User:Elizabeth Schott/Notebook/Biology 210 at AU

Zebrafish Lab

2/20- First Check Up (24hr)

All fish in experimental group alive, 2 dead in control. Each replaced with 10 mL of solution

2/23 Second Check up (Day 4) Control: 3 dead in control group, leaving 15 fish. 2 or 3 unhatched, all the rest hatched and immobile but heart beat visible. 1 mobile fish. About .5cm in length, heart rate at 90 beats/second. Experimental: 2 dead in control group, leaving 18 fish. 2 or 3 unhatched, all the rest hatched and very mobile with heart beat visible. More hyperactive in movement than control. Heart beat at 135 beats/sec.

2/26 Third Check Up (Day 7) Control: 10 fish dead in control group, leaving 5 fish. All hatched. Mobile with visible heart beat. About 1.2 cm in length, heart rate at 95 beats/second Experimental: 13 dead in experimental group, leaving 5 fish. One unhatched but dead. Mobile with visible heart beat, moving in circular motion as opposed to straight. Heart beat at 120 beats/second.

One fish from each group was euthanized for preservation. EMS 3/5 16S Sequencing Results

A few weeks ago, we ran samples of our 10^-2 from our tet plate and 10^-6 agar plated bacteria in a PCR machine to be sent off and sequenced to identify the bacteria living in our transect. We determined which samples to send off for sequencing after running our samples through a gel electrophoresis. After getting the results back from the sequencing lab, we used http://blast.ncbi.nlm.nih.gov/Blast.cgi to identify the species of our bacteria.

Materials & Methods: -Sterile tube -Heat block apparatus -Centrifuge machine -PCR tubes w/bead -Agarose gel

We selected one colony from each of the two plates that has the most complete characterization. We isolated the DNA from the bacteria in the colonies and used the primers & PCR to selectively amplify the 16S rRNA gene. Because the gene sequence for each is diverse and specific to each species, we would be able to better identify the inhabitant of our transect. First, we transferred a single colony of bacteria to 100 uL of water in a sterile tube. This tube was then incubated at 100C for 10 minutes, then centrifuged for 5 minutes at 13, 400 rpm. During the centrifugation, we added 20 ul of the primer/water mixture to PCR tubes. After centrifugation, we transferred 5 ul of the supernatant to the 16S PCR reaction and placed the tubes into the PCR machine. The third and fourth rows from the ladder proved to be the most successful, which are the ones we sent off for sequencing. Our PCR image & sequencing results can be seen here: https://docs.google.com/a/student.american.edu/document/d/1EqbsOM_CV_zgu0OrdILC9smHnhOldDS-8Tz4bk7EU3M/edit

Our results concluded that two of our organisms were a variovorax and chryseobacterium, closely related to the species we predicted would be living in our transect. EMS

2/12 Invertebrates & Vertebrates

The purpose of this lab was to examine the invertebrates and vertebrates that live in our transect. We predicted there to be worms and small vertebrate bugs living in our marsh-like transect. If we examined the contents of the Burlese Funnel, then we expected to find some small vertebrate organisms.

Materials & Methods: -Acoelomates, Pseudocoelomates, and Coelomate samples -Microscope -Dissecting Scope -The 50 mL content from the Burlese Funnel -Insecta Identification Sheet

The first thing we did in lab was examine the three types of worms: Acoelomates, Pseudocoelomates, and Coelomates. We tried to relate how their movement related to their body types. The type of acoelomates we examined, Planaria, were much smaller and less visible to the naked eye, so we had to examine them under a dissecting scope. Clear and very primitive organisms, they would stretch and retract in order to move. The pseudocoelomate sample, nematodes, were black and visible to the naked eye, but could only determine their movement under a dissecting scope, which showed that they use tail like appendages to propel themselves. The coelomate sample we had was the common earth worm, very large in comparison to the other worms and able to see its movement from the naked eye. The body had many segments, and used its head to pull the rest of its body behind itself segmentedly. Next, we took our content sample from our Burlese Funnel and poured samples into petri dishes to look for organisms under the microscope. We observed 5 common soil invertebrates, shown in figures 1 & 2, and fully outlined in the table here https://docs.google.com/a/student.american.edu/document/d/17E_70mPLG7OcQsfH5QhUc4YHDLyMOgoeWQTLicrnXFE/edit?usp=sharing The size ranged from .1mm-25mm, the smallest being the soil mite and the largest being the porturan. The most common in the assortment was Isoterpa, aka the common termite. Our group also identified five vertebrate species that most likely also reside in our transect: the Tufted Titmouse bird, the Yellow Rum Warbler Bird, a black squirrel, a Norway Rat, and although not spotted during our inspection, an Eastern American Toad. Their full descriptions can be found here: https://docs.google.com/a/student.american.edu/document/d/17E_70mPLG7OcQsfH5QhUc4YHDLyMOgoeWQTLicrnXFE/edit?usp=sharing There are biotic and abiotic features in our transect that benefit their inhabitants, such as the tall grasses for the rodents to hide from their predators, as well as a food source from various plant seeds. In the spring when the plants are flowering, the insects provide a good source of food for the birds. These organisms represent the ecological concepts of community because, as shown in the food chain (google doc), it is an endless cycle of coexistence within our transect. The carrying capacity of our transect is much smaller in comparison to a full range marsh, however the limited number of organisms of each species we have examined supports the idea that the population of a species is dependent on the size of its habitat. For example, while bacteria were abundant, only one squirrel was seen. The trophic levels favor the lower level organisms, such as bacteria and small invertebrates, because their food availability is much higher than that for the birds or rodents. Our transect is a very diverse habitat that ideally portrays a typical marsh environment.

EMS

2/5 Examining Plant and Fungi

In this two-part lab, we were to collect samples of different vegetation from our transect as well as a sample of leaf/ground matter. Examining the seeds and structure of the collected plants under the microscope, we used the lab manual to determine their vascularization, mechanisms of reproduction, and their particular classes. One of the unique features of our transect were the cat tails, formally known as Typha latifolia, which are monocot angiosperms. Typically found in wetlands, the cat tails in our transect we suspect to be successful as a result of the drainage that runs through the transect. The samples we collected can be seen here: https://docs.google.com/a/student.american.edu/document/d/1kdc6bZnhJ-xGlgXnexksJWa51cKv0gmvrisFs4hFthU/edit.

Methods & Materials: -Two large ziplock bags -Cone -Tape -Beaker -Plastic mesh

The leaf litter and vegetation samples were collected in the ziploc bags and brought back to the lab. We then proceeded to set up our Berlese Funnel to collect the invertebrates that live in our niche. The diagram is shown here: https://drive.google.com/a/student.american.edu/file/d/0B8FjnKLCYJxBaXJLRmV6VlZlV2c/view?usp=sharing

In the conical tube, we poured 25 mL of the 50% water, 50% ethanol solution in order to preserve the invertebrates that would fall into the tube. We then taped plastic mesh into the funnel, and attached the funnel to the tube using tape. After placing our leaf litter in the funnel, we placed the system under light. Because invertebrates are unattracted to light, the method is that they will move away from the light source and eventually fall into the tube. These will then be observed in the following lab.

Data and Observation: Most of the vegetation in our transect was dead or dormant due to the weather, but presumably the inhabitants should be accustomed to this environment and still be alive. We also observed a fungi (picture shown here: https://drive.google.com/a/student.american.edu/file/d/0B8FjnKLCYJxBcDFwT05lRVYxaVk/view?usp=sharing) which we correctly presumed to be a mushroom because it had gills on the underside, which contain the spores that allow for reproduction.

In conclusion, most of the vegetation had similar characteristics, likely due to the fact that plants tend to have similar structures when living in similar environments. Certain traits, such as the gills in the mushroom, help clearly identify organisms without knowing exactly what they are. In our next lab, we will examine the invertebrates from our Berlese Funnel.

EMS 1/28 Identifying Bacteria on Agar Vs. Tet Plates

In this lab, we were to examine the bacteria that had grown on agar plates and tet plates over the course of a week. We expected species to grow on the agar plates because we believed agar would be a good nutrient for the bacteria to feed on. We also did a final observation on our Hay Infusion, which we observed less water, more murky clarity, and more of the plant material had been broken down. We hypothesized that if the bacteria was breaking down the plants and starting to decay, then that would also attribute to the murky clarity of the water. After comparing the agar and tet plates, our group noticed the colony types were larger and more clustered on the agar plates than on the tet plates. The effect of the trracycline on the total number of bacteria was that it decreased the number of bacteria that grew in comparison to the agar plated bacteria. We decided to examine colonies from 4 plates, the 10^-2 concentration on both agar&tet plates, and the 10^-6 concentration on both agar&tet plates. Although our group wasn't able to get a good reading on a wet mount slide, all sizes ranged between 0-4 mm and were mostly round shaped with a yellow color and slight elevation above the surface. We also performed a gram stain sample to better identify the characteristics of our bacteria. At the end of lab, we ran the DNA of our bacteria through a PCR to be examined for next lab.

EMS 1/21- Hay Infusion Lab & Dichotomous Key

The purpose of this lab was to use the Dichotomous Key to identify the bacteria from our transect sample. We predicted that there would be bacteria living in the hay infusion because of the controlled environment (jar) that was supplied with food source. If there were bacteria in the jar from the transect, they would grow and multiply.

Materials & Methods: -Dichotomous Key -Hay Infusion -Microscope -Slides w/ samples from transect -Agar & Tet plates -Micropipetter -Glass spreader -Ethanol (& fire)

In lab, we took samples from our hay infusions, one from the top layer niche and the bottom layer nice, and put them onto slides which we observed under the microscope. Using the Dichotomous Key, we were able to identify the organisms living in our transect as Pelomyxa, Bursaria truncatella, Colpidium, and Paramecium. After identifying the bacteria in our transect, we wanted to see if we had missed any and see if any of the ones we did find were from an antibiotic resistant strain. To proceed, we used serial dilutions from our hay infusion into 10^-2,-4,-6,-8 concentrations. We then placed a 100μL of each dilution onto an agar plate using a micropipette, and spread the bacteria using a glass spreader (which was then sanitized by ethanol and burned over fire). We repeated this process using tetracycline (antibiotic) plates to see if any were resistant.

We are hoping to find no antibiotic resistant bacteria, and re-observe the bacteria in the agar plates after a week of growth. EMS

1/21- Transect 1

The purpose of this lab was to familiarize ourselves with our individual transects and identify the abiotic and biotic components of it. My group's transect, Transect 1, is located in front of the Kogod School of Business building. Our long term goal is to discover what kind of bacteria live in the environment, by which we would make hay infusions & observe them in the next lab.

Materials & Methods: -Abiotic materials: rocks, soil, trash, metal sign, snow -Biotic materials: grass, red cardinal flower, leaves, moss, cat tails -Test tube -.1gm Powdered Milk -500 mL Water -Jar

The general features include tall grasses, bushes, and mossy soil. To resource for our hay infusion, we took a 50 mL test tube and filled it with both abiotic & biotic materials from our transect. In the jar, we placed 10 g of the transect sample, .1gm of powdered milk, and 500 mL of water. This completed our hay infusion that we would observe in next weeks lab.

Hopefully if there are bacteria in our transect, the sample subset should be able to feed off the powdered milk and water and grow over the next week.

EMS

1/21 Hi! My name is Elizabeth Schott, this is my first post on OpenWetWare for my Bio210 lab :) EMS