Purpose- What plant materials, found locally, contain active ingredients that will inhibit the growth of bacteria?
Materials-
Procedure-
Part II: Preparing Plant Extracts (see Caution above)
4. Grind 2g of plant tissue (leaves) w/ 10 mL of deionized water in mortar and pestle, & let sit for 3 mins. Channel in 11cm pipe, sanitize extricate w/ syringe channel, & gather 1mL of concentrate in marked 1.7mL microtube.
5. Rehash Step 4, w/ the exception of supplant deionized H2O w/ methanol. Place 1.7mL tube w/ 1mL of methanol concentrate in 65*C warmth piece (tops open) for 24hrs to vanish methanol. Reconstitute dry matter in microtube w/ 1mL of deionized water.
6. Rehash Step 4 & 5 for six exs, mark them.
7. Drop channel paper circles in every sifted concentrate tube utilizing sterile forceps (disinfected by being flared in liquor).
8. Set up 3 (-) control circles of just methanol & sterile & refined water.
9. Set up 6 (+) control plates of ampicillin arrangement.
10. Permit plates to be soaked w/ the concentrate (maybe overnight).
11. Close tubes. Store all specimens at 4*C until prepared to utilize.
Part III:
12. Use a clean pipet to move 1ml of E.coli broth to middle of a petri dish. sterilize a spreading loop w/ fire & alcohol, to spread bacteria evenly. Cover, & leave for at least 15 mins.
13. Using forceps, place 1 dish in each quadrant, 2cm from edge of the petri dish. Place methanol samples in 1 dish & the H2O samples on another.
14. Repeat step 13, in order to end up w/ 3 methanol & 3 H2O replicates.
15. Place a (-) control disk in center of appropriate plate. Then a (+) control w/ amplicillin in a quadrant on each plate.
16. In the end you should have 6 petri dishes w/ a (-) control in the middle & a (+) control & 3 sample disks.
17. Guarantee that the disk hold fast to surface of agar. Alter plates & brood at 37*C for 24 to 48 hrs.
18. After incubation, search for at plates w/ plant concentrate circles for zones of inhibition, clear rasnge shaped by inhibitory (abatement in real life) activity of a substance in the plant material around circle. Photo plates, marking any restraint of bacterial development.
19. Make an information table for repeats & midpoints. Incorporate depictions of the bacterial garden around every circle. Record breadth & clarity of any cleared zones around circles in quantitative estimations.
Results
The plant samples I used had a negative result. The methanol disc had larger rings around it (1.1 cm), which seemed to have killed off some bacteria, but had a negative result due to the disk had some bacteria up against it.There are still signs of bacterial activity around the inner rims, meaning that the disc did not kill off any bacteria but instead pushed the bacteria farther out. The plant sample that had been doused in water clearly provided negative results. These disks were only 0.1-0.2 in width, and were most likely due to water.
Materials-
- Balance, weigh boat, lab scoops
- LB broth base
- Media bottles, 250 mL
- Sterilizer/autoclave
- Water bath, 37*C, shaking
- Sterile LB agar
- Laminar flow hood and disinfectant
- Plastic safety glasses
- Bunsen burner and gas lighter
- Inoculating loop, Ni/Cr wire
- Petri dishes, 60x15mm, sterile
- E. coli JM109 (stock plate)
- Plant specimen
- Mortar and pestle
- Pipet, 10 mL and pump
- Plastic funnels, short-stemmed
- Filter paper disks, 5mm 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: Preparing Plant Extracts (see Caution above)
4. Grind 2g of plant tissue (leaves) w/ 10 mL of deionized water in mortar and pestle, & let sit for 3 mins. Channel in 11cm pipe, sanitize extricate w/ syringe channel, & gather 1mL of concentrate in marked 1.7mL microtube.
5. Rehash Step 4, w/ the exception of supplant deionized H2O w/ methanol. Place 1.7mL tube w/ 1mL of methanol concentrate in 65*C warmth piece (tops open) for 24hrs to vanish methanol. Reconstitute dry matter in microtube w/ 1mL of deionized water.
6. Rehash Step 4 & 5 for six exs, mark them.
7. Drop channel paper circles in every sifted concentrate tube utilizing sterile forceps (disinfected by being flared in liquor).
8. Set up 3 (-) control circles of just methanol & sterile & refined water.
9. Set up 6 (+) control plates of ampicillin arrangement.
10. Permit plates to be soaked w/ the concentrate (maybe overnight).
11. Close tubes. Store all specimens at 4*C until prepared to utilize.
Part III:
12. Use a clean pipet to move 1ml of E.coli broth to middle of a petri dish. sterilize a spreading loop w/ fire & alcohol, to spread bacteria evenly. Cover, & leave for at least 15 mins.
13. Using forceps, place 1 dish in each quadrant, 2cm from edge of the petri dish. Place methanol samples in 1 dish & the H2O samples on another.
14. Repeat step 13, in order to end up w/ 3 methanol & 3 H2O replicates.
15. Place a (-) control disk in center of appropriate plate. Then a (+) control w/ amplicillin in a quadrant on each plate.
16. In the end you should have 6 petri dishes w/ a (-) control in the middle & a (+) control & 3 sample disks.
17. Guarantee that the disk hold fast to surface of agar. Alter plates & brood at 37*C for 24 to 48 hrs.
18. After incubation, search for at plates w/ plant concentrate circles for zones of inhibition, clear rasnge shaped by inhibitory (abatement in real life) activity of a substance in the plant material around circle. Photo plates, marking any restraint of bacterial development.
19. Make an information table for repeats & midpoints. Incorporate depictions of the bacterial garden around every circle. Record breadth & clarity of any cleared zones around circles in quantitative estimations.
Results
The plant samples I used had a negative result. The methanol disc had larger rings around it (1.1 cm), which seemed to have killed off some bacteria, but had a negative result due to the disk had some bacteria up against it.There are still signs of bacterial activity around the inner rims, meaning that the disc did not kill off any bacteria but instead pushed the bacteria farther out. The plant sample that had been doused in water clearly provided negative results. These disks were only 0.1-0.2 in width, and were most likely due to water.
Data Analysis
In the end none of the extracts gave me a positive outcome. My plant doesn't affect the E.coli bacteria. The controls turned out how we expected them. There is a possibility that error occurred due to being not accurate in placing them, and misplacing disks in wrong areas. I also had the negative and positive control slide into different areas over night. This can cause area in my results. Due to negative results I would use different solvents in the futrue. Also, due to the movement of my disk over night, I the future I would let the bacteria soak in more, to insure my data isn't read wrong. For the next steps i would test a new plant since this plant did not have any positive outcomes.
Thinking Like a Bio Technician Questions
In the end none of the extracts gave me a positive outcome. My plant doesn't affect the E.coli bacteria. The controls turned out how we expected them. There is a possibility that error occurred due to being not accurate in placing them, and misplacing disks in wrong areas. I also had the negative and positive control slide into different areas over night. This can cause area in my results. Due to negative results I would use different solvents in the futrue. Also, due to the movement of my disk over night, I the future I would let the bacteria soak in more, to insure my data isn't read wrong. For the next steps i would test a new plant since this plant did not have any positive outcomes.
Thinking Like a Bio Technician Questions
- We only tested our extract for E.coli, this means it could be antimicrobial for a different bacteria. So it is possible that the extract is an antimicrobial agent.
- It is a problem because you do not want the alcohol to tamper with the extract and have us test the alcohol instead. The alcohol could kill the bacteria as well.
- Through chromatography we can figure out what compound is causing microbial action. Each compound can have more than one compound in it so through this use we can identify it.