plant substances as potential medicines
Purpose
To find plant materials that contain active ingredients that will inhibit the growth of bacteria.
Materials
Procedure
PART II
PART III
To find plant materials that contain active ingredients that will inhibit the growth of bacteria.
Materials
- balance
- LB broth base
- media bottles, 250mL
- sterilizer
- water bath, shaking
- sterile LB agar
- Laminar flow hood and disinfectant
- safety glasses
- bunsen burner
- inoculating loop, Ni/Cr wire
- Petri dishes, sterile
- E. coli
- Plant specimen
- Mortar and pestle
- Pipet, 10mL and pump
- plastic funnel
- filter paper disks
- beakers, 100mL
- syringe and filter
- reaction tubes and rack
- methanol, absolute
- pipet, 1mL and pump
- dry blockheater/heat block
- forceps
- Ampicillin
- glass spreader
- incubator oven
Procedure
PART II
- Grind plant leaf with deionized water using the mortar and pestle. Filter the sample extract through the paper funnel. Then filter the sample using a syringe filter. Put 1 mL of the sample in a microtube.
- Repeat the step above, but instead of water use methanol. After you have the extract, put it in the heatblock with the cap open. After 24 hours reconstitute the sample with deionized water.
- Use sterile forceps to place paper disks in the sample tubes.
- Prepare negative and positive controls using water and ampicillin
- Allow the disks to soak in the samples.
PART III
- Use a sterile pipet to transfer E. Coli broth to the petri dish. Spread it around the plate using a sterilized spreader. Allow the broth to soak in.
- Place on disk in the center of each corresponding quadrant. Place the negative and positive control disks on the correct sign.
- Incubate the petri dishes for at least 24 hours.
- Examine the plates and the bacteria growth around the disks.
Unfortunately my partner and I accidently put the incorrect number disks in the water and plant extract tube. Therefore I only had one disk for this sample. All of the disks from each sample had negative results. The methanol extracts had rings around them that were about
4mm wide. At first this seemed to be a positive result, however when we looked closer there was bacteria right up against the disks. Around my one water and plant extract sample disk there was a ring of about 3mm, however like the methanol extracts, there was bacteria right up against the disk. Another thing that was strange was that our disks had more of a yellow color to them compared to other groups. Perhaps this is from the strong color of our extract.
4mm wide. At first this seemed to be a positive result, however when we looked closer there was bacteria right up against the disks. Around my one water and plant extract sample disk there was a ring of about 3mm, however like the methanol extracts, there was bacteria right up against the disk. Another thing that was strange was that our disks had more of a yellow color to them compared to other groups. Perhaps this is from the strong color of our extract.
Analysis/Conclusion
None of our extracts gave us a positive result. All of them had bacteria growing right up against the disks. Our controls did seem to turn out as we had expected them to. There were a few components that could have led to incorrect results. First there could have been pipetting errors. Next the equipment we used could have not properly been sterilized by us. Lastly when we placed the disks down there could have been too much liquid on them. In the future I would test multiple types of plants. I would also make sure we put the correct number of disks in each sample. If my plants had positive results, my next steps would have been to find out which component in the plant has these qualities that repel bacteria.
Thinking Like A Biotechnician
1. If an extract gave a negative result in the antimicrobial assay, it does not mean that the extract is not an antimicrobial agent. Since we only tested for one type of bacteria, we do not know if it kills or repels other types of bacteria.
2. It is a problem if he methanol extracts smell like alcohol because the alcohol will automatically kill the bacteria. This will mess up all of the lab results.
3. In order to identify each compound in an extract we can use chromatography. We have used this in a previous lab and it allowed us to separate out useful components of samples.
None of our extracts gave us a positive result. All of them had bacteria growing right up against the disks. Our controls did seem to turn out as we had expected them to. There were a few components that could have led to incorrect results. First there could have been pipetting errors. Next the equipment we used could have not properly been sterilized by us. Lastly when we placed the disks down there could have been too much liquid on them. In the future I would test multiple types of plants. I would also make sure we put the correct number of disks in each sample. If my plants had positive results, my next steps would have been to find out which component in the plant has these qualities that repel bacteria.
Thinking Like A Biotechnician
1. If an extract gave a negative result in the antimicrobial assay, it does not mean that the extract is not an antimicrobial agent. Since we only tested for one type of bacteria, we do not know if it kills or repels other types of bacteria.
2. It is a problem if he methanol extracts smell like alcohol because the alcohol will automatically kill the bacteria. This will mess up all of the lab results.
3. In order to identify each compound in an extract we can use chromatography. We have used this in a previous lab and it allowed us to separate out useful components of samples.