User:Nicole Bonan/Notebook/Biology 210 at AU

Lab 3: Microbiology and Identifying Bacteria with DNA Sequences

Nicole Bonan

TA: Alyssa Pedersen

Lab Section: D01

July 7, 2014

Introduction

There were two purposes of this experiment. The first was to observe the morphological differences between bacteria and their colonies on the agar plates versus the tetracycline plates using a wet mount preparation and gram staining. The second purpose was to set up a polymerase chain reaction (PCR) for each of three bacteria, two from the agar plates and one from the tetracycline plates, so that the DNA of those bacteria can be sequenced in the next lab if there is a PCR product for any of the bacteria. The hypothesis for this experiment was that if samples of the Hay infusion culture had been placed on the plates containing only the agar, then there would be visible growth of bacterial colonies on the plates, and if samples of the culture were placed on the plates containing both agar and tetracycline, then there would not be any observable growth of bacterial colonies, unless tetracycline-resistant bacteria was in the sample. In the following report, the methods used, results, data, and interpretation of the results of the experiment will be discussed.

Materials and Methods

First, the four agar and four tetracycline plates were observed by eye. Notes were taken on the number of colonies on each plate, the appearance of the colonies, and the morphological differences between the colonies on the agar and tetracycline plates.

Next, separate wet mounts of one bacteria colony from each of two agar plates and one tetracycline plate were prepared and observed under a microscope. The plates from which the colonies were observed were the 10^-3 dilution on the agar plate, the 10^-9 dilution on the agar plate, and the 10^-3 dilution on the tetracycline plate. The wet mount was prepared by first placing a small sample of each bacterial colony on separate slides using a sterile loop. Then, one drop of deionized water was dropped on each sample with a disposable pipette. The solution was then covered with a cover slip.

After that, gram stains of the same colonies were prepared and observed under a microscope. The gram stain was prepared by first using a sterile loop to place a sample of each colony on separate slides, and then dropping one drop of deionized water over the samples using a disposable pipette. A red wax pencil was used to circle the area underneath the sample. After air-drying, the samples on the slides were heat-fixed by using tongs to pass the slide over a Bunsen burner three times. The samples were then covered with crystal violet for one minute, washed with deionized water, covered with Gram's iodine for one minute, washed again with deionized water, and then decolorized with 95% alcohol for 20 seconds. The alcohol was rinsed off with deionized water, and then the samples were covered with safranin stain for 30 seconds before they were rinsed a final time with deionized water. The samples were then blotted dry with a paper towel and observed under a microscope.

Finally, PCR was set up for sequencing of the 16s gene of the colonies of bacteria. A sample of each of the same colonies that were previously observed were placed into separate sterile tubes containing 100µL water and then incubated at 100°C for 10 minutes. The samples were then boiled for 5 minutes at 13,400rpm. Next, 20µL samples of a primer/water mixture were pipetted into each of three tubes containing primer beads using a micropipette. 5µL of the supernatant from each of the bacterial samples were then pipetted, using a micropipette, into one of the three tubes, so that each tube contained a different bacterial supernatant. These three tubes were each labeled with the transect number (Transect 5) from which the bacteria came, the lab section (D01), and the sample of bacteria (either T3 for the tetracycline 10-3 sample, A3 for the agar 10^-3sample, or A9 for the agar 10^-9 sample). The tubes were then placed in the PCR machine.

Tables and Graphs

Table 1: 100-fold Serial Dilutions Results

Lab 2: Identifying Algae and Protists

Nicole Bonan

TA: Alyssa Pedersen

Lab Section: D01

July 2, 2014

Introduction

There are millions of different species on Earth ⁶, which can ultimately be grouped into either prokaryotes or eukaryotes². Prokaryotes are simple, unicellular organisms that do not have a nucleus or many organelles⁴, and they are surrounded by a plasma membrane⁵. They can be grouped into either the Domain Bacteria, which includes proteobacteria, chlamydias, spirochetes, gram positives, and cyanobacteria, or the Domain Archaea, which includes the euryarchaeota and crenarchaeota. Eukaryotes are more complex; these organisms can be multi or unicellular, and their cells have membrane-bound nuclei and more organelles than prokaryotes. Eukaryotes can be grouped into either algae, which photosynthesize, or protists, which consume nutrients instead of photosynthesizing¹.

Dichotomous keys can be used to help identify different organisms. These keys consist of a series of two morphological choices about an organism that is being observed, which, once answered, will allow a person to determine the identity of the organism¹.

The purpose of this experiment was to use a dichotomous key and a microscope in order to identify four organisms in the Hay infusion culture created in the previous lab. A serial dilution was then carried out in order to create samples of the culture that would be incubated in separate petri dishes of agar and tetracycline. It was hypothesized that if organisms existed near the top of the Hay infusion culture, then they would be able to photosynthesize, and that if organisms existed near the bottom of the culture, then they would be protists. In the following report, the methods used, results, data, and interpretation of the results of the experiment will be discussed.

Materials and Methods

First, a sample of the Hay infusion culture was taken from near the top of the culture using a disposable pipette. A wet mount was created using a drop of the culture sample, a drop of Protoslo, a cover slip, and a slide. The slide was placed under a microscope, and two organisms in the sample were observed and identified using a dichotomous key. The same procedure was then carried out using a drop of the Hay infusion culture taken from near the bottom of the culture.

Next, serial dilutions of the Hay infusion culture were made after swirling the culture to mix up all of the organisms in it. The first serial dilution was 10^-2, which was created by pipetting 100µL of the culture to a test tube containing 100mL sterile broth. The next serial dilution was 10^-4, which was created by pipetting 100µL of the 10^-2 dilution into a separate test tube of 100mL of sterile broth. The third serial dilution was 10^-6, which was created by pipetting 100µL of the 10^-4 dilution to a separate test tube containing 100mL of sterile broth. The final dilution, 10^-8, was created by pipetting 100µL of the 10^-6 dilution into a separate test tube containing 100mL of sterile broth. A 100µL micropipette was used for all of the pipetting in the serial dilutions. A diagram of the serial dilutions is shown in Figure 1 below.

Figure 1: Diagram of Serial Dilutions of Hay Infusion Culture

The above image is a diagram of how the serial dilutions of the Hay infusion culture were prepared. 100µL of the culture was added to 100mL sterile broth in the first test tube. Then, as the first arrow in the diagram shows, 100µL of this solution was added to 100mL sterile broth in the next test tube. This process continued until the final dilution. The serial dilutions prepared were 10^-2, 10^-4, 10^-6, and 10^-8, in that order.

A sample of each of the serial dilutions were then plated onto agar plates. 100µL of each serial dilution were pipetted, using a 100µL micropipette, onto separate agar plates and spread across the agar using a sterile glass rod. Then, separate 100µL samples of each dilution were pipetted onto separate agar plates that contained tetracycline and were spread across the agar using a sterile glass rod. All of the plates were covered, labeled with the initials of the lab members, and set on a windowsill to incubate at room temperature until the next lab. The tetracycline plates were also labeled with a "T" to distinguish them from the agar plates.

Results

After having incubated since the previous lab, the Hay infusion culture appeared to be about the same opaqueness as lemonade. There was sediment and plant matter at the bottom of the liquid, while the top of the liquid seemed relatively free of this type of matter. The liquid was a light brown color, and the matter at the bottom was very dark brown. The culture had an odor of algae and mildew.

Two organisms were identified from the top of the culture and from the bottom of the culture for a total of four identified organisms. The two organisms identified from the top of the culture were paramecium and amoeba. The paramecium was colorless and was the shape of an elongated oval. It had two vacuoles, a micro and macronucleus, and also had cilia surrounding its outer covering. The paramecium was 50µm long and was a protist. The amoeba had an irregular shape and was mostly colorless, except for the brown-green coloring that surrounded most of its numerous organelles. The amoeba also had a contractile vacuole that was constantly expelling water from the organism. The amoeba was 25µm in length and was a protist. The two organisms that were identified near the bottom of the culture in the sediment and plant matter were chlamydomonas and colpidium. Two chlamydomonas were observed in the same area; both were unicellular, colorless, and roughly cube-shaped. Both also had one pair of flagella, which were used to propel the organism in circular motions. The chlamydomonas were about 50µm in diameter each and were prokaryotes in the Domain Bacteria. Two colpidium were also observed near each other. Both were unicellular, colorless, and oval-shaped. Each organism was motile and had about six organelles. One colpidium was more motile than the other colpidium; this more motile organism tended to bounce around the other organism. At one point, the more motile colpidium seemed to engulf and then regurgitate the other. Both organisms were about 20µm in length and were protists.

Tables and Graphs

Figure 2: Image of Hay Infusion Culture

The above image is of the Hay infusion culture as it appeared just before samples of the culture were taken for observation. The top of the culture is lighter in color than the bottom; most of the plant matter and sediments are in the bottom of the culture. The culture is opaque, and only allows limited amounts of light to filter through the liquid.

Table 1: Characteristics of Identified Organisms in Hay Infusion Culture

The above table organizes information about each of the four organisms that were identified in the Hay infusion culture using a dichotomous key. The four organisms identified were paramecium, amoeba, chlamydomonas, and colpidium.

Figure 3: Illustration of Paramecium

The above image is an illustration of the paramecium observed in the Hay infusion culture. The paramecium was observed near the top of the culture. The organism was colorless and had a somewhat "fuzzy" outer covering, which was due to its cilia. Its micronucleus and micronucleus are shown as circles inside of the organism. The background coloring is the liquid from the Hay infusion culture.

Figure 4: Illustration of Amoeba

The above image is an illustration of the amoeba observed in the Hay infusion culture. The amoeba was observed near the top of the culture. The organism was mostly colorless, though some of its organelles, which are represented in the illustration as circles, were outlined in a brown-green color. On the top left side of the amoeba was a contractile vacuole, which was constantly expelling water from the organism. The background coloring is the liquid from the Hay infusion culture.

Figure 5: Illustration of Two Chlamydomonas

The above image is an illustration of the two chlamydomonas observed in the Hay infusion culture. The chlamydomonas were observed near the bottom of the culture in the sediment and plant matter. The organisms were colorless and unicellular. Each chlamydomonas had two flagella, which they were using to propel themselves in circular motions around each other. The background coloring is the liquid from the Hay infusion culture.

Figure 6: Illustration of Two Colpidium

The above image is an illustration of the two colpidium observed in the Hay infusion culture. The colpidium were observed near the bottom of the culture in the sediment and plant matter. The organisms were colorless and unicellular. Each organism had about six organelles, and both organisms were motile. The background coloring is the liquid from the Hay infusion culture.

Discussion

The millions of different species on Earth ⁶ can be grouped into either prokaryotes or eukaryotes². Prokaryotes are simple, unicellular organisms that do not have a nucleus or many organelles⁴, and they are surrounded by a plasma membrane⁵. Eukaryotes are more complex; they can be multi or unicellular, and their cells have membrane-bound nuclei and more organelles than prokaryotes.¹. Dichotomous keys can be used to help identify different organisms. These keys consist of a series of two morphological choices about an organism that is being observed, which, once answered, will allow a person to determine the identity of the organism¹.

The purpose of this experiment was to identify four organisms in the Hay infusion culture prepared from Transect 5 soil at American University. The two organisms identified from the top of the culture were paramecium and amoeba; the two organisms identified from the bottom of the culture were chlamydomonas and colpidium. The hypothesis of the experiment was that if organisms existed near the top of the culture, then they would be able to photosynthesize, and if organisms existed near the bottom of the culture, then they would be protists. This hypothesis was made because it was believed that organisms near the top of the culture would have more access to light and would thus be able to photosynthesize, while those at the bottom of the culture would have limited access to light but would be able to obtain nutrients from plant matter. However, the data from the experiment did not support this hypothesis. Both organisms that were taken from the top of the culture and also the colpidium, taken from the bottom of the culture, did not photosynthesize; in fact, they were protists. The chlamydomonas, taken from the bottom of the culture, could photosynthesize. One possible explanation for why the data did not support the hypothesis is that the Hay infusion culture did not incubate long enough for a clear division between photosynthesizing organisms and protists to develop. Another possible explanation is that because there was so much plant matter in the culture, most organisms could obtain nutrients from the plant matter instead of photosynthesizing.

While there did not seem to be clear differences between the organisms observed from near the plant matter and those far away from it, there are possible reasons for why these organisms could have differed under certain circumstances. Organisms near the plant matter most likely would not have gotten much light, but they would be surrounded by nutrient-rich plant matter. Thus, these organisms would most likely not photosynthesize, but they would consume the nutrients available from the plant matter. At the top of the Hay infusion culture, which was far away from the plant matter, there was more light but fewer nutrients from the plant. Thus, the organisms living in this part of the culture would likely photosynthesize rather than obtaining nutrients from plant matter, as the plant matter would have been more scarce than the sunlight.

If the Hay infusion culture had been allowed to incubate for two months, many changes might have occurred to the culture's ecosystem. As time went on, many of the nutrients in the plant matter would likely have been consumed by the organisms in the culture, so this resource would become scarce. As a result, a selective pressure would arise in that protists that had previously relied on the nutrients from plant matter would have to find other sources of nourishment. These protists might evolve into a species that could photosynthesize, as light would likely be more abundant than the plant matter, or they might evolve to be able to consume other microorganisms in the culture.

According to Freeman, Quillin, and Allison's Biological Science, Fifth Edition, most organisms share five fundamental characteristics: they take in and use energy, they are made of cells, they process and respond to information from their environment, they are able to replicate, and their populations evolve ⁴. The colpidium that was observed from the Hay infusion culture met all of these requirements. The colpidium took in and used energy in order to propel itself through its liquid environment. The organism was unicellular. The organism also processed and responded to information from its environment, as it was able to change direction whenever it bounced into something in its environment. While the colpidium was not directly observed to replicate, it is known that colpidium do replicate. Finally, while the colpidium population was not directly observed to evolve, it is known that its population will evolve over time if its environment changes; for example, if other organisms are introduced that take in the same types of nutrients as colpidium, the colpidium can begin to specialize in its nutrient intake so that the two organisms can coexist ³.

Of course, there were sources of error in this experiment. One source of error was that the Hay infusion culture was moved across the lab in order for samples of it to be taken; during its transportation, some of the plant matter and organisms may have gotten mixed up in the culture, causing some organisms that would normally only exist at the bottom of the culture to be near the top of the culture and vice-versa. A way that this error could have been prevented would have been to take samples from the culture without moving the culture. Another source of error was lack of experience among lab members in identifying microorganisms. While lab members were diligent in determining the identity of organisms, the lab members may have misidentified certain organisms. This error could have been minimized by consulting with other, more experienced lab members in order to confirm the identity of the observed organisms.

The results of this experiment could be applied to future experiments in order to further study the relationship between microorganisms in an ecosystem. For example, the populations of the paramecium, amoeba, colpidium, and chlamydomonas could be studied in order to determine how the populations evolve over time in order to adapt to changing ecological pressures, such as availability of resources. These results could also be applied to the real world in order to study how bacteria become immune to antibiotics and what can be done to overcome this issue. For example, the population of chlamydomonas, a type of bacteria, could be studied to see how the population evolves when faced with an antibiotic. If the chlamydomonas evolves so that it is antibiotic-resistant, changes in certain phenotypes of the organism could be studied to determine whether those phenotypes are linked to the organism's immunity to antibiotics. Then, ways to use that link to prevent future generations of the bacteria from becoming immune to antibacterials could be studied.

References

¹Bentley; Walters-Conte; Zeller. 2014. Bio 210 Lab Manual. AU Campus Store: Washington, DC. 11-14.

²Brooker, Robert J. et. al. 2011. Biology: Second Edition.McGrawHill: New York, NY. G-26.

³DeLong, John P.; Vasseur, David A. "Coexistence via Resource Partitioning Fails to Generate an Increase in Community Function". Coexistence via Resource Partitioning Fails to Generate an Increase in Community Function. January 10, 2012. PLOS One. (July 7, 2014) <http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0030081>

⁴Freeman, Scott, et. al. 2014. Biological Science: Fifth Edition. Pearson: Boston. 2; G:29.

⁵Gregory, Michael. “Prokaryotes”. Biology 2 (Bio 102). State University of New York: Clinton Community College. (July 7, 2014) <http://faculty.clintoncc.suny.edu/faculty/michael.gregory/files/bio%20102/bio%20102%20lectures/prokaryotes/prokaryo.htm>

⁶Sweetlove, L. 2011. Number of Species on Earth Tagged at 1.7 Million. Nature: International Weekly Journal of Science. 10:1038. (July 7, 2014) <http://www.nature.com/news/2011/110823/full/news.2011.498.html>

Lab 1: Biological Life at AU

Nicole Bonan

TA: Alyssa Pedersen

Lab Section: D01

June 30, 2014

Introduction

Every organism in an ecosystem has its own niche, or an environment that provides all the specific requirements needed for the organism to survive⁴. The study of how the organisms in an ecosystem interact with other organisms and with their environments can be described by the field of ecology¹. Ecosystems include all of the abiotic, or nonliving, and biotic, or living, factors in a specific area². Ecosystems can be broken down into communities, which are collections of species. These communities can be further broken down into populations, which consists of all of the same type of species that lives in a specific area. Populations are made up of individuals, which are each single organism¹.

Over time, populations can evolve through the process of natural selection. Three things that cause natural selection to occur are variability within species, heritability of higher fitness within species, and differing abilities among individuals in a species to survive and pass on their genes to their offspring¹. Populations are constantly evolving as individuals adapt to selective pressures in their environment, such as changing sources of food or climate change³.

The objective of this lab was to observe the characteristics of a niche in a transect of the American University ecosystem, Transect 5, and to create a Hay infusion culture from the soil in the transect. In the following report, the methods, results, data, and interpretation of the results of the experiment will be discussed.

Materials and Methods

First, a transect of about 20m x 20m at American University was observed. The area was noted as "Transect 5". Pictures of the transect were taken and notes about the abiotic and biotic components of the transect were written. Next, a sample of the soil was taken in a conical tube. 10g of this soil sample were then added to 0.1g dried milk and 500mL Deer Park water in a plastic jar in order to make a Hay infusion culture. The jar was labeled as "Transect 5 TJ NB".

Results

The area that was observed, Transect 5, was a garden near the entrance to AU's campus. The transect was surrounded by paved sidewalks and was situated in a gully between two roads and a dorm building. The transect was hilly and had many biotic and abiotic components, most of which were landscaped and not naturally-occurring. Most of the biotic components were plants, and most of the abiotic components were stones.

Tables and Graphs

Table 1: Biotic and Abiotic Components of Transect 5

Biotic Components	Abiotic Components
Birds	Rocks and Stones
Gnats	Clay, mud, and mulch
Bees	Benches
Ferns	Stone pathway
Spots of Mosses	Drainage ditch
Liriope
Trees
Junipers
Ants
Clovers
Plants

The above table includes all of the biotic and abiotic components of Transect 5 that were recorded. The biotic factors are listed on the left and the abiotic factors are listed on the right.

Figure 1: Aerial View of Transect 5

The above figure shows an aerial view of Transect 5. Transect 5 was surrounded by sidewalks, which are colored in yellow. The bushes and trees appear as green circles, the stones appear as grey circles, and the benches appear as brown rectangles. The grey area surrounding the sidewalks are roadways, and the brown structure to the bottom left of the transect is a dorm building. Light green areas represent grass; light brown areas represent mulch and soil.

Figure 2: Transect 5, Image 1

The image above is of Transect 5. The image shows part of the drainage ditch, made of stones, and a mulched area to the right that includes different types of plants. A path with patches of moss winds through the center of the image, stopping at the stones.

Figure 3: Transect 5, Image 2

The image above is of Transect 5, and the image was taken to the left of Figure 2. The image above shows the continuation of the mulched area from Figure 2, which includes a tree surrounded by ferns and other plants. The image also shows a second drainage path for runoff. A paved sidewalk, shown in the back of the image, surrounds most of the transect.

Figure 4: Transect 5, Image 3

The image above is of Transect 5. This image was taken 180 degrees from Figure 3. It shows the path, continued from Figure 2, which dead-ends into the bench. A large bush is situated next to the bench, and behind the bench are grasses and a sign describing the garden. Behind the grasses is a mulched area with junipers, which eventually meets a sidewalk on the edge of the transect.

Figure 5: Transect 5, Image 4

The image above is of Transect 5. This image was taken to the left of Figure 2 and to the right of Figure 4. The image shows a second bench, which sits along the path that runs through the transect. Behind the bench are grasses and a tree, and behind those is the continuation of the mulched area from Figure 4, in which junipers are planted. The drainage ditch from Figure 2 is continued in this image; it consists of sones and runs through the lefthand side of the image.

Discussion

Every organism in an ecosystem has its own niche, or an environment that provides all the specific requirements needed for the organism to survive⁴. Organisms with similar genetic makeups can be grouped into species, and collections of the same species in a given area can be classified as a population¹. Populations can evolve over time through the process of natural selection, as individuals adapt to selective pressures in their environment³.

The purpose of this experiment was to observe a transect (Transect 5) American University in order to learn about the types of biotic and abiotic factors in the transect, how they interact with each other, and how these interactions affect their niches. The result of this experiment was that many different abiotic and biotic components were observed in Transect 5 at American University. Most of the biotic components were plants, while most of the abiotic components were stones. The transect was landscaped and not naturally-occurring.

Sources of error existed in this experiment. One source of error was that the transect was only observed at one point on one day rather than multiple times over a longer period of time. Thus, observations about the transect only reflected that one point in time, so some outliers may have existed in the observations (for example, there may not normally be a bird in the transect, but as there was a bird in the transect at the time of observation, the bird was noted as one of the biotic factors in the transect). A way to overcome this error would have been to observe the transect multiple times over a longer period of time. Another source of error was that not every abiotic and biotic component was noted. A way to fix this source of error would have been to spend more time observing the transect and precisely identifying everything in it. A final source of error was that a soil sample was taken in only one small area of the transect, so it may not have accurately represented the majority of the soil and microorganisms in the transect. A way to overcome this error would have been to have taken multiple soil samples in the transect and compared them.

There were many implications of the data that was observed in Transect 5. One implication was that humans had shaped the land in order to suit their needs, and from there, an ecosystem was developed. For example, the drainage ditch that ran through the transect existed because humans needed a drainage system to conduct water away from roads during storms. The area around that drainage system was then landscaped in order to look appealing to humans. The landscaping included plants and trees, which helped to create an ecosystem for birds and insects in the area. Another implication of the data was that Transect 5 had very nutrient-rich soil in the areas where plants and trees were growing. These species were leafy and healthy, which indicated that they were getting the nutrients and water needed to sustain themselves. A final observation of this experiment was that the transect was very clean and organized, in that plants were planted in specific areas and there were few weeds that seemed to be growing randomly throughout the transect. The implication of this observation was that the niches of many species within the ecosystem was controlled by humans; humans decided where plants would be planted, and they worked to get rid of unwanted weeds.

The results of this experiment could be used by others to observe the difference between natural and artificially-created ecosystems. Organisms and their niches in each type of ecosystem could be compared. This data could be used to determine the role that the organisms play in each ecosystem and how those roles differ between naturally-occurring and manmade ecosystems. The differing contributions of organisms to their ecosystems could be used to help determine the health of the overall ecosystem, and whether a manmade ecosystem can be as healthy as a naturally-occurring one. Another use for the results of this experiment could be to help better design gardens so that the niches that organisms occupy within the gardens more closely match the niches that they would occupy in a naturally-occurring ecosystem. In this way, more "green" gardens could be designed that feature more native plants and species to the area in which the garden is planted.

References

¹Bentley; Walters-Conte; Zeller. 2014. Bio 210 Lab Manual. AU Campus Store: Washington, DC. 4; 7.

²Freeman, Scott, et. al. 2014. Biological Science: Fifth Edition. Pearson: Boston. G:11.

³Rivera-Guzman, Dr. Javier. (July 3, 2014). Evolutionary Processes and Speciation. Biology 210 Summer Session 2014 Lecture. Lecture conducted from American University, Washington, DC.

⁴Merriam-Webster, Incorporated. "Niche". Merriam-Webster Online Dictionary. 2014. Encyclopedia Britannica. (July 7, 2014) <http://www.merriam-webster.com/dictionary/niche>