You wish to study a variant [a close relative] of the Tobacco Mosaic virus. Specifically you want to characterize the capsid and genetic component as well as determine the viral load required to cause a disease state.
a. What methodologies might you use to isolate and characterize this microbe?
b. How will you determine the LD50 of this viral particle?
I have been searching the net, but I am not having much luck. I have found a few things on the LD50, such as inoculating mice maybe, but nothing on part a. Any help will be greatly appreciated.
Since Jiskha doesn't have any regular biology experts, I suggest you post your question at this biology forum.
http://biology-forums.com/index.php
To isolate and characterize a variant of the Tobacco Mosaic virus, you can employ several methodologies. Here are a few steps you can follow:
1. Isolation:
a. Identify a suitable tissue source: Determine a plant source that is known to be infected with the variant of the Tobacco Mosaic virus you wish to study.
b. Sample collection: Obtain plant tissue samples from infected plants. You can collect leaves, stems, or other plant parts showing symptoms of infection.
c. Homogenization: Grind the plant material to extract the virus particles. Use a buffer solution to maintain stability and prevent degradation of the virus.
d. Purification: Employ a combination of techniques such as filtration, centrifugation, and density gradient separation to isolate and concentrate the virus particles.
2. Characterization:
a. Electron microscopy: Visualize the purified virus particles using electron microscopy. This technique will allow you to observe their size, shape, and overall structure.
b. Immunological assays: Use antibodies specific to the Tobacco Mosaic virus or related viruses to examine the presence and distribution of viral particles in the isolated sample. Techniques like enzyme-linked immunosorbent assay (ELISA) or Western blotting can be employed.
c. Nucleic acid analysis: Extract the genetic material (RNA) of the virus from the isolated sample. Conduct nucleic acid-based techniques like polymerase chain reaction (PCR) or sequencing to determine the genetic component of the virus and identify any variations.
Determining the LD50 (Lethal Dose 50) of the viral particles involves assessing the dose required to cause a disease state in a specific percentage of the test population (in this case, mice). Here's how you can proceed:
1. Animal model selection: Choose an appropriate animal model, such as mice, that is susceptible to the variant of the Tobacco Mosaic virus.
2. Dose preparation: Prepare a range of virus dilutions or concentrations.
3. Inoculation: Administer different doses of the virus to groups of mice, either through intranasal or intravenous routes.
4. Monitoring: Observe the mice regularly for any signs of disease or adverse effects.
5. Endpoint determination: Determine the endpoint criteria that indicate the disease state, such as mortality or specific clinical symptoms.
6. Data analysis: Calculate the LD50 value based on the dose-response curve obtained from the experiment.
Obtaining specific protocols or published research on the methodologies described above may be helpful in conducting your studies. Consider consulting scientific articles, research papers, or specialized journals that discuss tobacco viruses, virology, or plant pathology. Libraries or university databases can be great resources for accessing scholarly articles in these fields.