User:Zachary Salas/Notebook/Biology 210 at AU

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7MAR14- Here is an update to the 16S rRNA gene DNA analysis that we collected in Lab3 on 30JAN14. The results from the DNA lab came in for our nucleotide sequences. The sequence codes for Sphingobacterium faecium which is a bacillus shaped bacteria as we predicted from our visual observation of our sample taken in Lab3. The sample came from the 10-5 dilution of the purple colony harvested from our agar plate. The results are captured in the table below.

http://openwetware.org/images/6/66/16S_results.png

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6MAR14-At the 14 day observation, all zebrafish hatchlings were dead and decomposing. No viable observations or measurements could be made on these zebrafish. The only good samples left were the samples fixed at day 7. A number of measurements were taken (length of tail, length of entire body, and eye diameter) for comparison between the control group and the treatment group. The results are captured in the table below:

http://openwetware.org/images/4/40/Zebrafish_measurements.jpg

From the data the following conclusions were made: Treated group overall (vs. control group): Slightly higher death rate (deformed one died) Were longer Were thinner Smaller eye diameter Body length and tail length did not follow historical precedent (treated longer than untreated)

Due to insufficient data we were unable to conclusively determine the negative effects of nicotine on zebrafish. A more thorough and longer in duration experiment would need to be conducted in order to see more clear results.

ZS

27FEB14-At the 7 day observation all 18 hatchlings from the control group were still alive and seemed to be developing normally. They appeared to be at stage F of development and a pectoral fin was clearly visible. They were about 3mm in length and swimming normally. From the treatment group 17 hatchlings were alive and the 1 that was malformed died at about stage C (big yolk). Of the 17 alive they appeared to be at the same development stage as the control group; stage F with visible pectoral fin. Length and swimming response were the same in both groups at this point. Next, 2 zebrafish from each group were removed from the petri dishes and fixed for later analysis. The fixing procedure was as follows: 1. Pipette two zebrafish from each petri dish and place in each of two labeled vials. 2. Treat with .02% tricaine solution to anaesthetize. 3. Fill vial with 4% formaldehyde. The next observation will be made at 14 days where another two samples from each group will be fixed for comparison to the samples from day 7.

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24FEB14-After 72 hours, the zebrafish had hatched. In the control group out of the original 20 embryos there were 18 living hatchlings and 2 dead eggs. In the treatment group their were 18 living hatchlings and 2 dead eggs. Out of the 18 living hatchlings in the treatment group, one had developmental issues and was deformed and swimming erratically. The next observation will occur at 7 days.

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21FEB14-At 24 hours of observation there was no change observed in the zebrafish development. All 20 embryos in both the control and treatment groups appeared to be viable. The next observation will occur at 72 hours.

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20FEB14-In the second portion of lab six we conducted an experiment with zebrafish to determine the effects of toxicity on embryo development. Building on previous studies on the subject, the zebrafish were developed with two groups: a control group in normal spring water and the test group in water treated with 25mg/L of nicotine. It is hypothesized that the group treated with the nicotine would experience decreased survival rates, smaller body length and eye diameter, as well as decreased startle response.

Lab6/Zebrafish set-up- On this first day, we set up the control groups and the treated groups in two separate covered petri dishes. 20 mls of deerpark spring water were placed in the control group petri dish and 20 healthy embryos were added. In the second petri dish, 20 ml of 25mg/L nicotine solution were added along with another 20 healthy zebrafish embryos. The embryos were observed to be at about 14-16 hours somite development stage.

The next observation will be at 24 hours to assess any changes in development.

ZS

20FEB14-Since we had to miss a lab day, both Lab5 and the first part of Lab6 had to be completed on the same day. In Lab5 we studied the invertebrates and started with identifying vertebrates in our transect. In Lab6 we again moved up in complexity and studied embryology and Zebrafish development. Since most animals reproduce sexually by the production of gametes, one male and one female, that fuse (fertilization) to produce a zygote, it is important to understand how this zygote develops. This lab provided an introduction to the development after fertilization referred to as embryology. One of the most important aspects of embryological development is how the organism gets its food and how long the development takes. The development of three egg laying (oviparous) species were compared for similarities and difference in embryological development.

Lab6/Procedure I,II, III- In this section the Starfish, Frog, and Chick were compared in development in relative yolk size, amount of yolk, distribution of yolk, blastulation, gastrulation, and larval stage. The results are captured in the table below.

http://openwetware.org/images/a/aa/Embryo_1.png

Ecological aspects were also compared for the three organisms. The environment of development, fertilization, mechanism of waste disposal, physical protection, and parental care were studied and the results captured in the table below.

http://openwetware.org/images/9/9b/Embryo_2.png

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20FEB14-For this week's lab we built on the complexity of the plants and fungi we studied last week and now moved on to the more complex invertebrates. We looked at body symmetry like radial symmetry where the organism has a top and bottom but no front, back or sides. Also bilateral symmetry where the organism body has a head and tail as well as a right and left side. We also studied the three layers of tissue that make up the body; ectoderm which makes up the skin and nervous system, endoderm which makes up the digestive tract, and mesoderm which makes up muscle, organs, and bones. As the invertebrates evolved, their internal organ structure became more complex. Those that lacked a fluid filled cavity for their digestive tract where referred to as acoelomates, those with an incompletely filled cavity were called psuedocoelumates, and those with a fully lined fluid filled cavity were called coelomates. The invertebrates are also divided into two groups based on embryonic development and today we focused on the Arthropods.

Lab5/Procedure 1- In this section we observed three types of worms that were representative of the three types of internal organ structure. The first was the acoelomate worm "Dugesia" which moved with a type of slither. The second was the pseudocoelomate "Nematoda" which moved their body in an S fashion like the movement of a snake. And finally we observed the coelomate "Earth Worms" which moved by expanding and contracting the individual links that make up their body.

Lab5/Procedure 2- In the second part went back to the Berlese funnel we built in lab4 and extracted the invertebrates we sampled from our transect. We were able to identify five distinct species that are common in leaf litter and record their size and a brief description. The data of which is captured in the table below. http://openwetware.org/images/2/21/Invertebrates_transect.png

The invertebrates ranged in size from 1-4 mm in length. The ground spider was the largest and the soil mite tended to be the smallest. The most common in leaf litter being the smaller arthropods or microarthropods like mites, springtails, and millipedes.

Lab5/Procedure 3- As we move on to study the most complex of organisms, the vertebrates, we once again looked at the transect due identify different vertebrate species. Five different species were identified; the bachmann sparrow, the Baltimore Oriole, the fox squirrel, the field mouse, and the raccoon. The species were identified from the phyla down and captured in the figure below. http://openwetware.org/images/f/ff/Phyla.png

There were plenty of trees and plants for the Oriole and Sparrows. The climate is also favorable for these species. There are also light posts that make great perches for birds. For the fox squirrel there is plenty of plant life and tree seeds to sustain them. The field mice have many diverse environments in the low shrubs and many food sources in centipedes, millipedes, and trash from pedestrian traffic. The raccoon has many opportunities to scavenge as trash from pedestrian traffic is abundant. Next a food web was constructed in order to show the food chain in this diverse transect. This is shown in the figure below. http://openwetware.org/images/3/36/Food_web.png

Next week we move on to study embryology and the Zebrafish.

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6FEB14-In this lab we studied plants and fungi in order to better understand their different characteristics and diversity as well as learn their function. We studied how plants evolved from the simpler aquatic green algae and slowly made their way on to land.

Lab4/Procedure 1- We began by collecting samples of plant life for analysis from our transect described in lab 1. First we collected about 500g of leaf litter in a plastic bag which was used in Procedure 6. Then we took five samples of different plant species from the transect. Photographs were taken (Figures below) and they were brought back to the lab for further analysis. In the lab, we examined the different samples and recorded different characteristics like location in the transect, description of the plant, type of vascularization, etc. The results of which are recorded in the table below. Also based on these observations using different plant reference manuals and internet resources made a determination as to what we thought the genera of the plants were (results also in table below).

http://openwetware.org/images/f/fb/Transect_plants_lab4.png

http://openwetware.org/images/7/77/-_1.jpg http://openwetware.org/images/a/aa/-_2.jpg http://openwetware.org/images/e/e0/-_3.jpg http://openwetware.org/images/4/40/-_4.jpg http://openwetware.org/images/a/ab/-_5.JPG

Lab4/Procedure 2- We now evaluated the type of vascularization of each of the five samples and found the first four plants to be dicot with vascular bundles in rings and the sample 5 as a monocot with vascular bundles that were scattered. This data was captured in the table above.

Lab4/Procedure 3/4- In this section we closely studied the leaves from our five samples in order to assist in determining speciation. The more complex the organism the more specialized cells will be present. We described the size, shape, and cluster of the leaves and used this primarily to determine species as well as to determine whether they were monocot or dicot. The results were captured in the table above.

Lab4/Procedure 5- In this section we studied Fungi. Fungi are extremely important for our survival as they decompose many materials and release carbon dioxide for plant consumption as well as nitrogenous material into the soil which is essential for plant growth. Critical for Fungi survival is the sporangia which are black globe like structures which house the spores that are released into the air and spread by the wind. This is how the Fungi reproduce. We were able to observe some different samples of Fungi under a dissecting microscope. We were able to identify one as being the Ascomycota group and draw a picture of it (figure below). Due to the visible black sacks (sporangia) we were sure it was a Fungi.

http://openwetware.org/images/e/e0/Fungi_ascomyta.jpg

Lab4/Procedure 6- In order to explore invertebrate species from our transect for next weeks lab, we prepared a Berlese Funnel to collect them. We used the 500g leaf litter sample we collected in Procedure 1. It was set up in the following steps: 1. Pour about 25 ml of the 50:50 ethanol/water solution into the flask or bottle. 2. Fit a piece of the screening material into the bottom of the funnel. Tape the sides of the screen if necessary, so that the leaf litter does not fall into the preservative. 3. Place the funnel into the neck of the square-sided bottle. 4. Carefully put the leaf litter sample in the top of the funnel. Place a lighted 40 watt lamp above the funnel with the incandescent bulb about 1-2 inches from the top of the leaf litter. Cover everything with foil. 5. Leave the lighted setup on the lab bench for a week.

In this lab we continued on our path of studying ever increasingly more complex organisms. We were able to study more complex plants and Fungi and practiced identifying them with several different criteria. Next week we continue with the invertebrates as we continue to the most complex vertebrates.

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30JAN14- As in last week's lab where we studied unicellular Eukaryotes, in this lab we studied the different types of bacteria that belong to the Prokaryotic Domain Bacteria. These include Proteobacteria, Spirochetes, Firmicutes and photsynthesizing Cyanobacteria among others. Along with studying morphology and nutritional requirements we studied the bacteria's resistance to antibiotics, specifically Tetracycline.

Lab3/Procedure 1- As in lab 2, we began by observing our Hay Infusion culture. The water appeared clearer and the smell was less marshy and less potent. Perhaps the nutrients in the culture are being depleted and the organisms are dying off. During last weeks lab we also prepared agar plates with 100 fold dilutions of the culture half of them inoculated with tetracycline. This resulted in eight agar plates 4 without tetracycline and 4 with. Since millions of bacteria multiply from one single bacteria we expected to see colonies formed on the surface of the plates. We observed the agar plates and counted the number of colonies that formed. The table below captures the results. http://openwetware.org/images/f/fb/Colonies_on_agar_plates_lab3.png

Lab3/Procedure 2- In this section of the lab we evaluated the effects of the antibiotic tetracycline on our bacteria in the agar plates. This is important because the rampant use of antibiotics to fight infection in modern society has accelerated the production of antibiotic resistant strands of pathogenic bacteria. Despite these resistant strains antibiotics are still extremely effective against many bacteria. As you can see from the table above, the plates that were treated with tetracycline had drastically less bacterial colonies than those that weren't, causing a major decrease in the amount of bacteria that survived. Despite tetracycline's effectiveness there were bacteria that still grew in the presence of the antibiotic. In fact, two species of bacteria on the plates were unaffected by the tetracycline and thus resistant. The tetracycline works by inhibiting protein synthesis by disrupting the ability of aminoacyl t-RNA to bind to the ribosome small subunit thus blocking translation.

Lab3/Procedure 3- This section a series of bacteria were studied in order to practice identifying by morphological observations. We first observed different bacterial species on prepared slides and practiced identifying them. We next prepared slides from our agar plate colonies by picking 3 different species; 2 bacteria from the agar plates not treated with tetracycline and one that was treated with tetracycline. For each sample we prepared wet mounts as well as gram stains. Wet mounts: 1. We scraped up a tiny amount of bacteria from the agar plate 2. We smeared it on the slide and placed a drop of water on it 3. We covered the smear with cover slip 4. We observed under the microscope at 10x and 40x magnification.

The wet mounts were difficult to see because they were not stained with any dye. Nevertheless, we were able to identify a few different bacteria. The results are in the figure below. http://openwetware.org/images/7/79/Wet_mount_lab3.png

Next we prepared gram stains of the same three colonies of bacteria. We followed the exact procedure from the lab 3 handout for preparing the gram stain. 1. Label the slides. 2. Heat fix the air dried slide by passing it through a flame three times with the bacterial smear side up! 3. Working with a staining tray, cover the smear with crystal violet for 1 minute. 4. Rinse the stain off using a wash bottle filled with water. 5. Cover the smear with Gram's iodine mordant for 1 minute. Rinse gently. 6. Decolorize by flooding the smear with 95% alcohol for 10-20 seconds. Rinse gently. Decolorization has occurred when the solvent flows colorlessly from the slide! 7. Cover the smear with safranin stain for 20-30 seconds. Rinse gently. 8. Blot excess water carefully with a paper towel and air dry. 9. Observe sample under 10x and 40x magnification.

After preparing the gram stains we observed the three samples under 10x and 40x magnification and then collected some data about the cell samples like: source colony, whether they were treated with tetracycline, colony morpholgy, # of colonies and bacteria/mL, cell description, and whether they were gram stain positive or negative. The results are in the table below. http://openwetware.org/images/f/fe/Gram_stain_lab3.png

Lab3/Procedure 4- In this last procedure for lab 3 we prepared PCR samples of the three colonies we observed in order to amplify the 16S rRNA gene for DNA analysis so that we can verify the species we predicted via visual observation. The steps are as follows. 1. Transfer a single colony of bacteria to 100 μl of water in a sterile tube. Incubate at 100°C for 10 min and centrifuge. Use 5 μl of the supernatant in the PCR reaction. In next weeks lab we will run the PCR samples on a agarose gel and if they work will be sent to a DNA lab for analysis. We will also study Plants and Fungi.

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23JAN14- This lab was crucial in learning to identify single celled eukaryotic organisms within the two large groups of unicellular eukaryotes: algae (photosynthetic) and the protists (feed). Lab2/Procedure 1- We began the process of identifying single celled eukaryotes by observing known organisms under the microscope in the 4X and 10X objective. We then compared our observations with a dichotomous keys (Ward's free-living protozoa key) which takes into account size, shape movement , and color to help identify it. The two samples identified were Eudorina, and Arcella.

Lab2/Procedure 2- In the second portion of this lab we observed our hay infusion culture created in lab 1 in order to draw microorganisms from it and identify them. After a week the culture had a marshy smell to it. It had a swampy appearance with brown murky water, mold floating on the surface of the water, and brown dirt/sand on the bottom. We took two samples from the culture; one from the surface mold and one from the bottom dirt. We then prepared wet slides for observation under a microsope. By taking samples from two different areas of the culture it was thought that we would find different organisms. This would be due to the constrains of the two different micro-environments. The organisms near the surface would have access to breathable air versus the underwater environment of the organisms at the bottom. From the two different samples we were able to discover and identify three different organisms from each of the different micro-environments. We observed and captured their characteristics in the figure and table below. http://openwetware.org/images/2/25/Hay_infusion_Salas.jpg

If you use the organism Colpidium which was taken from the surface of the water sample, you can easily determine that it meets all of the requirements of having life. It has cilia and is mobile so it is clear it uses energy to live and move. It is made up of at least one cell, it has genetic hereditary information, its capable of replication, and is the product of evolution. In creating the hay infusion in lab one, it was mixed with milk in order to provide a nutrient source for the organisms that feed. It is likely that after observing this culture over the next few months, those that need external nutrients to survive will probably die off as the milk source will have been depleted and those that photosynthesize might be able to survive longer.

Lab2/ Procedure 3- In the third part of this lab, we prepared for next weeks lab by preparing and plating a serial dilution. In order to more easily examine the mix of bacteria and fungi in the hay infusion samples we prepared 100 fold dilutions of the culture and then inoculated half of them with tetracycline. This resulted in eight agar plates 4 without tetracycline and 4 with. Concentrations of 10-3, 10-5, 10-7, and 10-9 as depicted in the figure below. http://openwetware.org/images/3/30/Serial_dilution_salas.jpg

In next weeks lab we will study bacteria and prepare PCR for DNA sequencing.

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16JAN14- Lab1/Procedure 1- Observed evolution by studying the volvocine line of Algae. Studied three types of algae in increasing genetic complexity. Chlamydomonas, Gonium, and Volvox. It is thought that Chlamydomonas, the simplest, is the origin of the volvocine line and Volvox, the most complex, the end of the line. From samples of the three algae a number of characteristics were observed: Number of cells, colony size, functional specialization, and reproductive specialization. The results are contained in the table below. http://openwetware.org/images/thumb/2/2a/Volvocine_Salas.JPG/800px-Volvocine_Salas.JPG

Lab1/Procedure 2- In the second part of this lab we were assigned a transect of land on the American University campus to study the biodiversity of that ecosystem. The transect assigned was in a green space/park next to the bookstore. It is roughly a 20' x 20' square bounded by a walking path. The area consisted of mostly low ground cover with a few medium to large sized trees. There were also two tall light posts. Some of the biotic elements in the transect were: small bushes, medium trees, large tress, birds, and a squirrel. The abiotic elements were: the sidewalk, light posts, sprinklers, milky way wrapper, and rocks. A rough sketch of the transect is below. http://openwetware.org/images/thumb/3/38/Transect_Salas.JPG/800px-Transect_Salas.JPG

A sample of soil and ground vegetation was taken in a sterile 50mL conical tube. This sample was used to create a hay infusion culture that we will use to observe/study protists and bacteria in next weeks lab.

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