User:Yale J.T. Friedman/Notebook/Biology 210 at AU

2/5/16

Final Observations of Hay Infusion Culture: Water has evaporated, There is a thick film at the top, Minimal smell, rather earthy smell, Water is much darker, Thicker consistency, Hypothesis for change in smell/appearance: Water evaporation each week in the Hay Infusion culture causes a change in smell and appearance. Archaea potentially could have grown on our agar plates, but a reason they most likely would not is that they typically grow in extreme environments such as hot springs or at the bottom of the ocean.

Serial Dilution Results Table:

The agar plates that had tetracycline had much less bacterial growth in general compared to the agar plates without antibiotics. There were much fewer types of colonies formed on the plates that had the tetracycline, and one of these plates actually had no bacterial growth. This indicates that most of the bacteria were affected by the antibiotic, but a few species are resistant to tetracycline. There was no fungal growth on any of the plates, so we did not see the impact tetracycline has on the growth of fungi. The agar plates without tetracycline had a variety of different colors and sizes of bacterial colonies on the plates. The agar plates with tetracycline however, had about only one or two types of colonies grown, so approximately 1-2 species of bacteria are unaffected by tetracycline.

According to primary research, the mechanism of action for tetracycline is a ribosomal binding mechanism, and the types of bacteria that are sensitive to tetracycline include chlamydiae, mycoplasmas, rickettsiae, as well as protozoan parasites. (Chopra & Roberts, 2001)

Chopra, I., Roberts, M. 2001. Tetracycline Antibiotics: Mode of Action, Applications, Molecular Biology, and Epidemiology of Bacterial Resistance. Microbiology and Molecular Biology Reviews. 65: 232-260.

Bacteria Characterization Table:

Materials and Methods The Gram Stain procedure began by sterilizing a loop with a flame from a bunsen burner, which was used to scoop some bacterial growth from the agar plate. Next the bacterial growth was smeared onto a microscope slide and a drop of water was added on top. A red wax pencil was used to draw a circle around the bacterial smear sample. With a set of tongs, the microscope slide was passed through the flame to evaporate the water on the bacterial sample. After fully cooling, a drop of Gram's iodine mordant was placed on top of the sample for one minute and then rinsed off with water. 95% alcohol was added on top for ten to twenty seconds to decolorize the area around the bacterial smear. Safranin stain was then added on top of the smear for twenty to thirty seconds and then rinsed off with water. Any excess water was removed with a kimwipe and the microscope slide was set aside to air dry. Lastly, the bacterial sample was observed under the microscope, and photographs were taken under 40x magnification. The bacteria were characterized, and this whole process was repeated for a total of four agar petri dishes.

For the DNA isolation and PCR amplification process, bacterial colony growth was scooped out of two agar plates with sterile toothpicks. One agar plate had tetracycline, and the other did not. 20μL of primer mixture was added to two PCR tubes and were mixed thoroughly to dissolve the beads. The two bacterial colonies were then placed in the PCR mixtures of separate tubes, and were placed in the PCR machine. The process was performed to amplify the 16S rRNA gene and allow the bacterial colonies to be further characterized.

Pictures of Colonies:

Figure 1. Gram Stain 10-5 tet (-); 40x

Figure 2. Gram Stain 10-5 tet (+); 40x

Figure 3. Gram Stain 10-7 tet (-); 40x

Figure 4. Gram Stain 10-7 tet (+); 40x

Figure 5. No Gram Stain 10-5 tet (+); 40x

Figure 6. No Gram Stain 10-7 tet (-); 40x

Y.J.T.F

1/29/16

Description of Hay Infusion and Initial Observations: The Hay Infusion culture was filled with fallen leaves, acorns, small pine cones, twigs and smaller branches, and some soil. After adding the deerpark water and the dried milk and waiting one weak, the culture contained a very brown colored solution inside. The collected biotic materials from the transect are distributed unevenly throughout the culture. There was a rather sulfuric or rotten egg like smell in the culture, but also a rather earthy smell. There were no green shoots on top of the liquid, but there appeared to be some mold growth and possibly the beginnings of fungal growth. The mold and fungus will probably grow much more overtime if we let the culture remain.

Serial Dilution/Plating Description: Four tubes were filled with 10mL of broth. The four tubes were labeled: 10-2, 10-4, 10-6, 10-8. 100μL of solution from the Hay Infusion Culture were pipetted into the 10-2 tube, then 100μl from 10-2 solution pipetted into the 10-4 tube, 100μL from 10-4 solution pipetted into the 10-6 tube, and finally 100μL from 10-6 pipetted into the 10-8 tube, as shown in Figure 1 below. Next the 100μL from each tube were placed on two sets of four petri dishes. The first set of plates contained tetracycline, and two plates were filled with blood while the other two were filled with nutrient agar. The other set of plates each contained nutrient agar but did not contain tetracycline. The plates were labeled: 10-3, 10-5, 10-7, 10-9. In both sets of plates, 100μL from the 10-2 tube was pipetted onto the 10-3 plate, 100μL from 10-4 tube was pipetted onto the 10-5 plate, 100μL from 10-6 tube was pipetted onto 10-7 plate, and 100μL from 10-8 tube was pipetted onto 10-9 plate, as shown in Figure 1 below. The petri dishes were set aside for one week to observe the bacterial growth.

Serial Dilution/Plating Diagram - Figure 1:

Niche Samples: The samples used for making wet mounts came from the Top Zone and the Middle Zone Depending on whether the organisms are algae or protists will determine whether or not they will be close to or away from plant matter. Algae has the ability to make its own food through photosynthesis, so it may not necessarily be found near plant matter. Protists however do not perform photosynthesis but they need to consume nutrients. They need a food source which is why they will most likely be found near plant matter.

Protists and Algae Found in Sample: Top Zone: Gonium: 30μm in diameter; Green in color; flat; not completely round, rather curvy edges; non-motile; algae; photosynthesizing Pandorina: ~25μm in diameter, colorless; appears to be 16 cells inside; quite round in shape; flat; non-motile; algae; photosynthesizing Peranema sp.: 40μm in length, colorless; appears to be one flagellum; very oval like in shape; motile, protist; not photosynthesizing

Middle Zone: Paramecium: 210μm in length; colorless; flat; quite large compared to other organisms found; oval like in shape; non-motile, protist; not photosynthesizing Eudorina:12μm in diameter; slight coloring; flat; smaller compared to other organisms identified; non-motile; algae; photosynthesizing Euglena: 40μm in length; Greenish color; round, oval like in shape; flat; non-motile; protist; not photosynthesizing

Images of Protists and Algae Under Microscope: Top Zone:

Middle Zone:

Meeting All the Needs of Life (based on page 2 of Freeman text) Organism: Gonium Energy: Gonium is a form of algae that has the ability to produce the energy necessary to survive by photosynthesizing Cells: As seen under the microscope, Gonium organism that forms colonies of multiple green cells Information: Gonium is going to respond to the information if finds in its environment. For example, in the Hay Infusion Culture, the Gonium may avoid being surrounding by leaves and other biotic material that may conceal it from natural light. It wants to position itself so that it can get sunlight to be able to photosynthesize. Replication: Gonium is isogamous and sexually reproduces offspring that is morphologically similar to the parent cell. Evolution: The textbook explains that the organisms we have today are a result of evolution, so therefore Gonium has evolved into the algae that it is today, and may continue to evolve overtime to adapt to potential influences such as the environment.

2 Months Later If we let the Hay Infusion Culture grow for another two months, there would probably be significant mold growth and fungal growth especially in the top zone niche. Some of the biotic materials such as the leaves may be very soggy or broken down due to being in liquid for a longer period of time. This may result in leaf matter or other biotic material moving around and forming a gunk like material at the top of the culture. These selective pressures may influence the communities of the sample based on how the organisms react to mold and fungus, and also many protists may have to change location to get nutrients from the plants that have moved location, and then the algae may move around as well away from the plants as they photosynthesis for nutrients.

Y.J.T.F

1/22/16

Aerial View of Transect 1:

Abiotic Factors: Light Post, Soil, Sprinkler Heads

Biotic Factors: Large Oak Tree, Smaller Fern Trees, Tall Grass, Fallen Leaves and Acorns, Fallen Oak and Fern Tree Branches, Squirrel, Moss

Description: The aerial diagram above shows the basic layout of Transect 1, which is a large soil plot consisting of a tall oak tree, ten smaller fern trees, small scattered sections of tall grass, a light post, as well as several sprinklers throughout the plot. Scattered all around the transect there are fallen branches/twigs, leaves, and acorns from the oak tree, and some fallen branches from the fern trees. There is a bit off moss grown on the trunk of the oak tree, and a squirrel was seen eating fallen acorns that surround the oak tree. Just outside the north border of the transect there is a cement wall.

Volvocine Line Table Image:

Y.J.T.F