Lab 6D
Purpose: What plant materials, found locally, contain active ingredients that will inhibit the growth of bacteria?
Materials:
Part 1:
7.we sterilized a 1 ml pipet, and put 1 ml of e.coli and spread it over the petri dish after with the sterilized glass spreader, we covered the petri dish and let it sit for 15 min. .
8. then, using sterilized forceps, we placed the filter paper disks where we were told. One methanol extract disc in each MeoH quadrant, one water extract disc in each water quadrant, one ampicillin disc on the +, and one water disc on the -.
9. they were incubated at 37C for 24 hours
10. Observe the petri dish. If their is bacteria growing against the disk then it came out negative. If there is a ring without bacteria around the disk then it is a positive sample.
Materials:
- Balance, weigh boat, lab, scoops
- Lb broth base
- Media bottles, 250 ml
- Sterilizer/autoclave
- Water bath, 37C, shaking
- Sterile LB agar
- Laminar flow hood and disinfectant
- Glasses, safety, plastic
- Bunsen burner and gas lighter
- Inoculating loop, Ni/Cr wire
- Petri dishes, 60 X 15 mm, sterile
- E. coli JM109 (stock plate)
- Plant specimen
- Mortar and pestle
- Pipet, 10 ml and pump
- Plastic funnels, short-stemmed
- Filter paper disks, 5 mm diameter
- Beakers, 100 ml
- Syringe, 10 ml and filter, 0.2 weird um
- 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, 37C
Part 1:
- \We used a mortar and a pestle to grind up 2 grams of our chosen plant product, mixed with 10 ml of deionized water. after it sat for 3 minutes, we filtered it through filter paper. the extract we received was then filtered with a syringe filter. we put 1 ml of the the extract in a microtube, and labeled it.
- this step is the same as the previous step except with methanol instead of deionized water. after the extract is put into the tube, it is placed in a heating block at 65C for 24 hours.
- we then used sterilized forceps to drop four filter paper disks into each extract.
- then, we prepared 2 negative control disks of methanol and distilled water
- then, we prepared 2 positive control disks of ampicillin.
- the tubes were closed and stored at 4C.
7.we sterilized a 1 ml pipet, and put 1 ml of e.coli and spread it over the petri dish after with the sterilized glass spreader, we covered the petri dish and let it sit for 15 min. .
8. then, using sterilized forceps, we placed the filter paper disks where we were told. One methanol extract disc in each MeoH quadrant, one water extract disc in each water quadrant, one ampicillin disc on the +, and one water disc on the -.
9. they were incubated at 37C for 24 hours
10. Observe the petri dish. If their is bacteria growing against the disk then it came out negative. If there is a ring without bacteria around the disk then it is a positive sample.
Questions:
1. no it doesn't always mean that the extract isn't an antimicrobial agent. we have only seen the extract interacting with e.coli, which limits our results.
2. this is a problem because the alcohol can kill the bacteria, which leaves us with incorrect data.
3. we would use chromatography to separate the individual compounds.
Conclusion:
in my petri dish, all results were positive, except for one of the methanol extracts. the controls worked as expected. alcohol could have interfered with our results,or just human error. we could improve by redoing the initial parts of the experiment and making sure the alcohol is completely off.
The next step would be to retest the methanol extract to see for sure if it is positive. If it is still positive, then we would use chromatography to isolate the part of the extract that causes it to be antimicrobial.
1. no it doesn't always mean that the extract isn't an antimicrobial agent. we have only seen the extract interacting with e.coli, which limits our results.
2. this is a problem because the alcohol can kill the bacteria, which leaves us with incorrect data.
3. we would use chromatography to separate the individual compounds.
Conclusion:
in my petri dish, all results were positive, except for one of the methanol extracts. the controls worked as expected. alcohol could have interfered with our results,or just human error. we could improve by redoing the initial parts of the experiment and making sure the alcohol is completely off.
The next step would be to retest the methanol extract to see for sure if it is positive. If it is still positive, then we would use chromatography to isolate the part of the extract that causes it to be antimicrobial.