User:Isabelle Rocca/Notebook/Biology 210 at AU

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Zebrafish Project 3/19

Purpose: The purpose of this lab was to determine the effect of the chemical Rhodamine on a zebrafish embryo.

2/19: Two groups of zebrafish embryos were made. A control group that consisted of a little more than 20 zebrafish embryos, and a Rhodamine group which also consisted of a little more than 20 embryos. The 20 embryos from each group were put in to two separate peatry dishes and water was added to each dish. The Rhodamine group also had a few mL of Rhodamine added to the dish.

2/20: This was the first observation after setting up the experiment. In the control group, 5 had died, leaving 17 alive. In the Rhodamine group 18 were alive and 4 had died. Most of the zebrafish at this point were 48 hours old. The control group appears to be more active than the Rhodamine group, but the Rhodamine fish are still moving without strain or abnormally, they just appear slower. There was a slight yellow color present in the embryos with Rhodamine. The Rhoda

2/23: There were 12 alive and 5 dead from the control group, and 16 alive and 2 dead from the Rhodamine group. The control group had less hatched zebrafish than the Rhodamine did. For the most part the two groups behaved the same. The control group appeared to swim faster, and the Rhodamine fish sometimes did not move straight. There is only a faint trace of Rhodamine dye on the Rhodamine group, this can only be seen under a microscope. Structurally both groups looked the same.

2/26: 3 fish had to be sacrificed from the control and the Rhodamine for observational purposes. The fish were put in an anesthetic to kill them and preserve the bodies. The remaining fish were fed a drop of food and new water was added to the dishes. The fish were about a millimeter larger and were all hatched at this point. There was not much of a difference between the control group and the Rhodamine group. Size, movement, appearance, all appear the same between the two groups.

3/6: This was the final check up day, and they day we euthanized the fish. The fish in the control group were about 3.5 mm long (able to be seen with the naked eye. Most fish had visible organs and large eyes. The fish moved less than before, only when the dish was shaken did they swim around. The Rhodamine dish was empty when we came in on the last day, so we do not know where our fish and water went, so we do not have any final data for the Rhodamine group.

Conclusion: The Control and the Rhodamine groups did not appear to have any major differences. Except the Rhodamine dye did not appear to have any tracer effects on the fish after the first two days.

IR


3/5/2015 Bacterial Sequencing Purpose: The purpose of this lab was to determine two species of bacteria that were present in our Hay Infusion Cultures using PCR to sequence the 16 S area on the bacterial DNA. Materials: The materials used can be found under the PCR section of the materials section of “2/4/15 Colonies of Bacteria found in Hay Infusions” open wetware entry. In addition to those, the genewhiz website: https://clims3.genewiz.com/links.aspx?oId=4p and the blast website: http://blast.ncbi.nlm.nih.gov/Blast.cgi were used on the computers. Methods: The methods used can be found under the PCR section of the methods section of “2/4/15 Colonies of Bacteria found in Hay Infusions” open wetware entry. On the genewhiz website, find the codes that were given for your specific bacteria and copy and paste the raw sequences given into the blast website to find a matching bacterial species. Results: The sequences that were identified from our bacterial samples can be found here: One of our species was Varivorax, and the second was chryseobacterium. The foreword direction did not place very concise results, but when put in the reverse direction, it was a likely match for chryseobacterium. The original gel sample pictures can be found at this link as well: Conclusion: The chryseobacterium are typically found in soil around water. They are characterized as gram negative bacteria. Varivorax in nature are categorized as biodegraders. They break down organic materials in the soil, to provide nutrients for other organisms. These bacterium are also gram negative (http://jcm.asm.org/content/42/1/445.full.pdf). The two gram stains that were performed in lab do not match with the known gram stains of the Varivorax or chryseobacterium ( http://jb.asm.org/content/193/5/1183.full). That probably resulted from experimental error, from doing the gram stain in lab.

IR 2/18/2015 Vertebrates found in the Transect Purpose: To determine and identify vertebrates that are found around the transect Methods: Transect was observed 4 times around the evening in various winter weather temperatures. Discussion: The vertebrates found in our transect were: The Tufted Titmouse bird, the Yellow-Rumped Warbler bird, a black squirrel, Brown rat, and although we did not see this, an eastern american toad can probably be found around this area in the summer. Tufted Titmouse Bird: • Phylum: Chordata • Class: Aves • Order: Passeriformes • Family: Paridae • Genus: Baeolophus • Species: B. bicolor Yellow-Rumped Warbler Bird: • Phylum: Chordata • Class: Aves • Order: Passeriformes • Family: Parulidae • Genus: Setophaga • Species: S. Coronata Black Squirrel: • Phylum: Chordata • Class: Mammalia • Order: Rodentia • Family: Sciuridae • Genus: Sciurus • Species: S. Scarolinensis Brown Rat: • Phylum: Chordata • Class: Mammalia • Order: Rodentia • Family: Muridae • Genus: Rattus • Species: R. Norvegicus Eastern American Toad: • Phylum: Chordata • Class: Amphibia • Order: Anura • Family: Bufonidae • Genus: Anaxyrus • Species: A. Americanus The wet nature of the transect would attract all the species because of the abundant water supply, and the birds and rodents would be able to feed on the insects around the area. Toads need to live in most areas, usually in the dirt. The plants found in the transect provide food and shelter for the animals. The food web based upon all the groups of organisms can be seen here https://docs.google.com/a/student.american.edu/document/d/1Rpg0Xp42Ou86xnqqwu_LxTJUKLkpMDzKY9SGj57i1O8/edit?usp=sharing. These organisms represent a biological community because they all rely on each other to survive for food. Since each level of the food web is used as food for the next level of the food web, it keeps the populations to a good level so that one species does not become over populated. All the plant matter and the dead organisms are the primary producers, the bacteria and ants are the decomposers and consumers, the soil mites and termites are secondary consumers, and the rats are the tertiary consumers. Conclusion: The organisms found in this transect are attracted to it because of the rich soil and water that can be found here. IR

2/17/2015 Observing the Invertebrates in the Transect Purpose: The purpose of the lab was to determine the types of invertebrates found in our transects. Materials: • Leaf litter collected from last lab • 25 mL of 50:50 ethanol/water solution • Berlese funnel • Large flask • Light fixture • Mesh/screening material • Dissecting microscope • Scanning electron microscope Methods: • Put the ethanol/ water mixture in a large flask • Fit a piece of mesh/screening material to the inside of the berlese funnel • Put leaf litter in the funnel • Put bottom of funnel into the flask and put under a lighting fixture • After one week, remove the leaf litter and examine the ethanol/water for invertebrates • Look at and classify invertebrates first under a dissection microscope, then a scanning electron microscope Data and Observations: After setting up the Berlese funnel, we let the insects fall into the alcohol for a week. For the first part of the lab we looked at various invertebrates that were placed around the classroom. Then we looked at three different types of worms and noted how each moved differently. The nematodes moved in a sideways S pattern, and since they were round, they probably were also rotating. The earthworms inched along to propel themselves through the soil, and finally the planaria elongated their bodies, then shrunk back to a small size to move around. When they were elongated the planaria appeared to be very flat (figure 1 https://docs.google.com/a/student.american.edu/document/d/1kKjEpQWzyLquYWhWBENu-gCOtpIKxqiLIg4Qsp0VQZ8/edit?usp=sharing). After observing the worms, we moved on to collect the invertebrates from the funnels. Our funnel did not contain many, so we had to use funnels from other classes that had our same transect. An ant was observed in ours, and a termite (Figure 2 https://docs.google.com/a/student.american.edu/document/d/1kKjEpQWzyLquYWhWBENu-gCOtpIKxqiLIg4Qsp0VQZ8/edit?usp=sharing), a flea, a proturan (figure 3 https://docs.google.com/a/student.american.edu/document/d/1kKjEpQWzyLquYWhWBENu-gCOtpIKxqiLIg4Qsp0VQZ8/edit?usp=sharing) , and a soil mite were observed in the other groups’. The table containing the invertebrate’s length, numbers, and descriptions, can be found here https://docs.google.com/a/student.american.edu/document/d/1kKjEpQWzyLquYWhWBENu-gCOtpIKxqiLIg4Qsp0VQZ8/edit?usp=sharing . The sizes of the organisms ranged from less than .01mm to about 5 mm, the smallest being the soil mites and the largest being the proturan. All the organisms were from the phylum arthropoda, and there were a lot of ants and termites observed. Conclusion: All the invertebrates found were of the arthropoda phylum, so most were pretty similar and small, found in our transect. IR

2/10/2015 Observing Different Types of Plants and Fungi Purpose: The purpose of this lab was to observe different plants and fungi that were growing in each individual transect. Materials: • Two large Ziploc bags • Leaf litter found in the transect • Flower, leaf, and stem samples from the plants growing in the transect Methods: • The Ziploc bags were taken out to each group’s individual transect, and the leaves and some soil found on the ground were put into one of the bags • In the other bag, parts of five different plants were collected to be examined back in lab • In lab, all the plants were examine with the naked eye as well as dissection microscopes to help characterize the vascularization, specialized structures, and mechanisms of reproduction • If the transect did not have any fungi growing, a mushroom and bread mold were to be examined under the dissection microscopes Data and Observations: First, we visited our transect. It is a marshy type of area that probably gets a lot of stagnant water since it is located by the water drain. Most of the plants besides the grass and moss were dead, so no live leaves or flowers were present. Instead, we collected leaves that were found on the ground. There were big leaves and straw like grass found all over the soil of the transect. There were many types of plants to be found around the transect, but we could not tell which were flowering and which weren’t. The pictures of the transect can be seen in figures 1-5 (https://docs.google.com/a/student.american.edu/document/d/1kdc6bZnhJ-xGlgXnexksJWa51cKv0gmvrisFs4hFthU/edit?usp=sharing). Five different plants were collected and brought back to the lab. All the plants that were brought back were examined to first see the physical features that could be seen without a microscope, like the shape and size and color of the plants. Then, cross sections were made so the plants could be examined in closer detail to observe the vascularization, specialized structures, and mechanisms of reproduction found in each plant. The Table for all these characteristics can be seen in Table 1 (https://docs.google.com/a/student.american.edu/document/d/1kdc6bZnhJ-xGlgXnexksJWa51cKv0gmvrisFs4hFthU/edit?usp=sharing). The cattail plant had seeds attached to it, the seeds were monocot. None of the other plants had seeds that could be seen. The fungi sporangia were observed by looking at the black bread mold. The sporangia is a cell containing spores which allows for the reproduction of more mold. The black mold is part of the zygomycota division, and the mushrooms are part of the basidiomycota division. A drawing of a mushroom which can be seen (https://docs.google.com/a/student.american.edu/document/d/1kdc6bZnhJ-xGlgXnexksJWa51cKv0gmvrisFs4hFthU/edit?usp=sharing ). I know this is a mushroom because it has gills on the underside, which contain the spores that allow for reproduction. Conclusion: Different plants and fungi were observed in each transect. Most of the plants were alike in their vascularization and reproductive patterns, probably because they were found in the same transect. IR

2/4/15 Colonies of Bacteria found in Hay Infusions Purpose: The purpose of this lab was to observe the different colonies of bacteria that have grown in the culture. Materials • 4 nutrient agar plates • 4 nutrient agar plus tetracycline plates • Hay infusion cultures • 4 tubes of 10 mL sterile broth • Micropipetter • Scanning electron microscope • Dissection microscope • Gram Stain: o Staining tray o Crystal violet o Water o Grams iodine mordant o 95% alcohol o Safranin stain • PCR: o Heat block o Centrifuge o Primer/water mixture o PCR tubes o PCR machine Methods: • Take four tubes with sterile broth and label them 10-2, 10-4, 10-6, 10-8 • Get 4 nutrient agar plates and 4 nutrient agar plates with tetracycline • Mix up hay infusion culture to mix up organisms, add 100 microliters from the culture to the tube of broth in the 10-2 • Add 100 microliters of the 10-2 mixture to the 10-4 tube • Repeat to make the 10-6 and 10-8 tubes • Spread 100 microliters of the 10-2 tube onto one of the agar plates • Repeat with the three other agar only plates with the rest of the tubes and label • Repeat the same steps for the agar plates with the tetracycline • After incubating for one week observe the plates and count the colonies present on each plate • Choose four different plates to observe the colonies under the microscope, gram stain, and perform PCR to • Look at each of the four plates under microscopes • Gram Stain o Scrape growth from agar and put in on a microscope slide, and add a drop of water o Dry the sample by passing it over a flame o Above a staining tray, cover the smear with crystal violet for 1 minute, then rinse off with water o Cover the smear with Gram’s iodine mordant for one minute, then rinse off with water o Cover smear with 95 percent alcohol for 10-20 seconds, rinse with water o Cover smear with safranin for 20-30 seconds, then rinse with water o Look at gram stains under microscope • PCR o Transfer a colony of bacteria to 100 microliters of water in a sterile tube o Incubate tubes at 100 degrees Celsius for 10 minutes o Centrifuge samples for 5 minutes o Add 20 microliters of primer/water to labeled PCR tube o Transfer 5 microliters of supernatant from sample into PCR tube o Put in PCR machine Data and Observations The hay infusion cultures were observed one last time. There was a less strong smell than last week. The plant life condensed at the bottom, there was residue on the sides of the container, and the liquid was clearer than last week. The smell or appearance might change from week to week because the plant life and microorganisms are decaying and changing, which could cause some changes in the container. From there, the attention shifted onto the agar plates. Bacteria were present on all except one of the plates. There was probably no archaea present because they tend to only grow in extreme environments like hot springs or really deep oceans. The number of serial dilutions on the agar plates can be seen in Table 1 (https://docs.google.com/a/student.american.edu/document/d/1ke_wnyoxLJJCQexk37r4BoIZ5iQZN15-9Cy5fWkMIoo/edit?usp=sharing). There was more growth on the plates that did not have the tetracycline plates. This signifies that the antibiotic had an effect on some species of bacteria, but not all. No fungus grew on the tetracycline plates. The Tetracycline inhibits some of a bacteria’s enzyme reactions, as well as its protein synthesis, because of this, the bacteria cannot function, and will die. Tetracycline affects mostly gram positive bacteria, and some gram negative bacteria (Klajn). For the gram stain and PCR reaction, four plates were chosen out of the eight: 10-2 with only agar, 10-6 with only agar, 10-2 with agar and tetracycline, and 10-6 with agar and tetracycline. The photos of these plates can be seen in figures 1-4 (https://docs.google.com/a/student.american.edu/document/d/1ke_wnyoxLJJCQexk37r4BoIZ5iQZN15-9Cy5fWkMIoo/edit?usp=sharing). The table that describes the characteristics of each of the colonies can also be found on the same link. Conclusion: The gram negative bacteria were mostly found on the tetracycline plates, because gram negative bacteria tend to be more resistant to the antibiotic than the gram positive were. IR

Eukaryote Biodiversity found in Hay Infusion Cultures 1/27/15 Purpose The purpose of this lab was to observe the life that grew from the culture that was created this past week. Samples were taken from the top and bottom to see if there would be any biodiversity present in the two different locations. The previous entry included a hypothesis that stated we thought the life present would be commonly found in wet marshy areas, so we will look under the microscope to see if this is true. Materials • Hay Infusion Culture that was created last week • Microscope slides and cover slips • Pipettes • Compound light microscope • Dichotomous key of microorganisms

Methods 1. Obtained our hay infusion culture that we created last week 2. Using the slides and cover slips, we created two wet mounts: one from the top of the culture, and one from the bottom 3. Using the microscope, the slides were examined on different magnifications to look for microorganisms found in the culture 4. Once a microorganism was found, it was identified using the dichotomous key Data and Observations

Upon obtaining the Hay Infusion Culture, a bad smell was coming from the jar. There was a brown, translucent liquid in the jar, with plant residue around the bottom. There was something growing on the top of the liquid that had an oil-like appearance. Samples of the culture were taken from two separate locations: one on the top, and one on the bottom. The top sample was taken from whatever was growing on the top of the liquid, and the bottom sample was taken from around some submerged straw. It was hypothesized that there would be more organisms around the straw or other plants because the organisms would have something else to feed on. Image:top of culture.jpg Organisms from top of culture: • Colpidium: 80-90 micrometers in length, oval shaped, small mouth, fast swimmer, ciliated body, colorless, protest, non-photosynthesizing Image:colpidium real.jpg • Bursaria Truncatella: large body, large open mouth, cilia, protest, non-photosynthesizing Image:bursaria truncatella isabelle 2.jpg • Pelomyxa: 1-5 milimeters, creeps with pseudopods, shape changes, colorless, protest, non-photosynthesizing Image:pelomyxa isabelle.jpg Organisms from bottom of culture: • Colpidium: 80-90 micrometers in length, oval shaped, small mouth, fast swimmer, ciliated body, colorless, protest, non-photosynthesizing • Paramecium: Colorless, moves with cilia, cigar shaped, longer than colpidium, protest, non-photosynthesizing • Unknown organism: 15 micrometers, colorless, fast moving, found around plant matter, colorless, so probably non-photosynthesizing

The colpidium meet all the needs of life because: they get energy by ingesting food, the colpidium is a single celled organism, it has a nucleus which stores all its genetic information, they reproduce asexually, and it is believed that, like other ciliates, the colpidium evolved from other eukaryotes early on in history. If the culture was to grow for another two months, I hypothesize that there would be more forms of life, whose populations would grow by being allowed to feed on the present life for a longer amount of time. Also, some populations would die out because they run out of food, or they are not used to eating the food that is present in the culture. Some variables that would affect the life growing in the cultures would be: water temperature, amount of sunlight received, and amount of plant matter present in the culture.

Conclusion At least one of the identified microorganisms, the colpidium, are commonly ofund in places that have water and soil present. This somewhat proves our hypothesis. Most organisms that were identified were found on both the top and bottom of the culture, so there was not much difference in the locations.

Hay Infusion Culture Lab

1/25/15 Purpose: In this lab, we are creating a culture in order to examine the forms of life that are present around the school. Each lab group was given a different transect of a different wildlife area around the school. We observed the living and non-living components that make up a biological niche. My group’s transect resembled a marshy/ swamp sort of land, because it was right by a water drain. I hypothesize that the types of organisms we will observe will be found commonly around areas of water and mossy life. Materials: • A wildlife transect found somewhere on the grounds of AU • Paper • Pencil • 50 ml conical tube • 500 mls water • .1 g of dried milk • Medium sized plastic jar Methods: 1. Go out to assigned transect and observe the biotic and abiotic substance found there 2. Draw an aerial view of the transect 3. Describe the location, and topography of the transect 4. In the conical tube collect 50% soil from the area, and 50% vegetation from the area 5. Back in the lab, weigh out 10-12 grams of the collected soil/vegetation, and place it in the jar 6. Add 500 mls of water to the jar with the sample 7. Finally add the dried milk to the water and the sample in the jar 8. Put the lid on the jar and gently mix for 10 seconds 9. Open the jar and place it somewhere safe in the lab Data and Observations: The transect of my group was on the north eastern side of campus, in the front of the Kogod building. It is supposed to have the characteristics of a marshy/ swamp land, because it is located by a water drain. Most of the plants found in the small garden were dead for the winter, but there was still green grass and moss present. Some of the biotic features that can be found in the transect are grass, moss, red cardinal flowers, cat tails, and straw plants. Some abiotic features that are there are rocks, snow, a metal sign, trash, and soil. The sample obtained for the culture consisted of soil, snow, moss, some leaves from the plants, grass, and some straw from the plant. Image:group 1 transect.PNG

Conclusion: Back at the lab, a hay infusion culture was made using the sample from the transect. It will incubate for a week, so that next week, the living organisms that are present in the transect will be observable under the microscope. This will demonstrate what sort of organisms grow in different environments around the school. IR

First Entry 1/22/15 I like the color orange. IR

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