User:Jennifer M Thelus/Notebook/Biology 210 at AU

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Analyzing the 16S Sequence from the Cultured Bacteria found in the Hay Infusion Culture from Transect 5 at AU

Purpose The purpose of the 16S Sequence was to double check the assumptions we made about the bacteria that was characterized using its physical features in addition to its motility, shape and arrangement in addition to whether or not it stained gram + or -. It was assumed that our predictions about the bacteria that was cultured from the Hay Infusion Culture would be correct.

Materials and Methods This week we simply analyzed the sequence we received from the lab. Our sequences were MB38 and MB31. The sequences were then copied into http://blast.ncbi.nlm.nih.gov/Blast.cgi and it was compared to the database. We then compared what the database found to our guesses.

Results This is a picture of our agrose gel prior to our samples being sent off for testing. IMG 0006.jpg


The sequence for MB31 was NNNNNNNNNNNNNNNNNCNNNNNNNCAGTCGACGGCAGCATGATCTAGCTTGCTAGATTGATGGCGAGTGGCGAACGGGT GAGTAATACATCGGAACGTGCCCTGTAGTGGGGGATAACTAGTCGAAAGATTAGCTAATACCGCATACGACCTGAGGGTG AAAGTGGGGGACCGCAAGGCCTCATGCTATAGGAGCGGCCGATGTCTGATTAGCTAGTTGGTGGGGTAAAGGCCCACCAA GGCGACGATCAGTAGCTGGTCTGAGAGGACGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAG CAGTGGGGAATTTTGGACAATGGGCGAAAGCCTGATCCAGCAATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGC ACTTTTGTCCGGAAAGAAATGGCTCTGGTTAATACCTGGGGTCGATGACGGTACCGGAAGAATAAGGACCGGCTAACTAC GTGCCAGCAGCCGCGGTAATACGTAGGGTCCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTTGTGC AAGACCGATGTGAAATCCCCGAGCTTAACTTGGGAATTGCATTGGTGACTGCACGGCTAGAGTGTGTCAGAGGGGGGTAG AATTCCACGTGTAGCAGTGAAATGCGTAGAGATGTGGAGGAATACCGATGGCGAAGGCAGCCCCCTGGGATAACACTGAC GCTCATGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGTCAACTAGTTGTT GGGGATTCATTTCCTTAGTAACGTAGCTAACGCGTGAAGTTGACCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAA GGAATTGACGGGGACCCGCACAAGCGGTGGATGATGTGGATTAATTCGATGCAACGCGAAAAACCTTACCTACCCTTGAC ATGCCACTAACGAAGCANAGATGCATTAGTGCTCGAAGANAAAGTGGNNNCNGNGCTGCATGGNTGTCNNCAGCTCNTGT CGTGAGATGTTNGGTTAAGTCCCGNCNACCNAGCNNCANNCN

and the sequence for MB38 was NNNNNNNNNNNNNNNNNANNNNGNNNNNNNNNGCCNGTANNNNAGAGCTTGCTCTCTGATTCAGCGGCGGACGGGTGAGT AATGCCTAGGAATCTGCCTGGTAGTGGGGGACAACGTTTCGAAAGGAACGCTAATACCGCATACGTCCTACGGGAGAAAG CAGGGGACCTTCGGGCCTTGCGCTATCAGATGAGCCTAGGTCGGATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCG ACGATCCGTAACTGGTCTGAGAGGATGATCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGT GGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTT TAAGTTGGGAGGAAGGGCATTAACCTAATACGTTAGTGTTTTGACGTTACCGACAGAATAAGCACCGGCTAACTCTGTGC CAGCAGCCGCGGTAATACAGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTTGTTAAGT TGGATGTGAAAGCCCCGGGCTCAACCTGGGAACTGCATTCAAAACTGACNAGCTAGAGTATGGTAGAGGGTGGTGGAATT TCCTGTGTAGCGGTGAAATGCGTANATATAGGAAGGAACACCAGTGGCGAANNNNNNNNCCTGGACTGATACTGACACTG AGGTGCGAAAGCGTGGGGNGCAAACAGGATTANATACCCTGGTAGTCCACGCCGTANACNATGTCAACTANCCGTTNGGN ANNCNTTGAGCTCTTANTGNCGCANCTNACGCATTAANTTTNACCGCCTGGGGANTACGAGNCNNCNNNGTTANGNCTCA NATGGANTTGACNGGGGCCNNGCACAAGCAGTGNAGCNGGTGGNTTGNANTTCNAANCANNACNNANACCNNACCGNGCC CTTGAGNTCCGATGNANCTGTGCANNANNNNNANTGGGTGGCCNGNNNGNNANCNTTGGNNNAGNNCTNNNNNNNCTGTC AGNNNNNNNNNNNNNNNNNANNNNTNNNNTNNNTTNNNNNTNNNNANNGNAACNNNNTNNNNTTNNNNNNNNNGNNNN.

MB31 was the sample from the white bacteria from the nutrient agar plate. It was matched to most likely be Ralstonia Pickettii Strain KTO-35. The MB38 was the sample from the white bacteria that was found on the tetracycline treated agar plate. It was matched to be Pseudomonas Flourescens Strain S2.

Conclusion With both samples, we originally identified them as paramecium. This was not correct. The sequence analysis did not correspond to the colony morphology, gram stain and motility studies at all. This huge error could be attributed to the fact that we had no idea what were looking for.

4.7.15 What can you say about the bacteria that was identified from your sequences? SK

JMT

Examining the Developmental Effects of Ethisterone on Zebrafish

February 19, 2015

Purpose

This section of the lab is where individual students devise their own experiments by testing the effects of different things on Zebrafish embryos. There were at least 10 independent variables, including salt, caffeine and fluoride. This experiment will test the effects of ethisterone on the embryos. As an estrogen mimic, this is relevant as more and more women are releasing estrogen into the water supply from their birth control, as was discussed in class. Therefore this experiment is necessary to see the effects that this increase in estrogen does to development. With this information it is hypothesized that by increasing the concentration of ethisterone in the solution, the developmental time frame will be slowed.

Materials and Methods

Prior to beginning the experiment, everyone was required to read a published paper about the effect on zebrafish development. The paper on nanoparticles that I read was entitled Quantum Dots Are Powerful Multipurpose Vital Labeling Agents in Zebrafish Embryos by Sandra Rieger, Rajan P. Kulkarni, Dan Darcy, Scott E. Fraser and Reinhard W. Koster. After the paper was presented to the class. Then pairs decided what variable would be used. My partner and I decided to used ethisterone and set up an experiment. We would have a control and two treatments, one with half of the full dosage that was prepare and another with the full dosage.

After deciding on a treatment, we began the experiment by having three petri dishes, one for each treatment and finding 20 live embryos to place in each of the dishes. After separating the embryos, observe and record the developmental stage on day 1 of the experiment. After this, the three dishes were placed in containers to prevent evaporation of the solutions.

On day two upon examining the embryos, it was determined that most of the embryos were dead and had started to form mold. In dish 1, which contained the control, 15 of the 20 embryos were dead. In dish 2, which was half of the treatment, 17 of the 20 were dead. Lastly, dish three which contained the full treatment only had 5 deaths. After some discussion, the experiment setup was redesigned and moved from petri dishes to 24 hole well plates. This was done because I didn't want to have to through away the old embryos that survived. At the same time, it would be difficult to track the new and old embryos if they were just mixed into the petri dishes. By having the wells, each individual well would hold one embryo that could then be labeled. Each embryo was then put into its individual well. After that 2 mLs of solution were placed in the well of its respective treatment.

On day five, the dead embryos were removed. The remaining fish were then fed. Data was recorded on whether or not they had hatched and if there were any deformations. Then around day 11, they were tested for response to outside stimuli. They were tapped with the point of a pipette tip. It was then decided if the were responsive or not. They were classified as responsive if they moved. The speed of their movement was compared to the movement of the control group. For the embryos that died, it was recorded whether or not they were deformed. The experiment was concluded with the measurements of the fish that had survived to day 14. They were then disposed of after the data was recorded.

Data and Observation

-Picture of trays included in google document- The number correlates to its treatment. 1 for the control. 2 is for half of the treatment. 3 is the full treatment. The letters coordinate to when they were added to the experiment. O stands for Old which means they were added on day 1. N stands for New which means they were added on day 2.

[1]

WIth this link, you can view the data that was collected about the developmental stage of the embryos.

-picture of zebrafish development can be in google document-

This is the chart that development will be compared against.


Conclusions and Future Directions

This experiment is two-weeks long. With that, the developmental process of the embryos were substantially affected by the presence of ethisterone. Some embryos were even deformed. One embryo's eyes were bulging even though the rest of the body was still resembling a pre-24 hour embryo. The control subjects suggested that there was a positive correlation between body size and organ size, but this was not true for the ethisterone treated subjects. This half-way proved the hypothesis. The developmental time frame was slowed for the everything but the internal organs.


2.24.15 Good entry but could benefit from some more detail in data and observations and conclusions sections. Food web is not accurate, includes rose & rose bush as two separate things? And you have producers feeding consumers. SK

Observing Invertebrates found in my Transect

February 12, 2015

Purpose

This week focused on observing more complex organisms that exist within the transect. From what was found in the Berlese funnel the invertebrates were examined. In addition to that, hypothesizing the vertebrates that inhabit and pass through the transect/

Materials and Methods

This weeks lab was divided into four sections. The first section dealt with acoelomates, pseudocoelomates, and coelomates, which are examples of invertebrates. The next section focused on arthropods and observing the five major classes, arachnida, diplopoda, chilopoda, insect, and crustacea. Following that, the invertebrates that were collected with the Berlese Funnel were observed. Finally, a food web was created for all of the organisms that have been observed in the transect, including the vertebrates.

Data and Observations

Procedure 1: The movements of acoelomates, pseudocoelomates, and coelomates were observed. Acoelomates were observed to move in rather simplistic manner in one direction. The pseudocoelomates moved in an s shaped fashion, similar to a slinky it it's vibrated. Finally the coelomates moved in the most complex fashion. They typical moved in a accordion like manner, but could move really any way. Procedure 2: The five major classes were observed. Procedure 3: The Berlese funnel was taken down and the ethanol was poured out. Only a sparse amount was left to move the soil and invertebrates around on the petri dish. The petri dish was then observed underneath a dissecting microscope. Only two kinds of organisms were discovered in the sample. The table below shows the data collected about them. IMG invert.jpg

The size range did not vary greatly since we only found two organisms. They ranged from .3cm to .1cm. The Springtail X was the largest observed organism, with the smallest being the soil mite. The soil mite was also the most common organism found in the soil.

Procedure 4: From the textbook, a food web was created as shown below.

IMG 0022 2.jpg

Conclusion and Future Directions

For the past weeks, transect 5 at American University has been observed starting with the most basic components and working up the food chain. This lab was the culmination of all of the observations, in the form of a food web.

JMT

2.19.15 Good entry but a little brief. Could include more detail. SK

Observing Plantae and Fungi found in my Transect

February 5, 2015

Purpose

Continuing with the examination of transect 5 at American University, this week's focus was on Plantae and Fungi that were found in the niche. After collecting samples, they were studied and characterized accordingly. This week fungi will also be observed.

Materials and Methods

For this lab, two samples were collected from the transect. The first was a leaf litter sample that would later be used to set up a Berlese funnel. The second was a collection of five plants that were found within the transect. After bringing the samples back to the lab, the leaves from the second sample were then characterized. The first sample was then used to set up a Berlese funnel to collect invertebrates for next week's lab.

Data and Observations IMG 0007.JPG Figure 1. The black box is a marker of where all of the plants samples that were found were collected.

Transect Photo.JPG Figure 2. These are the five samples that were collected.

IMG 6938.JPG Figure 3. This is the table characterizing the information that we found about the plants.

Conclusions and Future Directions

After examining the samples that were found in the transect, it was concluded that the transect is not very diverse, plant wise. There was only a rose bush and stray leaves that were blown into the niche by the wind. It is not a very diverse area. For next week, a Berlese funnel was set up using the first leaf litter sample that was collected from the transect. By setting up the funnel, we should be able to observe Invertebrates that are present within the sample.



2.10.15 The notebook entry is good with thorough description of methods and clear data. You do need to start using the Openwetware page and to learn how to upload pictures. SK

Quantifying and Observing Microorganisms found in my Hay Infusion Culture

1.29.15

I couldn't get any images to upload so I did it in a word document.

File:Lab 3 oww.docx

JMT

2.4.15 Good start but missing some information and detail. Include images, detailed observations and conclusions. SK

Using a Dichotomous Key to Identify Algae and Protist found in my Hay Infusion Culture

22 January 2015


Purpose This week using a dichotomous key I will be identifying different bacteria that are living within my transect on campus. By identifying the bacteria that live within the transect, I can then begin to understand my niche and what is taken place within it.


Materials and Method

After retrieving the hay infusion culture from last week, not the smell. Prepare two slides from different areas within the jar. These represent two different niches within the hay infusion culture. After making the slides, identify the protists and algae located within the slide. Draw pictures of the organisms. After observing the slides, create serial dilutions from your hay infusion to grow cultures on petri dishes. Three of the six dishes will have tetracycline to see if there are any resistant strains within the hay infusion culture.

Data and Observation

The hay infusion smelled awful. There was evidence of mold and there were some greenshots. The two locations that were chosen to make the slides from where the bottom of the jar and on the surface of the flower bud that we collected. On the slide that contained information for the bottom of the jar, we observed paramecium aurelia (150μm), paramecium bursaria (80μm), and eudorina (10μm). On the flower bud, we found euglena (40μm), chlamydomonas (10μm), and paramecium multimicronucleatum (200μm). 1.27.15 Excellent first lab book entry. Well structured. SK

Observations of the Volvocine Line and a Niche at American University

15 January 2015

Purpose

Natural selection drives the evolution of different species. The three different conditions that must be met in order for natural selection to occur are variability, differential capacity or survival and reproduction, and heritability. Once it has been established that natural selection is occurring, the changing traits can be observed to determine whether or not evolution is taking place by observing different frequencies. Therefore evolution is taking place if a specific frequency is increasing or another is decreasing.

Materials and Methods

-Part 1:The Volvocine Line

First observe Chlamydomonas. Then measure the number of cells, as well as colony size in μm. Be sure to answer whether or not the cells are specialized, the mechanisms of motility for each genera, and if they are isogamous or oogamous. Then draw a picture of the live specimen. Repeat this process for the Gonium and Volvox. Record all data in a table.

-Part 2: Observing a Niche at American University

Observe the assigned 20x20 transect and take note of five abiotic and biotic components of the transect. Draw an aerial picture of your transect. Using a 50 mL conical tube, collect soil/ground vegetation that is representative of the transect. Once back in the lab, make a Hay Infusion Culture. To do this place 10-12 grams of the soil/ground vegetation sample in a plastic jar with 500 mLs of filtered water. Then add .1gm of dried milk and mix for 10 seconds. Without the lid on the jar place it in the indicated area in the lab and be sure to label the jar. Let it sit for a week.

Data and Observations IMG 6823.png Table1: Evolutionary Specialization of Members of the Volvocine Line


It is evidenced by these three samples that over time they evolved from "chlamydomonas" to the "volvox" IMG 6824.jpg Aerial View of Transect 5

The transect is located in the middle of campus. It is located mostly on soil and grass, with a small portion being concrete. Most of the northeast portion is covered in grass. Most of south portion of the transect is made of soil, which holds a flower bed. The flower bed is bare as it is winter and has many thorns. Within the transect there are biotic and abiotic components. The five biotic components are grass, the flower bed, the flower bud, leaves, and a spider. The abiotic components are soil, snow, a fork, cement, and woodchips. Most of the items were located in the southeast quadrant.

Conclusions and Future Directions

The data found from observing the volvocine line supports the claim that evolution is taking place. As for the transect it is a very diverse area. All biotic aspects may not have been present due to the cold weather. Samples from the transect were taken and a hay-fusion culture was made.

JMT