User:Camilla Dagum/Notebook/Biology 210 at AU

July 9 2014

Introduction

The domain bacteria is one of the three domains of life. Bacteria are prokaryotes, single-celled organisms that lack complex membrane-bound organelles. Archaea is a second prokaryotic domain of life, and eukarya the third domain. Bacterial and archaeal lineages are the most ancient on Earth; the oldest fossils found, dating back 3.5 billion years ago, are carbon-rich deposits derived from bacteria. Eukaryotic life did not arise until 1.75 billion years ago. Bacteria are fantastically abundant and can proliferate in almost any environment. It is estimated that there are approximately [math]\displaystyle{ 5x10^{30} }[/math] individual bacteria and archaea alive today (Freeman et al, 2008).

In this lab we will be studying the prokaryotic life present in the Hay infusion made from a soil sample of Transect 3. Different dilutions of the Hay infusion were grown on nutrient agar and tetracycline plates then analysed. It is likely that the colonies present are bacteria rather than archaea, as archaea tend to prefer extreme environments. Tetracycline is a common antibiotic that can affect both gram-positive and gram-negative bacteria by inhibiting protein synthesis. When a bacterial cell attempts to translate an mRNA, the tetracycline prevents binding of the aminoacyl tRNA to the A-site of the large ribosomal subunit. Resistance to tetracycline, however, is an increasingly common occurrence in many bacterial strains (Chopra et al, 2001).

We predict that the bacteria that grow on the tetracycline plates are resistant, and there will be a greater variety and number of bacteria that grow on the agar-only plates. If this is true, we expect that samples of colonies taken from agar plates will contain different bacteria, and those colonies that grow in the presence of tetracycline will tend to be more similar as all the bacteria must be resistant.

Materials and Methods

The growth on the agar and agar plus tetracycline plates was observed with the naked eye. The number of colonies on each plate were counted, and the morphologies of the individual colonies were compared. Three colonies were chosen for further study: one from a tetracycline plate and two from different agar plates. Wet mounts of these colonies were prepared and observed under the microscope using the oil-immersion lens (1000x total magnification).

These same three colonies were then sampled to be gram-stained. To perform a gram stain, a sample of the colony is taken using a sterilized metal loop and smeared onto a slide. The location of the smear is noted by a wax pencil outline made on the underside of the slide. The slide is then passed through a flame three times to be heat fixed. Next, working above a staining tray, the smear is covered with crystal violet for 1 minute then rinsed off with water. The smear is then covered with Gram's iodine for one minute and again rinsed off, but with 95% alcohol. Once rinsed, the smear is covered with safranin stain for 20-30 seconds, and gently rinsed with water. The excess water is carefully blotted off with a paper towel. The sample is now gram-stained and ready to be observed under a microscope.

The three colonies were sampled for a third time to undergo a PCR reaction. A sample from each colony was transferred to a tube with [math]\displaystyle{ 100 \mu L }[/math] of water and incubated at [math]\displaystyle{ 100^oC }[/math] for ten minutes. The boiled samples are then centrifuged for five minutes at 13,400rpm. While the samples are centrifuging, [math]\displaystyle{ 20 \mu L }[/math] of primer mixture is added to each PCR tube with a PCR bead. [math]\displaystyle{ 5 \mu L }[/math] of the supernatant from the centrifuged samples is then transferred to the PCR tubes, and the tubes are placed in a PCR machine. The results of the PCR will be analysed in the following lab (Bentley et al, 2014).

Results

References

1. Freeman, S. et al. (2008). Biological Science (5th ed.). Boston, MA: Pearson.

2. 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. Retrieved July 9, 2014, from http://mmbr.asm.org/content/65/2/232.long

3. Bentley., Walters-Conte., & Zeller. (2014). Biology 210 Lab Manual. Washington: American University.

July 7 2014

Introduction

There are three domains into which all life is organized: archaea, bacteria, and eukarya. Archaea and bacteria are prokaryotes, lacking complex membrane-bound organelles. All prokaryotes are unicellular. Eukaryotes, however, can range in complexity from single-celled organisms to multi-celled plants, animals, and fungi. All multicellular organisms are eukaryotic (Freeman et al, 2008). In this lab we will be studying unicellular eukaryotes. Two large categories of such organisms exist; these categories are the algae, which are capable of photosynthesis, and the protists, which are heterotrophs (Bentley et al, 2014).

As described in the previous notebook entry, in the last lab we made a Hay infusion culture from a soil sample from Transect 3. The unicellular eukaryotes that will be identified are those that come from the soil sample Hay infusion. The Hay infusion has developed over the course of two days. We predict that within the Hay infusion there will be different ecosystems characterized by different organisms, based on the depth beneath the surface. If this is true, then different species will be identified at different layers of the infusion. We further predict that algae will likely be found exclusively in top areas of the Hay infusion.

Materials and Methods

The Hay infusion was carefully transported to the lab station, upon which time it was visually inspected. It is important to disturb the infusion as little as possible, as the infusion ecosystem changes with depth. Shaking the solution will disturb the protists and algae.

Two samples were taken from the infusion, one from the very top of the solution and one from the region near the bottom of the container. Two wet mounts were made from the samples, and each was studied under a compound microscope. Identification of two distinct unicellular eukaryotes from each region sampled was done using a dichotomous key, for a total of four organisms identified (Bentley et al, 2014).

After removing samples from the Hay infusion, a serial dilution of the solution was performed. The Hay infusion was mixed and a 100 [math]\displaystyle{ \mu L }[/math] sample taken. The 100 [math]\displaystyle{ \mu L }[/math] of Hay infusion was then added to 10mL of nutrient broth. This mixture, a 1:100, or [math]\displaystyle{ 10^{-2} }[/math] dilution was swirled to combine. 100 [math]\displaystyle{ \mu L }[/math] of the [math]\displaystyle{ 10^{-2} }[/math] dilution was then taken and added to 10mL of nutrient broth. This second mixture is now a 1:10000, or [math]\displaystyle{ 10^{-4} }[/math] dilution. The process is repeated two more times to make [math]\displaystyle{ 10^{-6} }[/math] and [math]\displaystyle{ 10^{-8} }[/math] dilutions (Bentley et al, 2014). A diagram of the serial dilution procedure is shown below.

After making the different dilutions, they were plated onto agar and tetracycline plates. To plate the dilutions, 100[math]\displaystyle{ \mu L }[/math] of solution was transferred to the agar plate and spread. This process was repeated for the four agar plates (one plate per dilution). For the tetracycline plates, the method was identical: 100[math]\displaystyle{ \mu L }[/math] of solution on each plate. The plates were capped and stored in the lab to incubate at room temperature for five days (Bentley et al, 2014).

Results

The Hay infusion was cloudy, with a yellow-green tinge. No green shoots or apparent life were seen at the surface of the infusion. The fibrous plant matter from the soil sample (wood chips and small sticks) had settled to the bottom of the infusion.

In the first sample, from the surface of the Hay infusion, an algae and a protist were identified. The algae was a deep blue-tinted green, and appeared to be composed of multiple cells. It was approximately 50[math]\displaystyle{ \mu m }[/math] across. The protist identified was a pair of Blepharisma, each approximately 25 [math]\displaystyle{ \mu m }[/math] across. The Blepharisma was a brownish pink and had cilia. It is shown in Fig. 1.

In the second sample, from the bottom of the Hay infusion, two distinct protists were identified. One organism identified was a Colpidium. The Colpidium was colourless, covered in cilia, and was quite small. It was approximately 12.5 [math]\displaystyle{ \mu m }[/math] across, and oval-shaped. Fig. 2 is a picture taken of the Colpidium through the microscope.

The second organism identified was a Chilomonas. It was approximately 37 [math]\displaystyle{ \mu m }[/math] across. It was an elongated cell with two locomotor flagella and was tinted green. We were unable to take a picture of the Chilomonas in the microscope; however, Fig. 3 is an image of Chilomonas taken from the Encyclopedia of Life (Feng et al).

All identifications were made using Ward's Natural Science dichotomous key for free-living protozoa (Ward's Natural Science, 2002).

Discussion

As predicted, different species of unicellular eukaryotes were found in the different layers of the Hay infusion observed. Algae was only found at the surface of the infusion. As algae are photosynthetic organisms, it stands to reason that they would be more successful in the sunnier environment. The protists observed at the bottom of the Hay infusion were different from those observed at the infusion's surface. This supports the initial prediction. The two regions of the Hay infusion had different qualities and components, and this was reflected in the protists that colonized the areas.

One of the organisms identified was a Blepharisma, shown in Fig. 1. This species, and the other organisms found, are qualified as life by meeting five fundamental characteristics. Blepharisma is a filter feeder, so it is able to acquire energy. It is made of cells, in this case, a single cell. It has a nucleus which stores genetic information, and although we did not see any Blepharisma actively dividing, they do reproduce. Finally, to be qualified as life an organism must be a product of evolution. We see similarities between Blepharisma and the other eukaryotes found in the infusion and documented by biologists; we know that Blepharisma is related to different species, thus it must be a product of evolution (Freeman et al, 2008).

It was difficult to locate the organisms under the microscope, which certainly influenced the results. Although the species found in the different layers observed were indeed different, so few organisms were found in the samples that it is possible some organisms were present in both layers. If the Hay infusion had been left to develop for a longer period of time, it is likely that we would have had more conclusive results.

If the Hay infusion had been observed for another two months, we expect to observe several changes. Initially, while the food source is still abundant, we expect a boom in protist population of many species. As time goes on, however, only some of those species will continue to proliferate, based on how well-adapted they are to the infusion environment. The protists that are more motile could be expected to thrive over those that are less motile, as they will be able to swim to different food sources as food grows scarce.

References

1. Freeman, S. et al. (2008). Biological Science (5th ed.). Boston, MA: Pearson.

2. Bentley., Walters-Conte., & Zeller. (2014). Biology 210 Lab Manual. Washington: American University.

3. (2002). Free-Living Protozoa. Rochester, NY: Ward's Natural Science. Retrieved July 7, 2014 from http://www.ocmboces.org/tfiles/folder1837/Free_Living_Protozoa%5B1%5D.pdf

4. Feng, A., Wie-song., & Patterson, D. Portrait of Chilomonas . Encyclopedia of Life. Retrieved July 7, 2014 from http://eol.org/data_objects/27474071

July 2 2014

Introduction

The ecosystems of an area as small as the American University campus are many and varied. One ecosystem present is an approximately 8mX6m transect of tall bushes. This region is characterized by several different types of bushes, both flowering and non-flowering, as well as some tall grasses and two different species of trees.

Materials and Methods

A macroscopical biological assay was performed simply by inspection of the transect. We documented the different varieties of plants present, noted their location as well as their individual characteristics.

A Hay infusion culture was also made using a sample of the soil, collected at the base of the Japanese maple tree. Approximately 10g of the soil sample was combined with 500mL filtered water and 0.1g of dried milk. The mixture was gently mixed until combined then left to develop in an uncapped jar for two days. The infusion was left undisturbed in the lab near a window (Bentley et al, 2014).

Results

An aerial-view diagram of the transect is shown in Fig. 1. Some plant varieties appeared in multiple locations of the transect, others only once. The two trees present were different species. The different colours are associated with a different plant, labeled accordingly. Fig. 2 shows actual images of the different plants found in the transect. There were abiotic components in the transect as well. A concrete sidewalk and four metal benches were present.

The transect was mostly flat, with some mounded soil where the plants were located. The soil was dry and composed primarily of wood chips. When collecting a sample for the Hay infusion, different insect species were found.

Discussion

The purpose of this lab was to characterize the macroscopic plant life in a transect of tall bushes. We predicted that there would be a number of different species present in even the small area (8mx6m) of the transect. As shown in Fig. 1, nine different species of plant life were found. Most of the plants were bushes of different heights and leaf shape; some were flowering and some were not. The different plant species are documented in images in Fig. 2. Interestingly, the conical flowers from one of the bushes are visually very similar to the flowers from the flowering tree. The morphology of the various bushes was similar as well.

Although the different species will all be competing for resources, it is actually beneficial to the ecosystem to have greater diversity. The results of a ten-year study performed by Dr.'s Tilman, Reich, and Knops showed that ecosystems with a greater number of plant species were more stable (Tilman et al, 2006). Transect 3 appeared to be a healthy ecosystem; none of the plants had dead branches or areas, and all were growing and flowering abundantly. The health of the ecosystem is certainly influenced by the variety of macroscopic plant life, and is likely influenced by the variety of microscopic life. In the following lab we will examine the unicellular life present in the soil of Transect 3.

References

1. Bentley., Walters-Conte., & Zeller. (2014). Biology 210 Lab Manual. Washington: American University.

2. Tilman, D., Reich, P., & Knops, J. (2006). Biosystem and Ecosystem Stability in a Decade Long Grassland Experiment. Nature, 441, 629-632. Retrieved July 6, 2014 from http://www.nature.com/nature/journal/v441/n7093/abs/nature04742.html