User:Maria Ochiai/Notebook/Biology 210 at AU

Plants and Fungi

February 13, 2016

Characteristics of Plants Collected from the Transect

1. Located in the Southeast side of the transect. The plant was found close to the ground. The leaves are paired and grows in even intervals on the stem of the plant. The leaves are oval-shaped and have rounded tips and net-like veins.

Figure 1: Picture of 1. plant with oval-shaped leaves.

2. Located in the Northeast side of the transect. The leaves are furry (almost have itchy feeling to touch) and have pointed tips and net-like veins.

Figure 2: Picture of 2. plant with furry leaves.

3. Located in the Northern edge of the transect. The plant bears red berries, which grows between two leaves at the tip of the stem. The leaves are deep green in color and have net-like veins. The leaves also have nice tough, hard, smooth layer like wax coating over them.

Figure 3: Pictures of 3. plant with red berries.

4. Located in the Western side of the transect. The leaves are pointy all around on the upper part of the leave. It almost looks like mistletoe leaves. The leaves have net-like veins.

Figure 4: Picture of 4. plant with pointy leaves.

5. Located in the Southwestern side of the transect. the branch has a grey color. There is a seed or flower bud growing on the tip of the branch. The leaves have net-like veins.

Figure 5: Picture of 5. plant with buds at the tip.

There were two seeds from the transect: the red berry from one of the plants collected and a black seed. The red berry was a dicot because it had two cotyledons. The black seed was a monocot because it had one cotyledon.

PCR amplification did not work because there was too much DNA in the sample that was put in the agarose gel. This resulted in blurry, undefined bar lines instead of clear bar lines that was supposed to appear when amplified in electrophoresis.

MO

Microbiology

February 9, 2016

In the Hay Infusion Culture, water evaporated, the film of mold that was growing on the surface covered only half of what it originally did, and the smell is not as strong as before.

From the table, the agar-filled petri dishes that included the nutrient and the antibiotics (tetracycline) have larger number of colonies growing on the plates. On the other hand, agar-filled petri dishes with no tet have fewer number of colonies on the plates. This indicates that the bacteria culture adapted to the antibiotic and therefore, grew out in large numbers as the bacteria reproduced in larger amount to counter the antibiotic. This could also mean that tetracycline becomes another food source or a stimulant that increase the reproduction of the bacteria culture. For both bacteria and fungi, they became resistant to the antibiotic, and so almost all the bacteria were unaffected by the tetracycline.

Tetracycline is used to treat human and animal infections, as growth promoters in animal feeds, and in prophlylaxis (Chopra). Tetracycline fight against gram-stain positive and negative bacteria, chlamydiae, mycoplasmas, rickettsiae, and protozoan parasites (Chopra).

Citation:

Chopra, I., & Roberts, M. (2001, June). Tetracycline Antibiotics: Mode of Action, Applications, Molecular Biology, and Epidemiology of Bacterial Resistance. Retrieved February 09, 2016, from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC99026/

Gram stain is performed by placing the bacteria onto the microscopic slide, creating a bacterial smear, and adding a drop of water. Heat the slide with a bunsen burner by waving the slide over the flame to evaporate the water. After it dries, place it slide on the staining tray and cover the bacterial smear with crystal violet for 1 minute. Then rinse the stain off using distilled water. Then, stain the bacterial smear with Gram's iodine mordant for 1 minute, and rinse off with distilled water. Then, discolor the bacterial smear using 95% alcohol for 30 seconds. Rinse off the alcohol using distilled water. After that, cover the smear with safranin stain for 30 seconds, and rinse it off using distilled water. Finally, using a wipe specific for microscopic slides, wipe off any excess water around the bacterial smear. Then the gram-stained slide is looked under the microscope (without cover slips) using 40X and 100X oil immersion objective lenses. The purpose of the gram stain is to see differences in the DNA sequence that encodes for an enzyme, peptidoglycan. Using gram stain is a plymerase chain reaction (PCR) that uses primer sequences specific for such genes that produce peptidoglycan and other non-coding areas. This method also provides information on how Archaea are different from other prokaryotes.

The PCR for 16S amplification was set up using 2 PCR tubes (labeled with our transect number and colony). 20µl of primer/water mixture was added to each tubes. Then, using toothpicks, we picked a small amount of bacteria from 10^(-5) tet and 10(-7) (using one toothpick each) and mixed them into the PCR tubes (one for each tube). Then, after discarding the toothpicks and the tubes are mixed by flicking the tube, the tubes are placed in the PCR machine. The PCR amplification on agarose gel will be observed next week.

Table 2: Bacteria Characterization _____________________________________________________________________________________________________________________________________________________________

10^(-7) tet: The colony is filamentous and has a color of pink. It does not exhibit motility and the colony is single or in clomps together. This bacteria is gram+ and it was measured 15µm at 40X magnification. _____________________________________________________________________________________________________________________________________________________________

10^(-7) no tet: The colony is irregular in shape and the edge is categorized as lobate. The colony is purple meaning that it is gram+. The bacteria is not motile, and they are not in clusters (cells all clomped together in a group) but separated as individuals. The cells measured from 4µm to 20µm at 40X magnification. The cells differs in size because all the cells are irregular in shape and size. _____________________________________________________________________________________________________________________________________________________________

10^(-9) tet: The colony is filamentous and coccus. The cells have no motility. The coccus parts of the cells are in groups, but the filamentous are tied together. This bacteria is gram+. The coccus measures 2µm and the filamentous varies from 20µm to 100µm both at 40X magnification. _____________________________________________________________________________________________________________________________________________________________

10^(-5) no tet: The colony is filamentous and are all in clomps together, as if there is not a lot of space for the cells to be by themselves. The cells are gram+ and measures 10µm at 40X magnification. _____________________________________________________________________________________________________________________________________________________________

Figure 1: Picture of 10^(-7) no tet. Measures from 4µm to 20µm at 40X magnification

Figure 2: Picture of 10^(-9) tet. The cells include both coccus - 2µm at 40X - and 20µm to 100µm at 40X magnification

Figure 3: Picture of 10^(-5) no tet. Measures 10µm at 40X magnification.

Figure 4: Picture of 10^(-7) tet. Measures from 15µm at 40X magnification.

MO

Protists and Algae

February 2, 2016

The Hay Fusion was set up using about 12 grams of the biotic and abiotic components collected from the transect, .1 grams of milk powder, and 500mL of water; all put into a plastic jar and mixed. Then the Hay Fusion was left for a week, to grow the protists. For the initial observation, there were layers of mold on the surface of the Hay Fusion, shaped like a surface of a leaf. The Hay Fusion also smelt like a porter potty and soil from decomposed things like leaves. The heavy objects like the wood chip, soil and leaves were at the bottom of the jar.

The protists might have come from the biotic components that was picked up from the transect. and possibly from frost because the frost might have trapped the protists as the water froze and turned into frost on the soil's surface . From the top layer of the Hay Fusion, there were two main protists that inhabited that layer. The protists were identified using the dichotomous key. One of them was Arcella. The protist was colorless and it was creeping or floating on the surface (no apparent movement by flagella or cilia) when viewed by the microscope. The protist was not spherical in shape. Although the picture looks as if the protist is spherical, according to the dichotomous key, if the protist is indeed spherical, then it would have radiating spines coming out of the protist. The shape of the protist remained constant meaning that it did not shape-shift. The protist did not have attached materials and had flat shell. So dichotomous key lead to Arcella, and the found Arcella was 50µm in diameter, which is within the range of 45-100µm in diameter of Arcella. From the dichotomous key, it is likely that Arcella is an amoeba since id does not use cilia or flagella to move around.

The other protist was of Paramecium Species, and the top layer particularly had Paramecium bursaria. This protist was moving around a lot; it span in circles and up and down. It was also colorless and it exhibited motions using hair-like structure: cilia, which covers the entire body. The protist had an elongated body with rounded ends, swam in corkscrew fashion, and was not attached to a substrate. The dichotomous key lead to Paramecium and by the measurement of 70µm and the shape of the protist, which was small and had a little hook-like or dent-like shape, told that the protist was Paramecium bursaria. Since this leaveing up on the deadline of summer break. Based on the motility and the dichotomous key, it looks like Paramecium bursaria is a protozoan. Also, since there is no color, it does not likely that they photosynthesize.

When the bottom layer of the Hay Fusion was observed under the microscope, the same protists that were identified on the top layer was the only protists that were identified. So in the Hay Fusion, the same protists appeared on both the top layer and the bottom layer of the jar.

The protists found describe how these species meets the needs of life. For example, arcella gets energy by feeding on the food, in this Hay Fusion the food was powdered milk and biotic components in the jar. Arcella is unicellular so it is made up of cell. It is encoded in units called genes to pass on the heriditary location, and so replication (protists are reproduced asexually) and evolution still occurs for Arcella.

If the Hay Fusion culture grow for another two months, then there will surely be a competition for food because food is not being added to the growing population. There will also be some kind of natural selection where one type of gene favors the environment compared to others.

MO

Transect Description

January 18, 2016

The transect was located near the Amphitheater on American University's campus. The transect had a circular shape and there were two lamp posts at each North and Northwest side of the circle. The transect had lots of shrubs, bushes, wood chips and dead leaves. The shrubs had dying leaves but the bushes grew healthy leaves. The ground was covered in dead leaves and wood chips and it was moist on the inside but was hard on the top soil with frost on top. The leaves were decomposed and so the soil created a layer of dead leaves and soil combinations. There was a berry-bearing tree right beside the transect and some berries dropped over to the transect.

Biotic: leaf from the shrub, wood chip, twig from the bush, berry, and black leaf collected from the ground

Abiotic: frost, aluminum foil, dirt, rock, and piece of gum

MO