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T4 bacteriophage

Submitted by amprovost on Sun, 04/30/2017 - 13:39

I recently experimented with T4 bacteriophage in a microbiology lab. My findings were as follows: 

This lab involved two experiments, the titration and isolation of a lytic phage and a phage typing experiment.

            In the experiment with the titration and isolation of a lytic phage, it was predicted that as the lytic phage became more diluted, it would produce less plaques on the Escherichia coli lawns, and that at least one plate would produce a countable number of plaques. This experiment was performed by creating a serial dilution of the T4 phage. This phage was diluted to 10^-8 dilution by initially combining 0.1 mL of T4 phage with 0.9 mL of TM buffer, then taking .1 mL of this solution and transferring it into another tube with0 .9 mL of TM buffer. This process was repeated until the 10^-8 dilution was achieved. Each of these solutions then had 0.1 mL transferred onto its own individual Luria agar bottom plate. Each of these plates also had 0.1 mL of E. coli B/r poured onto them. These solutions were swirled around the plate to make sure all of the surface area was covered. After these bacteria and phages were allowed some time to grow on the plates, the plates were inspected for plaques, which are small holes in the bacteria lawn caused by phage lysing. Plates with over 300 plaques were considered too numerous to count, and plates with less than 30 plaques were considered too few to count.

Dilution levels 10^-1 through 10^-5 were too numerous to count, and dilution level 10^-8 was too few to count. Dilution level 10^-6 had 205 plaques, and dilution level 10^-7 had 73 plaques. The plaque forming units/milliliter were then calculated using the equation average of PFU/ amount of phage added to the plate * dilution factor. The plate at dilution factor -6 was found to have 2050000000 PFU/mL, and the plate at dilution factor -7 was found to have 7300000000 PFU/mL.

The expected results of this experiment were confirmed, two plates with a countable number of plaques were cultivated.

            In the phage typing experiment, it was expected that the two E. coli strains would be infected by T4 phage, as E. coli have binding sites on their cell surface that T4 bacteriophage can bind to and infect the cell from. A LB plate was divided into four quadrants, and each was swabbed with its own individual bacteria (E. coli B/r, E. coli K12, Salmonella arizonae, and P. vulgaris). Each of these tiny lawns was then inoculated with T4 phage from the original plate, using a toothpick to stab the burst zone on the original plate and transfer it to the new plate. A new toothpick was used for each lawn in an effort to prevent cross contamination. E. coli K12 and E. coli B/r  both developed burst zones around the area of inoculation, and Salmonella arizonae, and P. vulgaris did not, showing that the T4 phage only affected E. coli K12 and E. coli B/r. The expected results were confirmed, as both strains of E. coli were infected with T4 bacteriophage.

Myopia

Submitted by amprovost on Fri, 04/28/2017 - 17:53

Myopia, also known as nearsightedness, is a visual impairment that causes one's vision to blur when looking at an object that is more than an armslength away. This condition has been around for a very long time, but has recently seen a very large increase in the percentages of populations that it affects. For some time scientists thought that this phenomenon may be caused by "near work", or work that requires visual focus on something at a close range, such as reading or writing. However, there has never been a firm scientific link made between near work and nearsightedness, so scientists are looking for other explanations. Another popular theory is the genetic theory, but this theory also has some holes in it. For example, one study showed that a community that only had a 2% rate of nearsightedness saw an increase to 50% in just two generations. This rapid increase is simply too much to be explained by genetics, so now popular convention believes that only a fraction of what controls myopia is genetically coded.

     After a series of different studies that ended up with no distinct results, researchers have decided that perhaps the possible link to levels of myopia are related to childhood exposure to sunlight. It is well known that children now spend less time outside than previous generations did, but this phenomenon may now be linked to nearsightedness. One study took chinese children living in Australia and compared them to Chinese children living in China. Asia is known to have some of the highest rates of nearsightedness in the world, so if it was a genetic factor it should show up in the Asian children who live elsewhere. However, despite the fact that the children in Australia did more near work than the children in Asia, the amount of myopia in the Asia group was still much much higher. The only major difference in the studies that researchers found was that on average, the children in the Australian group spent more than four times as much time outdoors than the chilldren in the Asian group. While the direct link to nearsightedness, if there is any, is unknown at this time, many scientists theorize that it may have to do with conditions such as vitamin D levels. Further research must be done on this topic before concluding whether or not natural light can help prevent myopia.

Nitrogen PP II

Submitted by amprovost on Fri, 04/28/2017 - 17:01

            In the denitrifying experiment, four different tubes of nitrate broth were used, one contained P. vulgaris, one contained P. aeruginosa, one contained rich soil, and one contained poor soil. The expected result was that it one would be able to determine the presence or lack of nitrite and nitrate in the broths using nitrate reagent A, nitrate reagent B, and zinc. After applying nitrate reagents A and B, a red color change indicated that nitrite was present. If there was no color change, zinc was added, if there was still no color change, this indicated that nitrate was broken down into a compound other than nitrite. If a red color change occurred after being exposed to zinc, this meant that nitrate was still present and never denitrified. The NB rich soil had nitrate break down into compounds other than nitrite. Both the NB poor soil and the P. vulgaris had the presence of nitrite. The sample with P. aeruginosa had nitrate broken down into compounds other than nitrite. The expected result was confirmed, as it was possible to determine the presence of nitrite or nitrate using these tests.

 

Research Project Discussion Final Draft

Submitted by amprovost on Thu, 04/27/2017 - 12:51

The results of this experiment show that dried Sphagnum was able to absorb a relative large amount of water, absorbing 6.125 grams of water for every one gram of moss before becoming saturated. This experiment also showed that dried Sphagnum absorbs liquids relatively quickly, as it only took 30 minutes for this moss to become saturated. This is useful knowledge as the rate of absorbency of a material has some practical applications, as certain conditions such as oil spills require a material that will absorb liquids as quickly as possible. Thus, this data may be useful for future production of liquid absorbing materials.

 

Antibiotics PP

Submitted by amprovost on Thu, 04/27/2017 - 12:20

            This experiment was performed by doing two things with a soil suspension. First, the suspension was streaked onto a casein-starch plate. Second, this suspension was diluted by taking one milliliter of suspension and transferring it into nine milliliters of sterile saline. This new solution was then also streaked onto a separate casein-starch plate. After the Streptomyces were isolated, three individual colonies were taken and streaked in a heavy band across the top quarter of an antibiotic-test plate. These heavy bands were then cross-streaked with S. aureus, E. faecalis, K. pneumoniae, and A. hydrophila. A control plate with all of these organisms and no Streptomyces, to show what uninhibited growth would look like. After allowing these organisms to grow, there appeared to be no inhibition by Streptomyces, as the cross-streaked organisms grew perfectly across the entire area inoculated, looking quite similar to the streaks in the control. One possible explanation for this is that Streptomyces does not produce any antibiotics that affect these types of organisms. Another possible explanation for this is simply that the Streptomyces were not given enough time to produce antibiotics, and perhaps the expected results would have been displayed had the plates been left undisturbed for a longer period of time.

Nitrogen PP

Submitted by amprovost on Thu, 04/27/2017 - 11:52

This graph shows that the expected results were confirmed in regards to growth, as the plant inoculated with bacteria and given nitrogenous fertilizer grew to be the tallest. The plant with nitrogenous fertilizer and bacteria also grew eleven nodules, the plant with only bacteria grew thirty nodules, the other two plants grew zero nodules. This does not confirm the original prediction as the plant with bacteria and no nitrogenous fertilizer still grew nodules, which are usually present when the organisms live in symbiosis and the bacteria fix nitrogen in the soil. A possible explanation for this result is contamination with nitrogenous fertilizer, as it is possible somewhere in the experiment the plant was either fertilized with the wrong fertilizer or with a tool contaminated with nitrogenous fertilizer.

Nitrogen Fixation

Submitted by amprovost on Wed, 04/26/2017 - 23:37

I was recently part of a team that studied how microbes cycle nitrogen in their environment. Our findings were as follows:

This lab had four different experiments performed regarding fixation, ammonification, nitrification, and denitrification.

            In the fixation experiment, it was expected that the plant with both nitrogenous fertilizer and bacterial inoculant would form nodules along its roots and grow better than the other three plants, as the nitrogen fixing bacteria would form a symbiosis with the plants. The results are displayed in the following graph.

 

This graph shows that the expected results were confirmed in regards to growth, as the plant inoculated with bacteria and given nitrogenous fertilizer grew to be the tallest. The plant with nitrogenous fertilizer and bacteria also grew 11 nodules, and the plant with only bacteria grew 30 nodules, the other two plants grew 0 nodules. This does not confirm the original prediction as the plant with bacteria and no nitrogenous fertilizer still grew nodules, which are present when the organisms live in symbiosis and the bacteria fix nitrogen in the soil. A possible explanation for this result is contamination with nitrogenous fertilizer, as it is possible somewhere in the experiment the plant was either fertilized with the wrong fertilizer or with a tool contaminated with nitrogenous fertilizer.

            In the ammonification experiment, three flasks were utilized. One flask contained rich soil, one flask contained poor soil, and one flask contained P. vulgaris. It was predicted that the flask containing bacteria would show the lowest levels of ammonia as this organism can break down ammonia. The ammonia levels in the flasks were tested using quantofix ammonia test strips. The results can be seen in the following graph.

The expected results were confirmed by this experiment, as P. Vulgaris had the lowest ammonia levels at the end of the experiment.

            In the nitrification experiment, it was predicted that the rich tubes would show a larger decrease in ammonia and increase in nitrate, as the rich soil would have more ammonia to convert to nitrite or nitrate. The results of this experiment are displayed in the following charts.

 

ASB Data

Week

Poor Soil Ammonia

Rich Soil Ammonia

Poor Soil Nitrite/Nitrate

Rich Soil Nitrite/Nitrate

1

0

0

0

0

2

0

0

10 mg/L NO3- & 1 mg/L NO2-

 

10 mg/L NO3- & 1 mg/L NO2-

 

3

0

0

10 mg/L NO3- & 1 mg/L NO2-

 

250 mg/L NO3- & 40 mg/L NO2

 

4

N/A (not enough mixture left to test)

N/A (not enough mixture left to test)

50 mg/L NO3- & 10 mg/L NO2-

 

500 mg/L NO3- & 80 mg/L NO2-

 

 

NIB Data

Week

Poor Soil Ammonia

Rich Soil Ammonia

Poor Soil Nitrite/Nitrate

Rich Soil Nitrite/Nitrate

1

N/A (not recorded by partner)

0

0

0

2

N/A (not recorded by partner)

0

10 mg/L NO3- & 1 mg/L NO2-

 

10 mg/L NO3- & 1 mg/L NO2-

 

3

N/A (not recorded by partner)

0

100 mg/L NO3- & 1 mg/L NO2-

 

25 mg/L NO3- & 40 mg/L NO2

 

4

N/A (not recorded by partner)

N/A (not enough mixture left to test)

50 mg/L NO3- & 10 mg/L NO2-

 

500 mg/L NO3- & 80 mg/L NO2-

 

 

            The fluctuation in levels of nitrite and nitrate can most likely be attributed to denitrifying bacteria. Denitrifying bacteria can reduce nitrite and nitrate into gaseous compounds, which may have escaped the flasks. No data was recorded on the levels of ammonia in the NIB poor soil by my partners in charge of this experiment, hence the lack of reporting on this section of the chart. The expected results were confirmed, as the rich soils produced more nitrite and nitrate than the poor ones.

            In the denitrifying experiment, four different tubes of nitrate broth were used, one containing P. vulgaris, one containing P. aeruginosa, one containing rich soil, and one containing poor soil. The expected result was that it one would be able to determine the presence or lack of nitrite and nitrate in the broths using nitrate reagent A, nitrate reagent B, and zinc. After applying nitrate reagents A and B, a red color change indicated that nitrite was present. If there was no color change, zinc was added, if there was still no color change, this indicated that nitrate was broken down into a compound other than nitrite. If a red color change occurred after being exposed to zinc, this meant that nitrate was still present and never denitrified. The NB rich soil had nitrate break down into compounds other than nitrite. Both the NB poor soil and the P. vulgaris had the presence of nitrite. The sample with P. aeruginosa had nitrate broken down into compounds other than nitrite. The expected results were confirmed, as it was possible to determine the presence of nitrite or nitrate using these tests.

Antibiotics

Submitted by amprovost on Tue, 04/25/2017 - 12:29

Recently I experimented with antibiotic producing bacteria and antibiotic resistant bacteria in my microbiology lab. The results were as follows: 

Two experiments were performed in this laboratory, a Kirby-Bauer test and an enrichment of Streptomyces and testing for its Streptomyces antibiotic production.

            The expected result of the Kirby-Bauer test was that this test would indicate whether or not the bacteria used was resistant or susceptible to the antibiotic discs used. This experiment was performed on two plates, one using S. aureus and one using E. coli. Each bacteria was individually mixed in a tube of sterile saline, both becoming concentrated enough to equal the 0.5 McFarland standard. These solutions were then spread across Mueller-Hinton plates that then had antibiotic discs placed on them. The zones of inhibition were then measured to see if the bacteria were resistant or susceptible to the antibiotics. The results were as follows:

 

 Kirby-Bauer Results

Antibiotic

S. aureus resistant, susceptible, or intermediate

E. Coli resistant, susceptible, or intermediate

Ampicillin

Resistant

Susceptible

Ciprofloxacin

Susceptible

Susceptible

Colistin

N/A

Susceptible

Erythromycin

Intermediate

Intermediate

Gentamicin

N/A

Susceptible

Penicillin

Resistant

Resistant

Rifampin

Susceptible

Resistant

Trimethoprim/sulfanilamide

Susceptible

Susceptible

Vancomycin

Susceptible

N/A

 

            Whether a bacteria was resistant, susceptible, or intermediate was determined by measuring the zone of inhibition in millimeters around the antibiotic disc. The measurement ranges determining this were supplied by the manufacturer of the discs. The results of this experiment were as predicted, as these discs allowed us to determine levels of susceptibility.

            In the experiment regarding the isolation and enrichment of Streptomyces, it was predicted that Streptomyces would be able to be isolated from soil and would produce antibiotics, which would be displayed by zones of inhibition in a cross-streaking experiment.

            This experiment was performed by doing two things with a soil suspension. First, the suspension was streaked onto a casein-starch plate. Second, this suspension was diluted by taking one milliliter of suspension and transferring it into nine milliliters of sterile saline. This new solution was then also streaked onto a separate casein-starch plate. After the Streptomyces were isolated, three individual colonies were taken and streaked in a heavy band across the top quarter of an antibiotic-test plate. These heavy bands were then cross-streaked with S. aureus, E. faecalis, K. pneumoniae, and A. hydrophila. A control plate with all of these organisms and no Streptomyces, to show what uninhibited growth would look like. After allowing for these organisms to grow, there appeared to be no inhibition from Streptomyces, as the cross-streaked grew perfectly across the entire area inoculated, looking quite similar to the streaks in the control. One possible explanation for this is that Streptomyces does not produce any antibiotics that affect these types of organisms. Another possible explanation for this is simply that the Streptomyces were not given enough time to produce antibiotics, and perhaps the expected results would have been displayed had the plates been left undisturbed for a longer period of time.

 

Fungi

Submitted by amprovost on Tue, 04/25/2017 - 11:48

I recently experimented with growing fungi in my microbiology lab. My results were as follows 

This lab had two separate experiments, one was the cultivation of fungus on both nutrient rich and nutrient poor agar, and one was on sugar fermentation by Saccharomyces cerevisiae.

            The expected results of the experiment on nutrient rich v. nutrient poor agar was that the fungus would grow better on the nutrient rich agar, as a better food source was available. The experiment was performed by inoculating a plate of FPDA (nutrient rich agar) and a plate of PCA (nutrient poor) with Fusarium. The results of this experiment showed a complete absence of Fusarium on the nutrient poor agar, and a large lawn covering about 75% percent of the nutrient rich agar. This lawn was red in the very center, with a white ring making up the outer half of the fungus. The expected results were confirmed by this conclusion,

            In the experiment with S. cerevisiae, the expected results were that glucose and sucrose would ferment, but that lactose would not. This experiment was performed by inoculating tubes of purple broth glucose, purple broth sucrose, and purple broth lactose with S. cerevisiae. Any sugars that were fermented would produce acidic byproducts, which would turn the tubes from purple to yellow. Each tube also contained a Durham tube, which would catch any gas produced in fermentation such as carbon dioxide; so one would be able to see if there were any gaseous byproducts. The results showed that S. cerevisiae is able to ferment glucose and produced a gaseous byproduct, but is not able to ferment either sucrose or lactose.

            The expected results did not match the results as sucrose was unable to be fermented. It was predicted that sucrose would be fermented as sucrose is a common sugar in many plants that share an environment with S. cerevisiae. The most likely explanation of this is simply that S. cerevisiae has no evolved mechanism to break down sucrose and must derive its energy from another food source in the environment, such as glucose.

Immunology PP

Submitted by amprovost on Mon, 04/24/2017 - 15:14

When an infection is spread, it is initally attacked by macrophages, which are also known as guard cells. These huge cells are the first to respond to an infection, and operate by using phagocytosis to ingest enemy organisms. These cells are larger than most human cells, and on average can ingest roughly one hundred bacteria per cell. Once ingested, these invaders are stored in a membrane until they can be degraded by enzymes from the macrophage. Macrophages also cause an influx of water from the bloodstream, which results in the swelling that we see around an infection. If the macrophages are unable to destroy the infection alone, they release messanger signals into the blood stream calling for backup. The next line of defense is the neutrophil. These cells are so deadly that they are saved for serious infection, as their attacks are somewhat indiscriminate that they also damage host cells. These cells also only have a lifespan of 5 days, as if they accumulate they can cause serious damage to the host. 

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