User:Michael Podielsky/Notebook/Biology 210 at AU

2/4/16 Exercise III- Microbiology and Identifying Bacteria

Purpose: Bacteria will be identified based on motility, gram stain, colony morphology and sequencing of the 16s subuniti gene. This is a continuation of last weks experiment with the hay infusion and will use the bacteria that has been groing on the agar and tetracycline petri dishes. This will help further identify and observe these bacteria.

Observation of Hay infusion culture before: The smell has remained just as terrible as it was the previous week. The plates without the tetrcycline smell considerable worse and this is likely due to less bacteria being present on the slides. Some of the non-tetracycline plates have no visible bacterial growth and this could be caused from the bacteria being too diluted in the serial dilution or from non of it being transfered. If left for another week we may see some bacteria form in the slides.

Materials and Metods: Each plate had the number and type of bacterial colonies counted to compare the overall growth in each. This was then converted to colonies/mL by the conversions based on the dilution factor for each plate. Next, an innoculation loop was used to scrape bacteria from two colonies not in the tertacycline plates and two from the tertacycline plates. These colonies where then deposited on slides with water on them. The slides where then passed back and forth over a flame to dry the water and leave the bacteria. the dried slides then had crystal violet saturate them for up to 1 minute before rinsing it off. Grams iodine was then applied to each of the slides for 1 minute and then rinsed off using water. The slides then where decolorized for 10 to 20 seconds by submerging the slide in 95% alcohol. When solvent stopped flowing from the slide a kimwipe was used to blot up any excess. These gram staine slides where then observed under a microscope at 40x magnification and 100x magnification to determine if they where gram positive or negative. A PCR amplification was hen set up for the most characterized slide. 2 PCR tubes where labeled with the transect number 2 and then 20 microliters of a primer water mixture where added. The PCR bead was dissolved and a toothpick was used to extract a sample of the desired colony and place it in the tube. The toothpick was submerged in the tube for 5 seconds.

Results:

Table:1 Dilution Agar Type # of colonies Conversion factor colonies/mL gram+/- 10^-3 nutrient 756 x10^3 756,000 10^-5 nutrient 64 x10^5 6,400,000 10^-7 nutrient 2 x10^7 20,000,000 10^-9 nutrient 0 x10^9 0 10^-3 +tet 70 x10^3 70,000 10^-5 +tet 0 x10^5 0 10^-7 +tet 0 x10^7 0 10^-9 +tet 0 x10^9 0

Note: It seems the tetracycline was effective at preventing the growth of bacteria with it only visibly growing on the highest concentration dilution and even then it was still considerable smaller at 70,000 compared to 756,000.

Cell description: 10^-5 nutrient- rhizoid, lobate, smooth glistening, convex. 10^-3 nutrient- filamentous, filamentous, pulvinate, rough, white.

2/4/16 Exercise II- Identifying Algae and Protis

Purpose: To identify protis found within the hay infusion in the previous experiment. This will be completed by using a dichotomous key which lists key characteristics of each organism.

Materials and Methods: separate samples where taken from the top and very bottom of the 500mL hay infusion. Micro pippettes where used to place the liquid on microscopic slides. Each was viewed under 40x magnification and photographic evidence was taken of each organism found on the slide. The dichotomous key was then used to provide identification for each of the protis found. Serial dilutions where then preparred with four tubes labeled 10^-2, 10^-4, 10^-6, and 10^-8. Each tube was filled with 10mLs of sterile broth and the first tube recieved 100 microliters of the hay infusion. Each test tube after that recieved 100 microliters from the previous test tube after it had been mixed with the one preceeding it. Four nutrient agar plates where then obtained and labeled 10^-3, 10^-5, 10^-7, 10^-9. The corresponding tube was then transeffered to the proper agar plate. This process was repeated for plates coated in tetracycline, an antibiotic. The plate where labeled with the dilution and the groups intials and then allowed to incubate for 1 week.

Observations: The hay infusion smells like raw sewage and is cloudy with a thick layer of bacteria and fungi growing on the surface of the water. When observing the protis that is further away from the plant matter as compared to the protis that is closer it may explain that the ones further away are smaller and photosynthesizers. The ones closer can use this plant matter as food.

Protis and algae present: Colpidium (found in both layers) paramecium (top layer)

Both of these organisms are motile protozoa and range in size from 100 micro meters to 120 micrometers.The colpidium meets 2 of the requirments for life by consuming enegy in the form of smaller bacteria and by expending energy by moving about its environment. If the hay infusion where allowed to grow for another 2 months we would see an increase in the number of bacteria present in the infusion, and maybe different forms of bacteria.

Photo:

1/28/16 Examining Biological Life at AU [Transect]

The Purpose: The purpose of this experiment is to observe the diversity of life in a 20x20 area at American University .It is important to study this so a better understanding of life and its diversity can be observed. It is likely that a large diversity will still be observed, despite it being a relatively small space.

Materials and Methods: - Notebook -Camera phone - 20x20 area - Test tube - Ziplock Bag

A small sample of water was taken from the streeam that ran through the transect and stored in the test tube. Leaves, sticks, dirt, and algae covered rocks where gathered into a ziplock bag. Next, a sketch of the area was produced so that the area could be appropriately mapped. Images of the area where taken on a camera phone. A list was composed of the visible abiotic and biotic things found inside the transect as well as the number of plants, trees, and general shrubbery. After gathering all of this information, a hay infusion was created from the contents gathered in our bag as well as 2ml from the test tube which contained the sample from the stream. The hay infusion was comprised of 500ml of water and all of the contents from the ziplock bag. The remaining sample from the stream was stored and observed independently under a microscope and compared to the results of the hay infusion a week later.

Data and Observations:

Abiotic: - rocks (estimated 400, ranging from 5cm to 50cm in size.) - dead sticks and brush (all over) - suspected dead treee - trash (1 styrofoam cup and a snack wrapper) - water - sporinkler boxes

Biotic: - 2 Squirrels (gray) - green colored algae on rocks - 3 trees - 10 bushes of an unknown type (estimated) - ferns - lichen (on many of the larger rocks) - small plants that resemeble flowers (tulips?)

Images: (Unable to attach)

Conclusions: This experiment would need to be repeated a few more times so more time can be given to colecting data. It will be hard to do this now since the transect is under a few inches of snow. It would be better if we had been able to observe the transect multiple days in a row and recorded it then.

1/28/16 Hay Infusion

The Purpose: To observe the ecosystem of the transect on a smaller scale and record the microgranisms in the infusion. It is predicted that the hay infusion will yield many of the same organisms found in the transect and should be similar to whatever is observed in the 2ml of stream water that was kept separate from the hay infusion, but more diversity will be present in the container.

Materials and Methods: Without distrurbing the contents of the hay infusions two separate samples where taken from the top and bottom of the infusion and observed under a microscope. These two slides where then compared to the stream water sample. A dichotomous key was used to identify each microorganism found in each of the slides. The slides have been observed at 4x, 10x, and 40x to ensure all organism could be observed.

Data and Observations:

Organisms found in the hay infusion: - colpidium (all slides) [100 micrometeres] - paramecium (only found in the hay infusions slides) [110 micrometers]

Images: (Could not attach them to this website)

Observations: - horrible smell and is similar to sewage. The appearance is cloudy and the large rock inside the container is barely visisble. The overall color of the infusion is green and seems to b teeming with bacteria and other forms of life. Shaking it up only made visibility worse and the smell somehow managed to become even more sewage like. - thin layer of mold or fungus on the tope of the infusion - If the infusion grew for another two months I would expect the same organisms to be present but in greater numbers so long as the enviornment is maintained and the essentials for life are still available. - could not attach the images of the organisms

Conclusions: The stream water had few organisms in it, but this could be because many of them could have died off during the week in between collection and observation. In the future it would be better to observe it immediatley. The hay infusion had a range of organisms and even a few vertabrates that could not be identified.