User:Sophie Kotik/Notebook/Biology 210 at AU

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Objective: To test the effect of Retinoid A on the embryonic development of zebrafish.

Hypothesis: If Retinoid A has an effect on the embryonic development of zebrafish than we predict that those treated with it will show signs of negative development/inhibited development.

Procedure: We had two petri dishes; one with 20 zebrafish embryo in plain deer park water, one with 20 zebrafish embryo in deer park water treated with Retinoid A. These acted as our "control" and "treatment" groups, respectively. Because Retinoid A is light sensitive both petri dishes were kept in a drawer over the course of the experiment except for the short amount of time that we observed and recorded data. We then observed the progression of their growth over the course of 14 days.

Data: Day 1 - Start of experiment. 20 zebrafish were plated in both petri dishes. Majority of embryos were in the 10-12 hour stage. Day 2 - One zebrafish had died in the treatment group and one had died from the control group, leaving 19 embryos in both petri dishes (a total of 38). Majority of embryos were in the 14-16 hour stage. Day 4 - No new deaths recorded. Majority of zebrafish in 48-72 hour stage. Day 7 - All zebrafish observed in 120 hour stage with the exception of one in the 16 hour stage. One death in the treatment group and three deaths in the control group leaving 18 treatment and 16 control. Day 14 - Final day of observations. In the treatment group I observed one recent death (fish was intact just not moving), 10 dissolved fish (died not as recently), 7 alive. Movement of alive fish was twitchy and spastic. In the control group I observed 2 dead (1 died recently and 1 dissolved) and 14 alive. There were no physical differences observed between the treatment fish and the control fish. Differences were observed in the behavior of the fish: treatment fish were twitchy and only moved when provoked, strained movement. Control fish were fluid in movement and kept active regardless of if I disturbed the water. Length of fish from head to tail was recorded as 100 microns at 4x for the treatment fish and 110 microns at 4x for the control fish. No physical differences observed. The main difference arose when I recorded the heart rates. The treatment heart rate was 140 beats/60 seconds and the control heart rate was 116 beats/60 seconds. This corresponds to the twitchy, spastic movement observed in the treatment fish.

Conclusions: Retinoid A has a significant effect on the internal functions of the cardiac system of zebrafish embryo.

Lab 5 conducted on 2/20/14... Entry on 3/8/14 Objective: This lab was performed on February 20th, 2014 on American University's campus. The lab objectives were to understand the importance of invertebrates and to learn how simple systems (including specialized cells and overall body plan) evolved into more complex systems. Due to a snow day on the intended day for this lab, our experiment was cut short to just observations of the invertebrates collected in our Berlese Funnels from our transect.

Procedure: The procedure for this experiment was simply observation. We collected our invertebrates from the top portion of the ethanol or the bottom portion and observed them in petri dishes under the dissecting microscope. Observations were noted.

Raw data: Observed in the top of the ethanol: all invertebrates are very, very small. Some small with antennas (presumed to be microscopic ants; 4 observed). A small worm (microscopic, may have been a branch fragment). Observed in the bottom of the ethanol: one spider, 2 insects: a smaller one and a larger, cockroach-y one (looked like a cockroach).

Conclusions: using these observations we were able to identify the kinds of invertebrates that inhabit our transect. This helps us to better understand and characterize the organisms living in this ecosystem/niche. SK

Lab 4 conducted on 2/6/14... Entry on 3/8/14 Objective: This lab was performed on February 6th, 2014 on American University's campus. The lab objectives were to understand the characteristics and diversity of plants and to appreciate the function and importance of fungi. By collecting and observing samples from our transects as well as prepared samples in the lab, we learned how to identify plant species. We then applied this knowledge to our transects to identify the plant samples we collected.

Procedure: Procedure I involved collecting five plant samples from our transect. We first obtained a leaf litter sample that consisted of soft soil and dead leaves and ground cover. We collected ~500 g of litter into a bag. Then we found representative samples of five different plants and put them in a separate bag. We then described the plant samples and were able to identify them based on their descriptions. Procedure II involved investigating plant vascularization. We observed a prepared moss sample and took notes on its physical appearance and height. We used this skill to then describe the vascularization of our plant samples from our transect. Procedure III involved investigating plant specialization. We examined the leaves of a moss sample and then the leaves of angiosperms. We then were able to identify the vascularization of the plant samples from our transect using this knowledge. Procedure IV involved investigating plant reproduction. We studied a diagram of a bryophyte reproductive cycle. Unfortunately we did not find any seeds at our transect so we were not able to identify them based on the skills we learned from this portion of the procedure. Procedure V involved observing fungi. We looked at prepared samples of fungi with a dissecting microscope and made observations about them. Procedure VI involved setting up the Berlese Funnel to collect invertebrates. We poured 25 ml of 50:50 ethanol/water solution into a plastic tube. Next we fit a piece of screening material into the bottom of our funnel using tape. We then placed the funnel into the neck of our tube and attached this to a ring stand. We added our leaf litter collected into the top of the funnel and placed a lighted 40 watt lamp above the funnel with the bulb about 1-2 inches from the top of the leaf litter. Everything was then covered with foil. This setup was left alone in lab for ~ a week.

Raw data: Procedure I: Table 1: Transect Plants (#1-5): File:Table 1 Transect Plants (-1-5).pdf Procedure V: Fungi sporangia are enclosures in which spores are formed. They can be unicellular or multicellular. They are important because they contain the spores which are pivotal for fungi reproduction. Of the observed samples, a bread mold was identified as being a fungi: File:Bread mold.pdf. We think this is a fungus because it possesses zygospores.

Conclusions: Because this was an experiment based largely in observation, our conclusions are represented in our descriptive raw data as demonstrated in table 1. This information helps us become more familiar with our particular transect and be able to identify the organisms that live in this niche. SK

Lab 3 conducted on 1/30/14... Entry on 2/17/14 Objective: This lab was performed on January 30th, 2014 on American University's campus. The lab objectives were to understand the characteristics of bacteria, to observe antibiotic resistance, and to understand how DNA sequences are used to identify species. By observing samples directly from our hay infusion culture we were able to apply classroom knowledge to a hands-on environment in which we were able to better understand these organisms.

Procedure: Procedure I involved quantifying and observing microorganisms. First we checked in on our hay infusion cultures and noted any changes we saw in the culture. The appearance or smell might change week to week as the micro-ecosystem has time to develop and different species that have greater fitness in this environment create colonies and populations. The dynamic changes as new species begin to overpopulate and emit certain smells as they digest their food and what not. The observation of our agar petri dishes involved observing each dish and counting/approximating the number of colonies visible and then calculating the total number of colonies per mL of our hay infusion culture by extrapolating the data. Procedure II involved observing antibiotic resistance. We noted differences/similarities between the tet agar petri dishes and the non-tet plates. Procedure III involved observing bacterial cell morphology. We created wet mounts from the different colonies on the different petri dishes and observed the cells, noting characteristics of the microorganisms using the normal 10x and 40x magnifications and the 100x oil immersion objective lens. We then created a gram stain of each of the four colony groups by heating the area on the slide with the water and colony sample and then staining it with crystal violet. We stained the smear for 1 minute and then rinsed it using water. Then we covered the smear with Gram's iodine for 1 minute and then rinsed with water. Then we decolorized the smear by flooding it with 95% alcohol for 10-20 seconds. We then rinsed with water until the solvent flowed colorlessly from the slide. Then we covered the smear with safarin stain for 20-30 seconds and rinsed with water. Finally we blotted the excess water with a paper towel and then observed the stained slide under the 10x and 40x objective lenses. Procedure IV involved preparing a PCR for DNA sequence identification. We transferred a single colony of bacteria to 100 microns of water in a sterile tube. Then we incubated the tube at 100 degrees Celsius for 10 minutes and centrifuged it. We then used 5 microns of the supernatant for the PCR reaction.

Raw data: Procedure I: Quantifying and Observing Microorganisms Screen Shot 2014-02-17 at 9.03.43 PM.png

Procedure II: The colonies with antibiotic, for the most part demonstrated less growth than those without. The exception is one type of bacteria that flourished in the tet environment, which means that it was antibiotic resistant. The tetracycline overall decreased the variety in strains of bacteria, though indicated to us which strain is antibiotic resistant. There was 1 noticeable species that was unaffected by the tetracycline. Tetracycline works by binding to a specific subunit of ribosomes and inhibits the protein synthesis by blocking the attachment of a tRNA to the A site on the ribosome. (1) It prevents the introduction of new amino acids to the peptide chain. (1) There are many strains of bacteria that are sensitive to tetracycline and they can be aerobic/anaerobic, Gram positive/Gram negative, or possess other varying characteristics. (1)

Procedure III: Cells appeared very, very small and round dots. They were so microscopic that identifying specific mechanisms was not possible under 40x magnification. The Gram-stain was helpful in observing the shapes of the organisms, but was not helpful in identifying other morphological characteristics such as methods of motility.

Conclusions: From this experiment we were able to further our understanding of our transect and the kinds of microorganisms that inhabit it. We found a significant presence of antibiotic resistant bacteria which would make sense considering that our soil sample for our hay infusion came from inside one of the planting boxes in the community garden which was most likely treated with fertilizers and insecticides. It would therefore make sense that there would be a lot of antibiotic resistant bacteria so that they could resist any and all treatments that were being imposed upon the plant life that was being harvested.

Citations: 1. Tetracycline. (n.d.). Retrieved from SK

Lab 2 conducted on 1/23/14... Entry on 1/26/14 and 2/7/14 Objective: This lab was performed on January 23rd, 2014 on American University's campus. The lab objectives were to understand how to use a dichotomous key and to understand the characteristics of algae and protists. By observing samples directly from our hay infusion cultures as well as provided samples we were able to apply classroom knowledge to a hands-on environment in which to better understand these organisms.

Procedure: Procedure I involved creating wet mounts of samples of the known organisms and observing them under the microscope at 4X and then 10X objectives. We focused on particular organisms and characterized them as well as measured them using the ocular micrometer. We then used the dichotomous key to identify the organisms. Procedure II involved observing our hay infusion cultures. We gathered samples from several niches within the culture and created wet mounts of them and observed them as we did with the known samples. Then we characterized them using the dichotomous key. Procedure III involved preparing and plating serial dilutions for our lab on bacteria. We obtained four tubes of 10 mLs sterile brother and labeled them 2, 4, 6, and 8. We then took four nutrient agar and four agar plus tetracycline plates and labeled one from each group 10^-3, 10^-5, 10^-7, and 10^-9. We then swirled the hay infusion to mix up all of the organisms. Then we took 100 microliters from the mix and add this to the 10mLs of broth in the tube labeled 2 for 10^-2 dilution. We then took 100 microliters from tube 2 and added it to tube 4. We did this successively until we made 10^-6 and 10^-8 dilutions. Then we took 100 microliters from the 10^-2 tube and placed it on the surface of the nutrient agar plate labeled 10^-3. We successively did this to each plate from the corresponding tube. We then incubated the agar plates at room temperature for a week.

Raw data: Procedure II: Hay Infusion Culture Observations: • No smell. There is a thin film on the top of the liquid. There is a light brown fuzz that is growing downwards from the top film layer towards the bottom of the jar. There are three main layers; a top film, a middle aqueous layer, and a compacted dirt layer on the bottom. The dirt is grey in color with apparent roots growing through it.

Organisms would differ near vs away from the plant matter in our hay infusions because those that lived near the plant matter would rely on the nutrients provided by them as sustenance, whereas the organisms that would live in, for example, the middle aqueous layer would probably be more simple and require less to survive.

Our samples were obtained from the top film and then from the liquid directly touching the compacted dirt layer on the bottom.

Observed in the top film sample: Didinium x 1 (90 microns; mobile; protozoa; not photosynthesizing), Bursaria truncatella x 1 (650 microns), Paramecium x 1 (130 microns; mobile; protozoa; not photosynthesizing), Chlamydomonas x 1 (6 microns; not mobile; algae; photosynthesizing).

Observed in compacted dirt/water sample: Paramecium bursaria x 1 (80 microns; mobile; protozoa; not photosynthesizing), Gonium x 2 (75 micron diameter; not mobile; algae; photosynthesizing), Peranema x 1 (55 microns; mobile; protozoa; not photosynthesizing), Difflugia x 1 (75 microns by 43 microns)

Paramecium meets all the needs of life because they acquire energy by eating bacterium, algae, and yeasts and use this energy to move and power its organelles... they are made up of a membrane bound cell... they process their genetic material and use it to function as itself... they reproduce asexually by binary fission... as one of the oldest organisms on Earth, they have evolved defense mechanisms and mobility as well as other features.

If the hay infusion culture had been observed for another two months, I would expect to observe an increase in the number of living organisms within the culture. The culture would evolve to become an even more expansive ecosystem.

A selective pressure that affected the compositions of our samples was whether or not the organisms photosynthesized and therefore required sun light (the lab does not receive as much direct sunlight as did our transect). We observed very few photosynthetic organisms, and this is because the organisms that didn't need to photosynthesize were favored over those that had to compete for sunlight in the small environment.

Conclusions: from our data it can not necessarily be concluded anything definitive. These are all observations about the quality and variety of life in our transect and are building our overall knowledge and understanding of the ecosystem within our small area of land. SK

'Lab 1 conducted on 1/16/14'... Entry on 1/30/14 Introduction: This lab, "Biological Life at AU," was performed on January 16th, 2014. The goal of this lab was to become familiarized with the concepts of evolution through observation as well as to understanding the concept of a "niche" and recognize a defined ecosystem on American University's campus.

Purpose: One purpose was to observe the Volvicine line of green algae and to recognize evolutionary differences. The second purpose was to become familiar with an assigned transect on American University's campus and record observations. A third purpose was to create a hay infusion culture for use in future experiments using a sample from our designated transect. The hypothesis regarding the first purpose was that the Volvox, due to its evolutionary advancement compared to Chlamydomonas and Gonium, would display more complex characteristics as an organism.

Materials/Methods: First, a slide of living Chlamydomonas was created by placing a drop of the sample onto a slide and adding a cover slip. This slide was then observed under the microscope and data such as number of cells, colony size, and functional and reproductive observations were made. The same was done for samples of Gonium and then Volvox sequentially and all data was recorded. The next part of the lab was performed outdoors at the assigned transect #4: the farm. Biotic and abiotic factors were recorded and general physical observations were made and recorded. An aerial picture of the transect was drawn. Finally, a sample of soil was collected in a 50 mL conical tube for use in the hay infusion culture. The hay infusion culture was created by weighing 10-12 grams of the soil sample and placing it in a plastic jar with 500 mLs of deerpark water. 0.1 grams of dried milk was then added and it was mixed for approximately 10 seconds. The jar was then left with the lid off to settle.

Data/Results: Table 1: Evolutionary Specialization of Members of the Volvocine Line Chlamydomonas: Number of cells (~500), Colony size (1 micron), Functional specialization (motile), Reproductive specialization (Isogamy). Gonium: Number of cells (2-3), Colony size (32.5 microns), Functional specialization (motile), Reproductive specialization (Isogamy). Volvox: Number of cells (1), Colony size (150 microns), Functional specialization (motile, large), Reproductive specialization (Oogamy). Transect observations Location: farm transect/community garden. Topography: includes six planting boxes constructed of wood with varying vegetation growing in each one. The boxes are aligned in two rows of three, with approximately two-foot wide pathways spacing between each box. The pathways are made up of wood chips, rocks, and dirt, with some dying vegetation (leaves, etc) spread around the area. Abiotic components: sun, rocks, structural wood, mesh wiring, sheet rocks, plastic planting labels. Biotic components: wood chips, soil, plant leaves, dried roots, vegetables, fruit plants.

Conclusions: Based on the data in Table 1, it can be concluded that Volvox is the more recently evolved of the three samples. An indicator of this is the fact that it is oogamous as opposed to isogamous as are Chlamydomonas and Gonium. Also, it is significantly larger in size than the other two. This data confirmed the experiment's hypothesis. It would be interesting to observe a similar concept in another series of organisms to gauge the levels of advancement that come with the various phases of evolution. No conclusions can be made as of now based off of data collected from the transect because all data was merely observational. In the future it would be interesting to gather a sample from one of the other planting boxes in the transect so as to see if the living organisms in that sample are similar or different to the ones we are observing now, and if so why that is. SK