User:Lisa White/Notebook/Biology 210 at AU

0701, T5 collection and survey

Purpose: To make observe the characteristics of a particular transect deemed a "Wildlife Sanctuary" by the university and to and create a sample for future protist/bacteria studies.

Material and methods: The group observed both biotic and abiotic components of the 20x20 meter transect. Using a 50ml sterile conical tube, the group collect a sample of the area's foliage (including a bit of plant life and also local soil) that was representative of the ground surface of the transect. Back in the lab, 10-12 grams of the mixture was weighed and then placed in a plastic jar with 500 mls of deer park water. Then, .1g dried milk was mixed with the collected soil/foliage and gently mixed for 10 seconds. The top of the jar was the removed and labeled for future reference.

Data and observations: This particular transect was deemed a "Wildlife Sanctuary" of the university. It was located in front of Hughes Hall. Biotic components include Topography: small trees, small day lilies/ mondo grass "monkey grass", patches of moss ; Living organisms: mosquitoes, ants. Abiotic components': soil, few rocks, hot and humid environment, bench

Above is a picture of transect 5

0703, T5, identify protists and preparation for next week's lab

Purpose: To practice using a dichotomous key to identify unknown protists in the transect sample under the microscope, and to also examine algae from the transect under the microscope.

Materials and Methods:First students made a wet mount of the mixture from the transect and evaporated milk. Its size was recorded using the micrometer and corresponding conversion for the magnification. For each organism found, the student practiced using the dichotomous key to identify protists in the mixture. Students also noted the smell of the mixture and took samples from top, middle, and bottom (different niches) of the sample. Students drew drawings of organisms.

Data and observations: The mixture had a smell similar to that of feces. Within the mixture the heavier filaments like heavy bits of moss, foliage, and dirt sunk to the bottom while the top had minimal foliage filaments and a clear milky film. I was able to find Colpidium, chilomonas, and perarena. The Colpidium was fast and roundish shape and about 60 micrometers. The chilomonas was characterized by its double tail and was about 20 micrometers. The perarema was about 40 micrometers and colorless and vibrated.

Lab manual questions: Organisms may differ in kind being close to and from the plant matter. It is likely that those that associate with plants may consume plants or photosynthesize, while those farther away do not. Organisms found at the bottom in the part with the soil may be the ones accustomed to living in soil as their natural niche. If the Hay Infusion had been continued for another couple of months it is likely that more organisms would have been found. Hay Infusions increase the density of protists as the dried milk adds sugars for the protists to thrive and allowing the solution to sit for longer would allow for the density of protists to increase. A selective pressure in this particular hay infusion is a lack of food for protists that determines the carrying capacity for this particular niche. Furthermore, the amount of foliage or dirt present determines what specific protists were chosen and how well they can thrive in the environment chosen for them. The Chilomonas meets the requirements necessary for life. This protist conducts photosynthesis (uses and acquires energy), it is made up of a single cells, has distributed nuclei (processes information), and like all protists represent the ancient organisms from which other species have derived and they themselves represent products of evolution.

Pictured above is the mixture from which the wet mount was made.

Pictured above are drawings of the protists observed.

Materials and Method II Prokaryotic organisms and fungi will be observed next week. 4 tubes of 10ml sterile broth were labeled 10^(-2), 10^(-4), 10^(-6), 10^(-8), respectively and were filled with the 100mls of transect mixture. 4 nutrient agar and 4 agar plus tetracycline plates were then given given the corresponding 100mls of mixture. They will be incubated at room temperature for a week.

In this manner a serial dilution was conducted of the ecosystem mixture.

Labeling the petri dishes with the correct labels.

0708, T5, Identify bacteria with DNA sequence

Purpose: To observe and quantify and conduct gram stain tests on bacteria that grew in different serial dilution petri dishes, including those that grew in the presence of the antibiotic tetracycline. Also to prepare a PCR reaction in order to identify the types of bacteria by the 16S gene.

Lab book question: It is highly unlikely that Archaea species will grow on the agar plates because archaea typically live in extreme environments with very hot temperatures like volcanoes. The appearance or smell of a bacteria culture might change from week to week. This is because in a petri dish that has not yet harvested colonies of bacteria, it is likely that the smell will be lacking. When the bacteria begins to colonize and increase in growth it is likely that the smell will increase. Furthermore, if the type of bacteria changes from week to week in a sample then its particular scent will also change depending on what type of bacteria it is.

Material and Methods I:

1. Sterilize loop over flame and scrape small amount from the surface. Mix it on a drop of water on slide. Circle area. Label slides.

2. Heat side over flame three times bacteria smear side up.

3. With a staining tray, cover smear with crystal violet for 1 minute

4. Rinse stain off using wash bottle with water

5. Cover smear with Gram's iodine mordant for 1 minute. Rinse gently

6. Flood smear with 95% alcohol for 10-20 seconds. Rinse. Decolorization occurs.

7. Cover smear with safranin stain for 20-30 seconds. Rinse.

8. Blot water with paper towel and air dry.

9. Focus on gram stained sample at a low magnification, observe under 40X and oil objective.

Materials and Methods II: Choose 4 plates with distinctive characteristics. Note the smell and appearance of each and count the number of colonies on each plate.

Do you see any differences in the colony types between the plates with vs without antibiotic?: The antibiotic has decreased the colony size of the bacteria by half. For example in 10^(-9) tet+ 35 colonies were counted and in dilution 10^-9tet- 61 colonies were counted. This indicates that for this particular species of bacteria 1/2 were antibiotic resistant and the other half were susceptible to the antibiotic. Overall the general trend was that tet killed about half of the bacteria cultures. 3 species of bacteria were able to grow in the presence of tet. The growth of bacteria on 10^-9tet was actually enhanced by tet as there are more orange colonies than in the 10^-9tet+ petri dish. It is not known what the effect of tet is on fungi growth since this experiment did not look into the growth of fungi. Tetracycline interferes with the bacteria's ability to produce proteins that are essential to them. Without these proteins the bacteria grow in large numbers (Princeton).

Data and observations:

Number of colonies & the number of colonies per ml.

Above is a photo of the cultures of bacteria.

This table describes the bacteria on a colony and cellular level.

Above are rudimentary drawings of the bacteria types examined under the microscope

Material and Methods III

Works Cited: Tetracycline. (n.d.). Princeton University. Retrieved July 8, 2014, from https://www.princeton.edu/~achaney/tmve/wiki100k/docs/Tetracycline.html