User:Student 56/Notebook/Biology 210 at AU

January 13,2016 20x20 Transect

Introduction Transect Description My group was assigned to transect #4. This transect consisted of a man-made pond that is surrounded by large rocks to add to the aesthetic of the pond. There are also several statues and signs that are around the pond periodically that were put there by AU staff. There are several small trees that are around the pond that give it shade, but that also deposit leaves all over the ground. The ground around the pond was coated in leaves and debris. There were not debris in the pond itself because there was a covering over it made of sticks and mesh. Behind the transect is a residence hall, which provides much artificial lighting to be able to see in the dark.

Data Photos of Transect

Biotic Components

-Water

-Trees

-Small Shrub-like Plants

Abiotic Components

-Rocks

-Soil

-Dead Leaves

Arial Diagram Of Transect

January 21, 2016 Protists and Algae

Purpose During this lab we identified different types of Protists and Algae in our hay infusions that were made the week prior. This was helpful because it allowed us to work with a dichotomous key and learn to identify and make observations on moving organisms. We also learned more about our transect by looking at and identifying the types of organisms that live there.

Materials and Methods First, we made observations about our hay infusion to see how it changed from when we originally made it. Then we made wet mounts for microscopic observation from three different niches in the hay infusion (one sample from the bottom of the jar, one from the middle, and one from the top). We then prepared and plated serial dilutions to be used the next week in the Microbiology Lab.

To make the serial dilutions four tubes filled with 10 mLs of sterile broth were labeled with 10^-2, 10^-4, 10^-6, and 10^-8 respectively. Four nutrient agar and four nutrient agar plus tetracycline plates were also labeled 10^-3, 10^-5, 10^-7, and 10^-9 respectively for both tetracycline positive and negative plates.

The hay infusion was then swirled with the lid on to mix up all the organisms. 100 microL was then added from the hay infusion to the 10 mLs of broth in the tube labeled 10^-2. The tube was then mixed thoroughly. 100 mLs of the broth from tube 10^-2 was then added to the 10^-4 tube and so on until all the dilutions were made.

The dilutions were then plated by pipetting 100 microLs from the 10^-2 tube onto the nutrient agar plate labled 10^-3. the sample was then spread carefully so that it was even across the plate. This was repeated with the 10^-4 dilution on the 10^-5 plates and so on. The agar plates were then placed on the rack with the agar side up.

Data

Hay Infusion Observations - smells mucky, much like a pond or a mud pool - brown color with leaves sticking out of the water - layer of soil on the bottom - defined layers between the top, middle, and bottom - mold growing on the top but no green shoots

Organisms on the Top of the Hay Infusion

- Peranema: cell elongated, colorless, with a broad, rounded edges. Highly plastic when stationary, often appeared to vibrate

- Amoeba: bloblike, small and creeped along not very fast. Had a single disc-shaped nucleus

- Actinosphaerium: Spherically shaped with radiating "spines"

Organisms at in the Middle of the Hay Infusion

- Colpidium: small body, oval shaped, with small mouth; fast swimmer

- Peranema: elongated, colorless cell with a broad, rounded posterior when moving

- Pelomyxa: Large creeping organism that moves using lots of small colored things (small nuclei)

- Oedogonium: Algae where the chloroplast forms a reticulated network extending from pole to pole of each cell

Organisms at the Bottom of the Hay Infusion

- Peranema: elongated, colorless cell with a broad, rounded posterior when moving

- Chlamydomonas: Cell is oval-shaped, fast moving, two observed flagella, colorless

- Chilomonas: Cell is elongated with a narrowed posterior, moves fast, colorless, two observed flagella

Pictures of the organisms

January 28, 2016 Microbiology

Our hay infusion had changed from the previous week. The water was much darker and the mixture as a whole was more compact. There was much less leaf litter sticking out of the water and taking up space in the container. The mixture also didn't smell as much as it had the previous week, which was surprising. There was still a separation in the layers of the mixture, with a definite line between the bottom middle and top parts of the water.

100-fold Serial Dilutions Results

Dilution Agar Type # Colonies on Plate Conversion Factor Colonies/mL

10-3 Nutrient 1040 x 10^3 1,040,000

10-5 Nutrient 60 x 10^5 6,000,000

10-7 Nutrient 2 x 10^7 20,000,000

10-9 Nutrient 3 x 10^9 3,000,000,000

10-3 Nutrient + tet 121 x 10^3 121,000

10-5 Nutrient + tet 1 x 10^5 100,000

10-7 Nutrient + tet 1 x 10^7 10,000,000

10-9 Nutrient + tet 0 x 10^9 0

The biggest difference in the plates with and without tetracycline was the ones with the tetracycline had much less growth than the ones without tetracycline. There were two or three species of bacteria/fungi that were unaffected by the tetracycline.

To do the Gram Stain, a loop was put over a flame to sterilize and then had time to cool so that the bacteria was not killed in the heat. The loop was then used to scrape up a tiny amount of growth from the surface of the agar. The bacteria was then mixed into a drop of water on the slide. All slides were labeled with their respective dilutions and weather tetracycline was present or absent. The slide was then passed through a flame to get all the water to evaporate. The slides were then placed on a staining tray and they were covered with crystal violet for one minute. The slides were then rinsed and then were covered with Gram's iodine mordant for one minute. The slides where then rinsed again and covered a third time with 95% alcohol for 10-20 seconds. The slides were then rinsed and covered with safranin stain for 20-30 seconds. The slides were then rinsed a final time with water. The excess water was carefully blotted with a kimwipe and the slide was then left to air dry. The slides where then observed under the microscope at 40x and 100x.

2 PCR tubes were then labeled with our transect number, colony identifier, and the group number. Then, 20 microL of primer/water mixture was added to the labeled PCR tubes. The primer/water mixture was then mixed to dissolve the PCR bead at the bottom of the tube. Using a sterile toothpick, a small amount of bacterial colony was picked up and mixed into the PCR tubes. The toothpick was then discarded and the cap was placed on the tube. The tube was then placed into the PCR machine and was left overnight.

Pictures