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

2/12/16 Plantae and Fungi Lab

Purpose The purpose of the lab experiment is to collect plant samples from our transect, and to analyze their characteristics as well as try to identify what they are exactly. We had to select five different plant samples that we found in different places throughout the transect. We are performing this experiment to not only determine the different plant life in our transect, but to understand how each plays a role in the ecology of the transect. Hypothesis: Plants from the transect influence the ecological environment of the transect and the organisms that inhabit it.

Materials and Methods -Collected 5 or more plant or fungal samples from different parts of our transect -Observed the 5 plant samples that we found under the dissecting scope -Used razor blades or scissors to cut open and analyze the inside of the plant materials and observed under dissecting scope again -Identified certain characteristics such as vascularization, specialized structures, mechanisms of reproduction, shape, and size, and recorded that information in the plant characterization table -Determined if the plants were monocots or dicots using the key provided in the lab -Used a field guide from the library to try to identify what the plant samples were (Source cited down below)

Data and Observations The five plants that were found in this transect include a dandelion leaf, a branch from a Virginia pine tree, a common milkweed, a white clover leaf, and half a walnut, as shown in Table 1. The dandelion leaf was found between one of the sprinkler heads near some tall grass, the branch from a Virginia pine tree was found near the pine/fern trees nears the back wall of the transect, the common milkweed was found near the front of the transect growing around some tall grass, the white clover leaf was found on the soil underneath the tall oak tree, and the half of a walnut shell was found near the lamp post on the western side of the transect.

Plant Identifications and Descriptions: After thoroughly analyzing the plant samples and recording the data in the Plant Characterization Table (Table 1 below) we went to the AU library and examine a field guide consisting of plants, animals, and other organisms around Washington D.C. The first identification was of the dandelion leaf. The leaf itself is much longer than it is wide, and its has rather sharp cut pointy edges. When examining the field guide, the this leaf found in the transect matched the dandelion leaf pictured and described in the guide exactly. Next, the the fern or pine branch that was found in the transect has many similar characteristics of the Virginia pine tree that was found in the field guide. There are some rather distinct differences between the branch sample found in the transect and the picture of the pine found in the field guide, but it appears that they are most likely closely related with similar short and smooth needles as well as detailed pinecone structures attached. Prior to examining the field guide in the library, we identified that the twig like plant with several smooth spherical berries was most likely a milkweed. After examining the field guide, we determined that the features of the plant found in the transect and the picture and description in the guide were very similar in their features. Regardless of whether or not this plant is a milkweed, it seems to be very closely related. The next the plant sample was a very small, light green leaf that was made up of multiple round leaves and pointy leaves. After examining the field guide, there are some similarities between the leaf sample found in our transect and the white clover leaf pictured and described in the field guide. There are several distinct features between the sample and the field guide picture in terms of leaf shape and slightly different coloring, but overall it appears that perhaps the leaf we found in the transect is part of the clover family. Lastly, we identified in lab that the brown half of a shell found in the transect best resembles a walnut. We could not find this walnut in the field guide, but after asking about it in class, we determined that it most likely is some form of walnut. It has very elaborate details on the outside shell, and it is somewhat hollow on the inside where there most likely used to be a nut or seed inside.

Plant Descriptions and Images: Dandelion Leaf: This is the dandelion leaf that was found between one of the sprinkler heads near some tall grass in the transect.

Branch from Virginia Pine Tree: This is the Virginia pine tree branch that was found near the multiple pine/fern trees near the back wall of the transect.

Common Milkweed: This is the common milkweed that was found growing near the front of the transect surrounded by tall grass in the transect.

White Clover Leaf: This is the white clover leaf that was found underneath the tall oak tree in the transect.

Walnut: These are two pictures of the walnut shell that was found near the lamp post on the western end of the transect. The first picture is of the elaborate front of the shell, and the second is of the inside where a seed most likely used to exist.

Table 1: Plant Characterization Table

Conclusions and Future Directions After thoroughly collecting and examining the plant samples in our transect, we can conclude that there is a lot of plant diversity that makes up part of the living things in our transect. In particular we noticed several different types of weeds and wild flowers (milkweed, clover, and dandelion leaf) and a couple of plant material fallen from the trees. These plants have the ability to reproduce throughout the transept and grow new plants, which therefore allows us to conclude from our hypothesis that the plants in our transect do indeed influence the environment of the transect.

Field Guide Source: Youth, Howard. 2014. Field Guide to the Natural World of Washington, D.C. Johns Hopkins University Press: Baltimore 285-302.

Y.J.T.F

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