User:Katrina Wakeman/Notebook/Biology 210 at AU

Observing Bacteria Using the Microscope and Gram Staining

January 28, 2016

Final Hay Infusion Observation

The hay infusion from transect 1 smells like musty and moldy water, which is a change from last week when there was no detectable smell. There was dry detritus on the sides above the water line, suggesting that the water line had receded due to evaporation. The water had a darker/ murky color. There were more fuzzy organisms growing on sediment. The blade of grass was still green. Finally, there were no more organisms growing on the surface of the water.

It is hypothesized that if there are microorganisms growing in the hay infusion, then they are producing a musty smell through either aerobic, anaerobic respiration or fermentation. Additionally, it is possible that the microorganisms growing on the surface of the water perhaps ran out of food sources, and died off.

Procedure I and II: Quantifying and Observing Microorganisms in Nutrient Agar and Nutrient Agar + Tetracycline

Table 1: 100-fold Serial Dilutions Results

The 10^-3 Nutrient plate had more diversity in the types of bacteria than the 10^-3 Nutrient + Tetracycline plate. On the nutrient plate there were purple, white, white-clear, yellow and orange colonies. The colonies were, on average larger than those on the tetracycline plate of the same dilution. On the Tetracycline plate of the 10^-3 dilution there are smaller orange colonies and one small, pale yellow colony. There was one similar orange colony viewed on the Nutrient 10^-3 dilution. On the 10^-5 dilution with just nutrient there are ten colonies (although only 4 are visible in the picture). They include the yellow, white and white-clear colonies that were observed on the Nutrient 10^-3 plate. The Nutrient + Tetracycline plate of the same dilution has no colonies. The rest of the plates at greater dilutions have no visible colonies. This shows that a more diverse population of bacteria was able to live in the plate with just nutrients. Also, less overall colonies of bacteria survive when tetracycline is present. Furthermore, the small orange colonies were scarce in the nutrient10^-3 plate and non-existent in the rest of the nutrient plates. However, once competition from other bacteria colonies was eliminated by the tetracycline, the small orange bacteria thrived and produced many colonies.

Pictures of the first and second dilution plates are below. Pictures of the second and third dilution plates were not included as there were no visible bacteria colonies. *Note: the pictures appear too large but if they are clicked on then it is possible to view them in a normal size.

There were most likely not any Archaea species growing in the hay infusion or the cultures made from it because Archaea tend to grow in harsh environments. A transect on AU and its subsequent hay infusion is not a harsh enough environment.

Nutrient Agar Plate: Serial Dilution 1

Nutrient + Tetracycline Plate: Serial Dilution 1

Nutrient+Tetracycline Plate Dilution 2 (left), Nutrient Agar Plate Dilution 2 (right)

Tetracycline is a broad range antibiotic that functions against gram-positive bacteria, gram-negative bacteria, chlamydiae, mycoplasmas, rickettsiae, and protozoan parasites. It has been used in treatment, as well as in animal feed, and because of this overuse, many bacteria and parasites have developed immunity to it. Tetracycline works by preventing bacterial protein synthesis. It does this by stopping the aminoacyl-tRNA from associating with the bacterial ribosome. Several methods of antibiotic resistance have been documented including bacteria with new genes that code for energy-dependent efflux of tetracycline or that code for a protein that protects bacterial ribosomes. Some bacteria have had gene mutations that alter the permeability of the out membrane, which prohibits tetracycline from entering the cell (Chopra, 2001).

Procedure III: Bacteria Cell Morphology Observations and Gram Staining of Bacteria

Table 2: Bacteria Characterization

White Bacteria Colony, Unstained

White Bacteria Colony, Stained

Purple Bacteria Colony, Unstained

Purple Bacteria Colony, Stained

Orange Bacteria Colony, Unstained

Orange Bacteria Colony, Stained

Yellow Bacteria Colony, Unstained

Yellow Bacteria Colony, Stained

Observing Protists and Algae in the Hay Infusion from Transect 1

January 21, 2016

Purpose: The purpose of this experiment was to observe the hay infusion that was taken from transect 1, one week ago. The hay infusion was observed on a macro scale, and cultures were made from the hay infusion in order to observe the protists and algae present. Finally, serial dilutions were plated in agar plates in preparation for observing prokaryotes and fungi next week.

Materials and Methods: First, the hay infusion was observed and hand written notes as well as pictures were taken to record its appearance. The scent, and the presence of life at the surface, in the middle and near the bottom were noted. A drop of water was taken from the surface of the water, from just above the bottom of the jar and from the water surrounding some suspended sediment that was observed. Each drop was taken with a different transfer pipet and placed on a separate slide. Then a drop of Proto Slow was added to each slide and a cover slip was put on top of the liquid. Each slide was examined using a microscope and several protists and algae were identified using the dichotomous keys provided. All the observations and identifications were recorded with hand written notes and pictures.

After the hay infusion was observed, eight serial dilutions were made. This was done by adding 100 microliters of the hay infusion water to 10 milliliters of the broth. This was mixed and then 100 microliters were taken from it and added to another tube with 10 milliliters of broth. This was repeated four times resulting in four tubes that contained 10^-2, 10^-4, 10^-6 and 10^-8 dilution. Two samples of 100 microliters were taken from each tube and one sample was deposited on a nutrient agar plate and the other was deposited on nutrient plus tetracycline (antibiotic) plate. Each was labeled and put away to be observed next week.

Data and Observations: Description of Hay Infusion—The hay infusion had no detectable smell. There was a gray film on the surface that was distributed in a way that looked like colonies of bacteria. All the sediment including soil, sticks and grass had settled to the bottom except for a few pieces of sediment and one blade of grass that were suspended from the surface of the water. The suspended sediment had fuzzy looking organisms growing on it. There were red-brown organisms growing on the bottom centimeter of the jar walls. The water had a murky green tint. On the sides of the jar above one centimeter there were green organisms growing, probably algae.

Description of Slide 1— Slide 1 was taken from the water just above the sediment at the bottom of the jar. 3 organisms were identified. A Paramecium multimicronucleatum, 300 micrometers in length was observed and more than four Paramecium multimicronucleatum were observed in the samples. These organisms moved fast, were a green color, oval in shape and had cilia around the edge used to propel them. A Gloeocapsa, 300 micrometers in length was also identified. A larger organism, Oscillatoria, surrounded this organism. The Gleocapsa and Oscillatoria were both non-motile. All three organisms are protists. Description of Slide 2— Slide 2 was taken from the surface of the water in the hay infusion. A Paramecium bursaria, 50 micrometers in length was identified. This organism was also very motile. Description of Slide 3— Slide 3 was taken from the water surrounding the suspended sediment. A Peranema, 50 micrometers in length was identified and a Euplotes, 100 micrometers in length was identified. Both are protists.

Paramecium multimicronucleatum- This organism meets all the needs of life, including acquiring energy, containing a cell/cells, being able to store information, being able to replicate and being able to evolve. The Paramecium acquires energy by ingesting other organism like algae and bacteria. It has a cytostome which allows food to enter the cell. A Paramecium is a single celled organism, therefore it does have a cell, including a cell wall and protoplasm inside. Inside the paramecium cell, DNA is contained which both stores genetic information and allows for this information to be passed on to offspring. Paramecium reproduce through binary fission, meaning everything inside the cell duplicates, including the DNA, and then the cell splits down the middle to form two separate Paramecium. Because Paramecium reproduce and their DNA can mutate, this means that natural selection can act on the genetic variability within a population and cause evolution over generations (http://www.fcps.edu/islandcreekes/ecology/paramecium.htm).

Conclusions: Based on the observations made, there are many different forms of protists present in the Hay Infusion. In future studies it would be prudent to make a larger effort to identify algae. Furthermore, if the hay infusion were to grow for another two months, it is predicted that more plants would start to grow. Also, since, it is a limited space with limited resources, some species my out compete others in the hay infusion. This could cause a loss of diversity.

-KW

Observing Biodiversity in an Ecosystem: AU Transect 1

January 14, 2016

Purpose: The purpose of this experiment was to observe the biodiversity present in a 20 foot by 20 foot transect on American University’s grounds. In addition to observing the transect, a hay infusion was taken in order to see the different protists and invertebrates in the transect. It is hypothesized that there will be protists and invertebrates present in the hay infusion.

Materials and Methods: First, transect 1 was observed on the macro scale and hand written notes were taken to record the data. The number and type of both biotic and abiotic items were recorded. 19 pictures were also taken of the landscape to record the data. In addition, a sketch of an aerial-view diagram of the transect was drawn.

After observations were made, a sample of the topmost layer of soil, leaves, rocks and other material was put into a plastic bag and brought back to the lab. There, 12 grams of the soil sample was put into a plastic jar with 500 mLs of deerpark water, and 0.1 grams of dried milk. The deerpark water was measured using a graduated cylinder and the dried milk powder and soil sample were weighed separately, using a scale. The mixture was shaken up and left with the lid open to sit for 1 week.

Data and Observations: Description of Transect 1-- Transect 1 is located between Hurst building and Ward Circle building. The north side of the transect is bordered by a concrete wall, the east side is bordered by a concrete sidewalk, the south side is bordered by pavement and the east side is an extension of the plant bed that is the transect. The topography of the transect is relatively flat. There are two large trees, about four stories high, that have nearly bare branches with some dead leaves that appear to be oak leaves. on the trunks of these trees there are large nodules that could possible be growths of some kind. There are also many smaller trees in the transect. They are characterized by thin bare branches that all grow out of the ground in one cluster rather than sprouting from a shared trunk. Additionally in the southeast corner, and around the middle of the transect there are patches of green grass that is about 6 inches long. The ground is littered with half eaten acorns, maybe by squirrels, dead leaves, a pine branch, several rocks and branches. Underneath this is soil. There are also small, green, leafy plants that grow close to the ground around the east side of the transect as well as these dead looking root tufts. The tufts are short and round with branches sticking out. There are also several sprinkler heads spaced throughout the transect. On the east side, concrete sidewalk makes up part of the transect.

Biotic Organisms Observed: -Clover like plants -Acorns -Roots -Large trees -Small bush trees (about 2 meters high) -Grass clumps -Eaten seed chunk -Pine tree branch -Growth on tree possibly -Sticks -Dead leaves -Bark

Abiotic Items Observed: -Soil -Rocks -Sprinkler heads -Lamp post -Sidewalk

Possible Competitive Relationships in Transect 1: -Squirrel eating the acorns (squirrel competes with oak tree)

    -- could also be symbiotic 

-Plants competing for space and nutrients in soil

Possible Symbiotic Relationships: -Squirrel living in oak tree and spreading the acorns -Fungus/bacteria decomposing detritus making soil richer

Transect 1 Aerial Diagram

Link to photos

https://drive.google.com/a/student.american.edu/folderview?id=0B4ZH8rpczezCMjdiaXFkbkVJdW8&usp=sharing_eid&ts=569d320a

Conclusions Based on the observations made, there is a large amount of biodiversity in the plot even without the results of the hay infusion. It would be interesting to do further investigation into what types of organisms exactly are in the transect rather the just describing them. Also, it would be interesting to see if there is a growth on the large oak tree and if so what it is. Our hypothesis cannot yet be addressed because the hay infusion won't be examined until the next lab.

-KW

1/17/16 First Post

Hi, just doing my first post.

-KW