| 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.
Tuesday, April 29, 2008
Daily Log
Microbiology Log
Day 1: Tuesday April 15
A. Enumeration of a urine sample:
In order to determine the number of organisms per ml in a urine sample, the first thing that has to be done is a dilution of the original sample. The following dilution was carried out: 1ml of the urine sample was added to 99ml of water which created the 10^-2 dilution. From this sample, 1ml was taken out and added to a 9ml water blank creating a 10^-3 dilution and then one last time a 1ml sample was taken out of this sample and added again to a 9ml water blank giving a final dilution of 10^-4.
To count the number of bacteria in these dilutions two methods were used; 1ml of each sample was placed in a petri dish and then TSA was poured over the samples for a standard pour-plate count procedure. The second method utilized the Petrifilm Aerobic count method, which takes 1ml of each sample and is placed underneath the top film. The film is then placed down onto the sample and then the sample is distributed evenly with a plastic spreader that is pressed down onto the petrifilm recessed side down in order to create a circle of the sample that is solidified. Plates and petrifilm were place in the 37degree room till next period.
B. Isolation and Identification of all M/O’s
My unknown number obtained was #7. A gram stain was performed of the mixture and I saw the following organisms: one gram positive cocci in clusters, and two gram negative bacilli that I couldn’t differentiate.
The next step was to dilute the unknown mixture by adding a few drops of it to a 9ml water blank and then streaking this diluted mixture onto BAP, MAC, and EMB plates in order to obtain isolated colonies. All three organisms should grow on the BAP plate, but only the gram negatives should grow on the MAC and EMB plates since they have components (bile salts and crystal violet, methylene blue respectively) to inhibit gram positive organisms. These plates were then incubated at 37degrees, except the BAP plate which was placed in the CO2 chamber for 24hrs and then put into the cold room.
Day 2: Thursday April 17
Note: The day in between classes, plates (mainly BAP) were observed for possible S. pneumonia: since this organism was not present, they were placed in the cold room until Thursday.
A. Enumeration
Plates and petrifilm were counted. The 10^-2 diluted plate and petrifilm were TNTC and the 10^-4 plate and petrifilm were TFTC. Both the plate and petrifilm had a count of approximately 42 colonies on the 10^-3 samples. Counts between 25 and 250 are considered statistically significant. Therefore the urine sample has about 42,000 CFU/ml. This count indicates insignificant bacteriuria because it is not over 100,000 CFU/ml.
Pros and Cons of each method:
Standard Pour Plate Count:
This method is standard just like the name implies. It works and can be useful if there is no other method available. Cons for this method can be aspects such as: having to make sure the bacteria sample is mixed well with the agar, differentiate the different colonies when counting, hoping that the colonies actually grow, etc.
Petrifilm Aerobic Count:
I prefer this method over standard plating method because it’s quick and reliable. The counting is easier because the colonies are red and in the center of the petrifilm vs. trying to resolve between two colonies stuck together, identifying colonies at the bottom or edge of the plate, etc. The only con about this method that seems to be of any importance is the fact that it’s meant for aerobic organisms only.
B. Isolation and Identification
The plates went as follows:
BAP: there seemed to be gamma and some beta hemolysis. White, medium sized, raised colonies were very distinctive and these I decided to name as number 1. However, the two other colonies were hard to decipher and seemed grayish and white, and very small and clumped.
MAC: after 24hrs there appeared to be one colony type that was purple, but after time, the colonies turned clear and made the purple agar turn yellow. The gram negative appeared to be a lactose fermentor.
EMB: at first glance there seemed to be two types of colonies; a purple big colony (named #2) and pink big colonies (originally #3). However these became one type of colony after time and made me realize that my initial idea of there being two gram negative bacteria in my sample was probably wrong.
I gram stained three colonies I thought were different and grew the gram positive on TSA and the two gram negatives on KIA agar. The gram stains showed the same results as before. A gram positive cocci and two gram negative bacilli (turns out these two were the same organism), so I thought I had isolated my organisms correctly.
Day 3: Tuesday April 22
B. Isolation and Identification
My streaked plates were checked for technique.
In checking my KIA slants, the two gram negatives showed the exact same results: A/A with gas production so I knew that it was probably the same organism. With this knowledge I knew that my #2 organism was my gram negative bacilli probably from the Enterbacteriaceae family. My TSA slant grew my gram positive cocci, my #1, and so I had only one problem, what was my third organism? I searched over all my plates: the BAP plate was too hard to distinguish a colony besides my white one that I had already determined, the MAC plate only grew one gram negative which I also had determined; and in taking another glance at my EMB I saw a small pink colony hidden amongst my large gram negative, purple colonies. I gram stained this colony and it turned out to be gram positive; which should not have grown on an EMB plate. With that I was given a virtual organism for my #3.
Tests to be preformed:
Organism #1: Gram positive cocci (clusters)
TSA slant showed white streak, BAP showed white colonies and possible Beta hemolysis
Tests ordered: MSA plate, coagulase test, and catalase
Organism #2: Gram negative bacilli (random)
KIA: A/A, gas formation, BAP showed possible gamma hemolysis
Tests ordered: Citrate slant, LIA, MIO, and Urease
Organism #3: Virtual, Gram positive cocci (chains)
BAP plate showed gamma hemolysis
Tests ordered: Catalase and Bile esculin
Place in appropriate incubation areas.
C. Other Tests: API 20E and Enterotube
A purple colony from my EMB plate was used to inoculate an API-20E strip and an Enterotube. The organism should be gram negative bacilli.
The API-20:
The middle of a purple colony on my EMB plate was isolated and placed in a water sample to dilute the sample. Then samples were placed into each compartment on the API strip. The compartments with no lines were filled to the bottom line with the sample, the compartments underlined were filled to the bottom line with sample and then oil was added to fill the open oval, and the compartments boxed in with lines were filled to the top with sample.
Enterotube:
Each side had a cap and these were twisted off. The white side’s needle was inoculated and then the blue side pulled the entire needle thru to inoculate each test and then it was broken off. Holes were poked thru the tests that required oxygen.
Day 4: Thursday April 24
B. Isolation and Identification
Organism #1: Staphylococcus aureus
Gram Reaction
Gram positive
Purple color
The cell wall stayed intact due to the thick peptidoglycan layer and crystal violet was maintained.
Morphology
Cocci, clusters
BAP: Hemolysis
Beta
White colonies, yellow hemolysis
Complete lysis of RBCs.
MSA plate
Positive, yellow agar
Pink agar to yellow and growth
7.5% sodium chloride is hard to grow on. Selective for staphylococci, and ferments mannitol.
Coagulase
Positive
White clumping
Bound/free coagulase is attached to the cell wall and reacts with fibrinogen in plasma.
Catalase
Positive
White compound; fizzes
Catalase present. Hydrogen peroxide is converted into water and oxygen gas.
Organism #2: Enterobacter aerogenes
Gram Reaction
Gram negative
Red color
Thin peptidoglycan layer and crystal violet was not maintained therefore secondary stain is present.
Morphology
Bacilli, random
BAP: Hemolysis
Gamma
Grayish colonies
No lysis of RBCs. No change in medium except for growth.
KIA
A/A, gas formed
All yellow, air bubbles
Lactose and Glucose were both fermented.
Citrate Slant
Positive
Green to Blue
Can use citrate as sole carbon source.
LIA
Positive, K/K
All purple
Lysine decarboxylase is present. Lysine deaminase is not present.
MIO
Positive, Negative, Positive
Cloudy, stayed clear after Kovac’s reagent, purple color
Is motile, no indole formation, lysine decarboxylation is present.
Urease medium
Negative
No color change
Does not hydrolyze urea.
EMB/MAC
Purple/Purple & clear
Purple colonies/clear
Lactose fermenter
Organism #3: Enterococcus faecalis
Gram Reaction
Gram positive
Purple color
The cell wall stayed intact due to the thick peptidoglycan layer and crystal violet was maintained.
Morphology
Cocci, chains
BAP: Hemolysis
Gamma
Grayish/white colonies
No lysis of RBCs. No change in medium except for growth.
Catalase
Negative
No change.
No catalase present. Hydrogen peroxide is not converted into water and oxygen gas.
Bile esculin
Positive
Slant turned black.
Hydrolysis of Esculin occurred in the presence of bile.
C. Other Tests
API-20E results: see paper
Number: 430 5773
Classified: Enterobacter aerogenes
Enterotube results: see paper
Number: 36361
Classified: Enterobacter aerogenes
D. Another method to identify Enterococcus faecalis:
Enterococcus faecalis is very antibiotic resistant whether naturally or through R-plasmid transmission. Vancomycin is an example of an antibiotic that it could possibly be resistant to. In this case, other tests that can be performed to distinguish this bacterium can include antibiotic disks inhibiting of growth.
Reference: http://web.mst.edu/~microbio/BIO221_2005/E_faecalis.htm
Day 1: Tuesday April 15
A. Enumeration of a urine sample:
In order to determine the number of organisms per ml in a urine sample, the first thing that has to be done is a dilution of the original sample. The following dilution was carried out: 1ml of the urine sample was added to 99ml of water which created the 10^-2 dilution. From this sample, 1ml was taken out and added to a 9ml water blank creating a 10^-3 dilution and then one last time a 1ml sample was taken out of this sample and added again to a 9ml water blank giving a final dilution of 10^-4.
To count the number of bacteria in these dilutions two methods were used; 1ml of each sample was placed in a petri dish and then TSA was poured over the samples for a standard pour-plate count procedure. The second method utilized the Petrifilm Aerobic count method, which takes 1ml of each sample and is placed underneath the top film. The film is then placed down onto the sample and then the sample is distributed evenly with a plastic spreader that is pressed down onto the petrifilm recessed side down in order to create a circle of the sample that is solidified. Plates and petrifilm were place in the 37degree room till next period.
B. Isolation and Identification of all M/O’s
My unknown number obtained was #7. A gram stain was performed of the mixture and I saw the following organisms: one gram positive cocci in clusters, and two gram negative bacilli that I couldn’t differentiate.
The next step was to dilute the unknown mixture by adding a few drops of it to a 9ml water blank and then streaking this diluted mixture onto BAP, MAC, and EMB plates in order to obtain isolated colonies. All three organisms should grow on the BAP plate, but only the gram negatives should grow on the MAC and EMB plates since they have components (bile salts and crystal violet, methylene blue respectively) to inhibit gram positive organisms. These plates were then incubated at 37degrees, except the BAP plate which was placed in the CO2 chamber for 24hrs and then put into the cold room.
Day 2: Thursday April 17
Note: The day in between classes, plates (mainly BAP) were observed for possible S. pneumonia: since this organism was not present, they were placed in the cold room until Thursday.
A. Enumeration
Plates and petrifilm were counted. The 10^-2 diluted plate and petrifilm were TNTC and the 10^-4 plate and petrifilm were TFTC. Both the plate and petrifilm had a count of approximately 42 colonies on the 10^-3 samples. Counts between 25 and 250 are considered statistically significant. Therefore the urine sample has about 42,000 CFU/ml. This count indicates insignificant bacteriuria because it is not over 100,000 CFU/ml.
Pros and Cons of each method:
Standard Pour Plate Count:
This method is standard just like the name implies. It works and can be useful if there is no other method available. Cons for this method can be aspects such as: having to make sure the bacteria sample is mixed well with the agar, differentiate the different colonies when counting, hoping that the colonies actually grow, etc.
Petrifilm Aerobic Count:
I prefer this method over standard plating method because it’s quick and reliable. The counting is easier because the colonies are red and in the center of the petrifilm vs. trying to resolve between two colonies stuck together, identifying colonies at the bottom or edge of the plate, etc. The only con about this method that seems to be of any importance is the fact that it’s meant for aerobic organisms only.
B. Isolation and Identification
The plates went as follows:
BAP: there seemed to be gamma and some beta hemolysis. White, medium sized, raised colonies were very distinctive and these I decided to name as number 1. However, the two other colonies were hard to decipher and seemed grayish and white, and very small and clumped.
MAC: after 24hrs there appeared to be one colony type that was purple, but after time, the colonies turned clear and made the purple agar turn yellow. The gram negative appeared to be a lactose fermentor.
EMB: at first glance there seemed to be two types of colonies; a purple big colony (named #2) and pink big colonies (originally #3). However these became one type of colony after time and made me realize that my initial idea of there being two gram negative bacteria in my sample was probably wrong.
I gram stained three colonies I thought were different and grew the gram positive on TSA and the two gram negatives on KIA agar. The gram stains showed the same results as before. A gram positive cocci and two gram negative bacilli (turns out these two were the same organism), so I thought I had isolated my organisms correctly.
Day 3: Tuesday April 22
B. Isolation and Identification
My streaked plates were checked for technique.
In checking my KIA slants, the two gram negatives showed the exact same results: A/A with gas production so I knew that it was probably the same organism. With this knowledge I knew that my #2 organism was my gram negative bacilli probably from the Enterbacteriaceae family. My TSA slant grew my gram positive cocci, my #1, and so I had only one problem, what was my third organism? I searched over all my plates: the BAP plate was too hard to distinguish a colony besides my white one that I had already determined, the MAC plate only grew one gram negative which I also had determined; and in taking another glance at my EMB I saw a small pink colony hidden amongst my large gram negative, purple colonies. I gram stained this colony and it turned out to be gram positive; which should not have grown on an EMB plate. With that I was given a virtual organism for my #3.
Tests to be preformed:
Organism #1: Gram positive cocci (clusters)
TSA slant showed white streak, BAP showed white colonies and possible Beta hemolysis
Tests ordered: MSA plate, coagulase test, and catalase
Organism #2: Gram negative bacilli (random)
KIA: A/A, gas formation, BAP showed possible gamma hemolysis
Tests ordered: Citrate slant, LIA, MIO, and Urease
Organism #3: Virtual, Gram positive cocci (chains)
BAP plate showed gamma hemolysis
Tests ordered: Catalase and Bile esculin
Place in appropriate incubation areas.
C. Other Tests: API 20E and Enterotube
A purple colony from my EMB plate was used to inoculate an API-20E strip and an Enterotube. The organism should be gram negative bacilli.
The API-20:
The middle of a purple colony on my EMB plate was isolated and placed in a water sample to dilute the sample. Then samples were placed into each compartment on the API strip. The compartments with no lines were filled to the bottom line with the sample, the compartments underlined were filled to the bottom line with sample and then oil was added to fill the open oval, and the compartments boxed in with lines were filled to the top with sample.
Enterotube:
Each side had a cap and these were twisted off. The white side’s needle was inoculated and then the blue side pulled the entire needle thru to inoculate each test and then it was broken off. Holes were poked thru the tests that required oxygen.
Day 4: Thursday April 24
B. Isolation and Identification
Organism #1: Staphylococcus aureus
Gram Reaction
Gram positive
Purple color
The cell wall stayed intact due to the thick peptidoglycan layer and crystal violet was maintained.
Morphology
Cocci, clusters
BAP: Hemolysis
Beta
White colonies, yellow hemolysis
Complete lysis of RBCs.
MSA plate
Positive, yellow agar
Pink agar to yellow and growth
7.5% sodium chloride is hard to grow on. Selective for staphylococci, and ferments mannitol.
Coagulase
Positive
White clumping
Bound/free coagulase is attached to the cell wall and reacts with fibrinogen in plasma.
Catalase
Positive
White compound; fizzes
Catalase present. Hydrogen peroxide is converted into water and oxygen gas.
Organism #2: Enterobacter aerogenes
Gram Reaction
Gram negative
Red color
Thin peptidoglycan layer and crystal violet was not maintained therefore secondary stain is present.
Morphology
Bacilli, random
BAP: Hemolysis
Gamma
Grayish colonies
No lysis of RBCs. No change in medium except for growth.
KIA
A/A, gas formed
All yellow, air bubbles
Lactose and Glucose were both fermented.
Citrate Slant
Positive
Green to Blue
Can use citrate as sole carbon source.
LIA
Positive, K/K
All purple
Lysine decarboxylase is present. Lysine deaminase is not present.
MIO
Positive, Negative, Positive
Cloudy, stayed clear after Kovac’s reagent, purple color
Is motile, no indole formation, lysine decarboxylation is present.
Urease medium
Negative
No color change
Does not hydrolyze urea.
EMB/MAC
Purple/Purple & clear
Purple colonies/clear
Lactose fermenter
Organism #3: Enterococcus faecalis
Gram Reaction
Gram positive
Purple color
The cell wall stayed intact due to the thick peptidoglycan layer and crystal violet was maintained.
Morphology
Cocci, chains
BAP: Hemolysis
Gamma
Grayish/white colonies
No lysis of RBCs. No change in medium except for growth.
Catalase
Negative
No change.
No catalase present. Hydrogen peroxide is not converted into water and oxygen gas.
Bile esculin
Positive
Slant turned black.
Hydrolysis of Esculin occurred in the presence of bile.
C. Other Tests
API-20E results: see paper
Number: 430 5773
Classified: Enterobacter aerogenes
Enterotube results: see paper
Number: 36361
Classified: Enterobacter aerogenes
D. Another method to identify Enterococcus faecalis:
Enterococcus faecalis is very antibiotic resistant whether naturally or through R-plasmid transmission. Vancomycin is an example of an antibiotic that it could possibly be resistant to. In this case, other tests that can be performed to distinguish this bacterium can include antibiotic disks inhibiting of growth.
Reference: http://web.mst.edu/~microbio/BIO221_2005/E_faecalis.htm
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