hours.
18. After incubation, look for at the plates with plant extract disks for zones of inhibition, clear area formed by inhibitory (decrease in action) action of a substance in the plant material around the disk. Photograph the plates, labeling any inhibition of bacterial growth.
19. Create a data table for the replicates and averages. Include descriptions of the bacterial lawn around each disk. Record the diameter and clarity of any cleared areas around the disks in quantitative measurements.
Data Analysis/Conclusion:
Thinking Like A Biotechnician:
- All of my extracts gave me positive results, which means my plants did have anti-bacterial properties.
- My controls did not not work as expected. Both my negative and positive controls were positive.
- Some reasons for this are possible cross-contamination or an incorrect application of the filters.
- Some further experiments that could be improved are testing different types of bacteria.
- The next step would be to repeat this experiment and then test for other bacteria.
Thinking Like A Biotechnician:
- Yes, this negative result means that the extract is not an antimicrobial agent.
- This is a problem because if it smells like alcohol there is alcohol in the filter, and alcohol is antimicrobial. Therefore, you are testing the antimicrobial properties of the alcohol and not the extract.
- To test each compound for its antimicrobial properties, you have to separate them and test each one individually using the process we used for this lab.