| 1. Was the initial E. coli/R factor recipient culture sensitive or resistant to ampicillin? Why? Sensitive; because it wasn’t able to grow on the MAC + AMP plate. It has the ability to uptake the R-factor plasmid by conjugation which would give it the resistance characteristic against AMP, but until it does this, E. coli will be sensitive. 2. Was the initial S. typhimurium/R factor donor culture sensitive or resistant to ampicillin? Why? Resistant; it has the R-factor plasmid already present and so it can therefore grow on the MAC + AMP plate. In our results this fact proves to be right. 3. After conjugation was E. coli/R factor recipient culture sensitive or resistant to ampicillin? Why? Resistant; S. typhimurium and transfer a copy of the R-factor plasmid to E. coli and therefore E. coli was able to grow on the MAC + AMP plate. 4. Why did the two cultures need to be incubated together? Incubation allows the two organisms to come into contact and combine; the heat simulates the temp. that each grow more readily in. 5. Which organism is lactose positive? How do you know this? E. coli ferments lactose and therefore turned the MAC plates pinkish and had these colored colonies. 6. Which organism is lactose negative? How do you know this? S. typhimurium doesn’t ferment lactase and therefore turned the plates yellowish and had tan colonies grown on the plates. 7. Were you able to obtain pure cultures of ampicillin resistant E. coli and S. typhimurium bacteria?Yes; tan and pinkish colonies were observed. |
Friday, May 2, 2008
Bacterial Conjuation Questions
Thursday, May 1, 2008
Vaccines
1. Describe the Salk and Sabin vaccines and identify how they differed.
Jonas Salk's vaccine is a clear, colorless sterile suspension for subcutaneous injection. It contains strains of the 3 types of polioviruses originally grown in monkey kidney cell culture and inactivated by exposure to formaldehyde; the vacccine consisted of an injected dose of inactivated (dead) poliovirus. Albert Sabin’s vaccine was taken orally and used attenuated poliovirus; it contains the three serotyped strains of poliovirus. He attenuated the wild type poliovirus by passaging the virus in monkey kidney epithelial cells.
2. Give three advantages and three disadvantages of each vaccine.
Salk’s Advantages:
1. The virus is not live, thus it is easier to manage than Sabin’s.
2. There is no risk of vaccine-associated polio paralysis (VAPP).
3. Immunization triggers an excellent immune response and long-lasting immunity to all 3 poliovirus types.
Salk’s Disadvantages:
1. Offers no protection to the mucosal lining of the intestine, so people vaccinated can still carry the disease and spread it to unvaccinated individuals; ultimately heightening the risk of viral circulation within the community.
2. The price of Salk’s is over 5 times that of Sabin’s.
3. Administering of Salk’s vaccine requires trained health workers.
Sabin’s Advantages:
1. Provided longer lasting immunity than Salk’s vaccine.
2. Sabin’s also indirectly protects other susceptible individuals by secondary vaccination, which means that vaccinated individuals may spread the vaccine virus in the community and thereby inhibit the spread of the wild type virus if it occurs in the population.
3. The Sabin’s vaccine is easily administered by giving children a sugar cube or sugar liquid containing the vaccine, neither of which requires extensive medical training to be administered.
Sabin’s Disadvantages:
1. The risk of vaccine-associated paralytic polio can be a result of the vaccination.
2. In Third World countries, other gastrointestinal viruses interfere with the replication of the attenuated polio vaccine viruses in the intestine of the vaccines.
3. Immunosuppressed individuals shouldn’t take this vaccine and are pushed to take Salk’s if need vaccination.
3. Polio has been eradicated from the US, though it has not been completely eradicated from the rest of the world. Vaccination is still necessary to prevent the return of the disease. Which vaccine do you think should be used for immunizations and why?
The oral vaccination founded by Albert Sabin should be used to prevent Polio because it provides longer lasting immunity, is cheaper and easier to administer, and this method protects other individuals from being susceptible to you. Salk’s vaccine allows the virus to still be active through the fecal/oral route transmission. If however, you are immunosuppressed and need the vaccine, you should take Salk’s because Sabin’s vaccine can cause the virus to be found in respiratory secretions for several days and in stool for several weeks after vaccination; which would be bad for that type of person.
Reference:
http://www.brown.edu/Courses/Bio_160/Projects1999/polio/vac.html
Jonas Salk's vaccine is a clear, colorless sterile suspension for subcutaneous injection. It contains strains of the 3 types of polioviruses originally grown in monkey kidney cell culture and inactivated by exposure to formaldehyde; the vacccine consisted of an injected dose of inactivated (dead) poliovirus. Albert Sabin’s vaccine was taken orally and used attenuated poliovirus; it contains the three serotyped strains of poliovirus. He attenuated the wild type poliovirus by passaging the virus in monkey kidney epithelial cells.
2. Give three advantages and three disadvantages of each vaccine.
Salk’s Advantages:
1. The virus is not live, thus it is easier to manage than Sabin’s.
2. There is no risk of vaccine-associated polio paralysis (VAPP).
3. Immunization triggers an excellent immune response and long-lasting immunity to all 3 poliovirus types.
Salk’s Disadvantages:
1. Offers no protection to the mucosal lining of the intestine, so people vaccinated can still carry the disease and spread it to unvaccinated individuals; ultimately heightening the risk of viral circulation within the community.
2. The price of Salk’s is over 5 times that of Sabin’s.
3. Administering of Salk’s vaccine requires trained health workers.
Sabin’s Advantages:
1. Provided longer lasting immunity than Salk’s vaccine.
2. Sabin’s also indirectly protects other susceptible individuals by secondary vaccination, which means that vaccinated individuals may spread the vaccine virus in the community and thereby inhibit the spread of the wild type virus if it occurs in the population.
3. The Sabin’s vaccine is easily administered by giving children a sugar cube or sugar liquid containing the vaccine, neither of which requires extensive medical training to be administered.
Sabin’s Disadvantages:
1. The risk of vaccine-associated paralytic polio can be a result of the vaccination.
2. In Third World countries, other gastrointestinal viruses interfere with the replication of the attenuated polio vaccine viruses in the intestine of the vaccines.
3. Immunosuppressed individuals shouldn’t take this vaccine and are pushed to take Salk’s if need vaccination.
3. Polio has been eradicated from the US, though it has not been completely eradicated from the rest of the world. Vaccination is still necessary to prevent the return of the disease. Which vaccine do you think should be used for immunizations and why?
The oral vaccination founded by Albert Sabin should be used to prevent Polio because it provides longer lasting immunity, is cheaper and easier to administer, and this method protects other individuals from being susceptible to you. Salk’s vaccine allows the virus to still be active through the fecal/oral route transmission. If however, you are immunosuppressed and need the vaccine, you should take Salk’s because Sabin’s vaccine can cause the virus to be found in respiratory secretions for several days and in stool for several weeks after vaccination; which would be bad for that type of person.
Reference:
http://www.brown.edu/Courses/Bio_160/Projects1999/polio/vac.html
Kosh's Postulates
1. Show how each of Koch’s Postulates were proven or disproven.
The first step was successful in that we isolated the ‘soft rot’ from our diseased carrot and grew a pure culture on a N/A plate. Koch’s Postulate’s was disproven because we didn’t grow any ‘soft rot’ on our carrots when we inoculated them with the disease and therefore couldn’t isolate any pure colonies.
2. What causes soft rot in carrots? Explain.
A bacteria called, Erwinia carotovora, is responsible for ‘soft rot’ in carrots. It is a gram negative bacillus, plant pathogen that invades plants and causes decay. It weakens the cells, destroys pectin and degrades cellulose (Wikipedia).
3. Why will the addition of water speed the disease process?
Water allows the diffusion of the diseased particles to migrate faster throughout the carrot/infected specimen. Also, due to waters osmosis effects, water probably went into the intercellular spaces of the carrot and caused a hyper or hypotonic effect; which results in cell death.
4. When would it be inappropriate to apply conventional Koch’s Postulates to an infectious disease?
It would be inappropriate to use Koch’s Postulates when the infectious disease is human based. If you find a disease in a human, it would be unethical to isolate it and then inject a healthy human in order to see if the disease is in fact what you thought. I would assume that if you did isolate a pathogen from a human and tried to do Koch’s Postulates, but with an animal being the recipient of the disease, the data would be inefficient because a different subject was used and the pathogen would not infect the animal in the same way as a human.
5. What are Molecular Koch’s Postulates?
Identify gene (or gene product) responsible for virulence determinant
Show gene present in strains of bacteria that cause the disease
Not present in avirulent strains
Disrupting the gene reduces virulence
Introduction of cloned gene into avirulent strain confers virulence.
The gene is expressed in vivo
Specific immune response to gene protects (http://www.microbiologybytes.com/introduction/b2.htm)
6. Give a specific application of Molecular Koch’s Postulates.
Wasn’t really sure what you wanted here, so I found a summary of when someone used Molecular Koch’s Postulates specifically.
“Salmonella pathogenicity island 1 (SPI1) encodes the components of a type-III-secretion apparatus, which allows the delivery of bacterial effector proteins to host cells. The effector genes in SPI1 are primarily expressed during the initial gastrointestinal phase of the infection. Mutations in SPI1 are attenuated for oral infection; however, SPI1 is not necessary for the systemic phase of the infection because SPI1 mutants are not attenuated when they are administered in mice intraperitoneally, which bypasses the gastrointestinal phase of the infection. In macrophages, one SPI1-encoded effector protein, SipB, induces host-cell death through its interaction with a host cysteine protease, caspase-1. Caspase-1 is a proinflammatory enzyme, and its activation by SipB not only induces cell death, but also leads to caspase-1 cleavage of the inactive precursors of interleukin (IL)-1 and IL-18 to generate the mature proinflammatory forms. Therefore, Salmonella kills the phagocytic cells it encounters but, paradoxically, at the same time 'deliberately' induces an inflammatory response that is destined to bring even more phagocytic cells into its immediate vicinity. A direct role for SipB in SPI1-mediated cell death is supported by a number of observations. However, single mutations in the sipB gene do not lead simply to the loss of the ability to induce caspase-1 and cell death, but also to a loss of all SPI1 functions — presumably because disruption of the SipB protein leads to disruption of the mechanism by which effector molecules are translocated to host cells. So, although SipB, has been proposed to be the main activator for caspase-1 during in vivo infection, it has been difficult to directly address this possibility owing to the multiple roles of SipB, in addition to directly activating caspase-1 (Molecular Koch's postulates applied to bacterial pathogenicity — a personal recollection 15 years later).”
7. Eighty-one patients became ill and 14 died in an outbreak at St. Elizabeth’s Hospital, Washington, D.C., in 1965. Epidemiologic evidence suggested a link between infection and wind-blown dust from excavations on hospital grounds. In 1968, 144 cases of self-limited illness occurred in employees at the health department in Pontiac, MI. Investigations at that time demonstrated that the etiologic agent was present in the condenser of a malfunctioning air-conditioning system. In 1974, at least 20 persons attending a convention at the Bellevue Stratford Hotel in Philadelphia developed pneumonia, and two died. In 1976, 182 people became ill and 29 died at another convention at the Bellevue Stratford hotel. Explain, in detail, how you would identify the causative agent of these outbreaks by applying Koch’s Postulates.
I would isolate the disease in all the people that were infected as well as the people who died to see if the organism was found in abundance; this would be done by growing the organism that is found in the ill people and grow it in a pure culture. This organism would be labeled and verified. In order to identify the causative agent, with this organism identified, it would need to be compared to the other samples and then have tests run to determine where the pathogen grows and what the likely culprit was that gave it to these people; something in the dust or self-employed disease. You could also inoculate a breed of animal and see what happens to the animal; grow a pure culture from the sampled animal and see if the same organism grows.
The first step was successful in that we isolated the ‘soft rot’ from our diseased carrot and grew a pure culture on a N/A plate. Koch’s Postulate’s was disproven because we didn’t grow any ‘soft rot’ on our carrots when we inoculated them with the disease and therefore couldn’t isolate any pure colonies.
2. What causes soft rot in carrots? Explain.
A bacteria called, Erwinia carotovora, is responsible for ‘soft rot’ in carrots. It is a gram negative bacillus, plant pathogen that invades plants and causes decay. It weakens the cells, destroys pectin and degrades cellulose (Wikipedia).
3. Why will the addition of water speed the disease process?
Water allows the diffusion of the diseased particles to migrate faster throughout the carrot/infected specimen. Also, due to waters osmosis effects, water probably went into the intercellular spaces of the carrot and caused a hyper or hypotonic effect; which results in cell death.
4. When would it be inappropriate to apply conventional Koch’s Postulates to an infectious disease?
It would be inappropriate to use Koch’s Postulates when the infectious disease is human based. If you find a disease in a human, it would be unethical to isolate it and then inject a healthy human in order to see if the disease is in fact what you thought. I would assume that if you did isolate a pathogen from a human and tried to do Koch’s Postulates, but with an animal being the recipient of the disease, the data would be inefficient because a different subject was used and the pathogen would not infect the animal in the same way as a human.
5. What are Molecular Koch’s Postulates?
Identify gene (or gene product) responsible for virulence determinant
Show gene present in strains of bacteria that cause the disease
Not present in avirulent strains
Disrupting the gene reduces virulence
Introduction of cloned gene into avirulent strain confers virulence.
The gene is expressed in vivo
Specific immune response to gene protects (http://www.microbiologybytes.com/introduction/b2.htm)
6. Give a specific application of Molecular Koch’s Postulates.
Wasn’t really sure what you wanted here, so I found a summary of when someone used Molecular Koch’s Postulates specifically.
“Salmonella pathogenicity island 1 (SPI1) encodes the components of a type-III-secretion apparatus, which allows the delivery of bacterial effector proteins to host cells. The effector genes in SPI1 are primarily expressed during the initial gastrointestinal phase of the infection. Mutations in SPI1 are attenuated for oral infection; however, SPI1 is not necessary for the systemic phase of the infection because SPI1 mutants are not attenuated when they are administered in mice intraperitoneally, which bypasses the gastrointestinal phase of the infection. In macrophages, one SPI1-encoded effector protein, SipB, induces host-cell death through its interaction with a host cysteine protease, caspase-1. Caspase-1 is a proinflammatory enzyme, and its activation by SipB not only induces cell death, but also leads to caspase-1 cleavage of the inactive precursors of interleukin (IL)-1 and IL-18 to generate the mature proinflammatory forms. Therefore, Salmonella kills the phagocytic cells it encounters but, paradoxically, at the same time 'deliberately' induces an inflammatory response that is destined to bring even more phagocytic cells into its immediate vicinity. A direct role for SipB in SPI1-mediated cell death is supported by a number of observations. However, single mutations in the sipB gene do not lead simply to the loss of the ability to induce caspase-1 and cell death, but also to a loss of all SPI1 functions — presumably because disruption of the SipB protein leads to disruption of the mechanism by which effector molecules are translocated to host cells. So, although SipB, has been proposed to be the main activator for caspase-1 during in vivo infection, it has been difficult to directly address this possibility owing to the multiple roles of SipB, in addition to directly activating caspase-1 (Molecular Koch's postulates applied to bacterial pathogenicity — a personal recollection 15 years later).”
7. Eighty-one patients became ill and 14 died in an outbreak at St. Elizabeth’s Hospital, Washington, D.C., in 1965. Epidemiologic evidence suggested a link between infection and wind-blown dust from excavations on hospital grounds. In 1968, 144 cases of self-limited illness occurred in employees at the health department in Pontiac, MI. Investigations at that time demonstrated that the etiologic agent was present in the condenser of a malfunctioning air-conditioning system. In 1974, at least 20 persons attending a convention at the Bellevue Stratford Hotel in Philadelphia developed pneumonia, and two died. In 1976, 182 people became ill and 29 died at another convention at the Bellevue Stratford hotel. Explain, in detail, how you would identify the causative agent of these outbreaks by applying Koch’s Postulates.
I would isolate the disease in all the people that were infected as well as the people who died to see if the organism was found in abundance; this would be done by growing the organism that is found in the ill people and grow it in a pure culture. This organism would be labeled and verified. In order to identify the causative agent, with this organism identified, it would need to be compared to the other samples and then have tests run to determine where the pathogen grows and what the likely culprit was that gave it to these people; something in the dust or self-employed disease. You could also inoculate a breed of animal and see what happens to the animal; grow a pure culture from the sampled animal and see if the same organism grows.
Case Study
Case study: Alleged bacteria, Campylobacter jejuni
Approximately three days ago from 3/13/2008 Martha Stewart, alleged twin sister according to client, got out of prison and therefore Margret Stuart had a celebration. A turkey was prepared, but since Martha Stewart didn’t show up to the party, the turkey wasn’t cooked properly and had visible pink areas exposed giving the concern of a possible undercooked turkey. After digestion of the raw turkey, the client felt the following symptoms: cramping in stomach/intestines, nausea/vomiting, possible fever of a couple degrees over, and her stool was in the form of green/watery diarrhea. Client had no previous contact with anyone due to no job, neighbors, or family. The diagnostics of this causative agent is Campylobacter jejuni. It’s a human pathogen carried in turkey, chicken, and water fowl and is accountable for approximately 4-35% of acute bacterial diarrheal disease. Humans are infected by raw or contaminated milk and partially cooked poultry; possible symptoms can be, but are not limited to, cramping/ abdominal pain, diarrhea, chills, and fever. Campylobacter jejuni is usually self-limiting (3-7 days) and may be treatable with oral erythromycin.
Approximately three days ago from 3/13/2008 Martha Stewart, alleged twin sister according to client, got out of prison and therefore Margret Stuart had a celebration. A turkey was prepared, but since Martha Stewart didn’t show up to the party, the turkey wasn’t cooked properly and had visible pink areas exposed giving the concern of a possible undercooked turkey. After digestion of the raw turkey, the client felt the following symptoms: cramping in stomach/intestines, nausea/vomiting, possible fever of a couple degrees over, and her stool was in the form of green/watery diarrhea. Client had no previous contact with anyone due to no job, neighbors, or family. The diagnostics of this causative agent is Campylobacter jejuni. It’s a human pathogen carried in turkey, chicken, and water fowl and is accountable for approximately 4-35% of acute bacterial diarrheal disease. Humans are infected by raw or contaminated milk and partially cooked poultry; possible symptoms can be, but are not limited to, cramping/ abdominal pain, diarrhea, chills, and fever. Campylobacter jejuni is usually self-limiting (3-7 days) and may be treatable with oral erythromycin.
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