User:Hannah C. Goldbach/Notebook/Biology 210 at AU

January 28th, 2016

Week Three: Microbiology

Table 1: Agar Plate Results

Table 2: Cell Observations

Figure 4: Colonies for Gram Stain

Figure 5: Gram Stain Results

Purpose:

The purpose of this week's lab was to observe what, if any, populations of bacteria are present in the representative sample from Transect 4. It's predicted that incubated agar plates, which were created a week prior using samples from the hay infusion culture, will have large quantities of bacteria present given the location of Transect 4 near the duck pond and given previous observations.

Materials and methods

A week prior to this lab period, two identical serial dilutions were created. The initial dilution for both sets was made with 100μL of water from the hay infusion culture, and 10mLs of broth. From there, 100μL were transferred from each dilution to the next. The product was a set of dilutions with concentrations 10^-2, 10^-4, 10^-6, and 10^-8. Two sets of nutrient agar plates, one with tetracycline and one without, were both inoculated with 100μL of of broth for final concentrations of 10^-3, 10^-5, 10^-7, and 10^-9. These inoculated plates were allowed to incubate at room temperature for one week before observations were made. Upon returning to the incubated plates, colonies were counted and observed under a dissection microscope. Four individual colonies were chosen to undergo a Gram stain procedure which would indicate the presence of peptidoglycan in the bacteria's cell walls. Using a sterile loop, a small amount of bacterial growth was transferred onto a slide and then heat-fixed. The smear was covered with crystal violet for one minutes, rinsed, covered with Gram's iodine mordant for one minutes, rinsed, covered with 95% alcohol for 10-20 seconds, rinsed, covered with safranin stain for 20-30 seconds, and rinsed a final time before being allowed to dry. The dry slide was then observed under a microscope at 40x - a pink color indicated a gram-negative bacteria while a blue-purple color indicated a gram-positive bacteria. A wet-mount of each colony was then made so that cell size, motility, and shape could be observed. Based on the observations of the wet-mounts and the results of the gram-stain, two colonies were chosen to go through PCR for 16S amplification. The PCR tubes were prepared with 20μL of primer/water and a very small amount of bacterial growth. They were then placed in the PCR machine so that next lab period the products may be run on an agarose gel.

Data and Observations:

The hay infusion culture appeared much as it did last week; the smell was the same and there was still a heavy film on top of the water. The water appeared clearer and the layer of sediment on the bottom was thicker. The hay infusion culture was discarded after this lab. The results of the serial dilutions may be found in Table 1. From the thousands of colonies that grew, four main types of colonies were identified (Figure 4). Colony 1 was a medium sized, smooth, round, orange colony, colony 2 was a very small, smooth, round, green colony, colony 3 was a medium sized, smooth, round, tan colony, and colony 4 was a large, filamentous, white colony. These four colonies were stained and observed. The results of both the gram stain and the wet-mount may be found in Table 2. Colony 4 contained very large, highly motile, oddly-shaped cells.

Conclusions and Future directions:

As can be seen in Table 1, there was far less growth in the nutrient agar plates with tetracycline. While bacterial growth was greatly inhibited, fungal growth was allowed to strive. Tetracycline is an antibiotic which functions by inhibiting protein synthesis within the bacterial cell. As a broad-spectrum antibiotic, Tetracycline works on both gram-positive and gram-negative bacteria. However, because of it's availability and low side-effect rate, the antibiotic became a regular additive to cattle feed. The continual use of tetracycline to promote growth in meet-animals has led to a large resistance to the drug- it is now starting to work on continually fewer strains.[1] As mentioned, four individual colonies were chosen for additional analysis. Based on the observations, it was determined that Colony 4 was not a bacterial colony, but a fungus. It was also very difficult to observe individual cells from Colony 1, so the only two remaining colonies were processed for PCR, which will be completed next lab period.

--Hannah C. Goldbach 23:11, 9 February 2016 (EST)

January 21st, 2016

Week Two: Algae and Protists

Figure 3: Week two: Hay Infusion

Figure 2: Hay Infusion - Paramecium

Purpose

The purpose of this lab was to observe and record what, if any, algae and protists were present in the hay infusion made last lab period. It's predicted, given the location and features of Transect 4 as well as the physical appearance of the hay infusion, that both algae and protists will be present in the samples taken from the hay infusion.

Materials and Methods

The hay infusion used for this lab was created using 12g of organic matter from Transect 4, 500mLs of Deerpark water, and 0.1g dried milk. The components were mixed and allowed to settle in an open jar for a week prior to this lab period. At the start of this lab period, the obvious physical characteristics were observed and recorded before wet mounts were made from three different niches in the hay infusion for microscopic observation. Observations made from these wet mounts were used in conjunction with a dichotomous key to identify what, if any, algae and protists were present in the hay infusion, and therefore also present in Transect 4.

Data and Observations

Upon returning to our hay infusion this week, we found that the sediment had settled to the bottom of the jar, the water had turned cloudy, and a thick layer of gray scum had formed on top of the water. Other than the layer of scum, which is believed to be bacteria, there was no other apparent life. We took three samples, one from the top layer of scum, one from the water in the middle of the jar, and one from the sediment on the bottom. The top layer of scum was all bacteria, there was no other evidence of protists or algae. The sample from the bottom layer of sediment produced little to no protists or algae as well. We were able to find paramecium in the water from the middle of the jar, as well as more bacteria.

The paramecium we were able to identify were clear in color and roughly oval shaped with visible organelles, cilia, and oral grooves. The bacteria from the top layer were so numerous that looking at the prepared slide was like watching static – a solid wall of tiny, tiny vibrating dots.

Conclusions and Future Directions

The paramecium we found in our hay infusion produce energy by consuming other organisms, most likely the bacteria we also found, they contain DNA and reproduce asexually, and although they are unicellular they are capable of evolution; by these criteria the paramecium can be characterized as living. Given the duck pond, it was predicted that there would be more algae and protists then there were. However, it was later brought up that the lack of protists or algae could be because the layer of bacteria on top of the water was so thick that it suffocated anything living in the water. The water our sample was taken from was stagnant, which could cause the high bacteria count.

If the hay infusion were allowed to sit for another two months, the bacteria would probably continue to multiply until carrying capacity was reached. After that point, the bacteria population would remain fairly constant until all available resources in the hay infusion jar were consumed. The bacteria population would then start to die off.

--Hannah C. Goldbach 17:12, 3 February 2016 (EST)

January 14th, 2016

Week One: Transect Description

Purpose

The purpose of this week's lab was to observe and record obvious physical characteristics of a given plot of land on American University's campus. These observations will then be used to make more detailed hypotheses and conclusions about the transect in relation to the rest of Campus life. American University is an urban campus, and it is expected that the effects of daily city activity will be evident in the recordings and samples taken from even a small plot of land like Transect 4 (Roper Hall Duck Pond).

Figure 1: Transect 4 - Aerial Map

Materials and Methods

The lab group visited the transect and recorded all evident abiotic and biotic components and other significant features were noted. In addition to these lists, photographs were taken and an aerial map was drawn to further document the transect's features. Samples of soil and plant matter were also collected for creating a hay infusion.

Data and Observations

Transect 4 is a plot of land located in the South-East corner of campus. Bordered by Roper Hall on two sides, the transect features a storm drain, a stone walkway, and a duck pond, which is covered with netting at this time of year. As a National Wildlife Federation Certified Wildlife Habitat, the transect also provides cover for wildlife in the forms of tall grasses, seven bushes, and two trees. The transect probably receives the most sunlight in the early to mid morning, after then the sun is blocked by Roper Hall. Since the transect is located next to a dorm building, students often cross through using the stone walkway - a few pieces of garbage were also scattered here and there.

• Abiotic Features: Frame over the pond, big rocks (5), stepping stones (51), medium pond rocks (39), little rocks in and around the pond (70-100), pond water - partially ice over, duck sculptures, fake rock, storm drain, rodent traps, signage, benches (3), sprinkler, gas/water pipes, wrappers (3)

• Biotic Features: People (4), squirrels (3), hibiscus plants (4), leafless bushes (2), Dogwood tree, Oak tree, acorns, dead plants, leaves, budding bulbs, dead rat

Conclusions and future directions

The physical characteristics were documented and observations were recorded. Signs of daily city activity were evident, as the transect received heavy foot traffic and a fair amount of pollution. Organic matter was collected to create a hay infusion, which will lend more insight in next week's lab period.

--Hannah C. Goldbach 17:12, 3 February 2016 (EST)

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