User:Student 67/Notebook/Biology 210 at AU

Thursday January 28th, 2016

Characterizing Colonies Using Gram Stain and Wet Mounts

Purpose:

The purpose of this experiment was to characterize the colonies and cells of bacterial species found in Transect 3 after having left the serially diluted agar plates to grow over the course of a week. We did so by observing the motility and behavior of the organisms using a wet mount that allowed to see the beings alive. We also employed the use of the Gram stain technique that allowed us to see whether the cell walls of the bacteria contained small or large amounts of peptidoglycan. It was hypothesized that the bacteria found on the regular agar plates/tet(-) would be far more abundant and diverse that the bacteria observed on the agar plates with tetracycline/tet(+), not to mention that the tet(-) would have some species that the tet(+). Tetracyclines inhibit bacterial protein synthesis by preventing the association of aminoacyl-tRNA with the bacterial ribosome and thus function as great antibiotics that have many applications. The fact that the plates treated with tetracycline had far fewer and diverse colonies indicates that those colonies we found were in fact antibiotic resistant. In general, tetracycline plates grew exponentially less than the regular ones. It would seem that there were approximately 2-3 species of bacteria that were resistant, while there were about 10 or so thriving on the tet(-).

Chopra,I. June 2001 "Tetracycline Antibiotics: Mode of Action, Applications, Molecular Biology, and Epidemiology of Bacterial Resistance". Journal of Microbiology and Molecular Biology Review. Volume 65(2).

The Hay Infusion Culture itself still smells rather horrible; however, it is not as acrid and revolting as it was the week prior. It also does not have as thick of a film on the surface and has gotten somewhat clear. This is possibly due to the presence of bacteria that have begun to break down and metabolize some of the other microorganisms in the culture.

Materials and Methods:

Characterizing Bacteria: glass slides and cover slips, bunsen burner, agar plates, microscope, Crystal Violet, Iodine, 95% Alcohol, Safranin Stain, distilled water, PCR tubes,

1) Retrieve the 8 agar plates from previous week and observe colony morphology and take samples from 2 of each (+) and (-) and place on glass slides.

2) Begin Gram stain process by evaporating excess water using a bunsen burner and continue with Gram stain protocol using Crystal Violet, Iodine, Alcohol, and then Safranin Stain, rinsing with deionized water between.

3) Let dry fully and observe bacteria under a microscope, recording whether or not the organisms are Gram positive or negative based off of the color of stained peptidoglycan.

4) Prepare a wet mounts of the same aforementioned samples from agar plates and observe behavior of bacterial species.

5) Take sample of bacteria, (-) and (+), and place in 2 PCR tubes for genetic analysis

1) coccus, Gram negative, wave-like motion; 2) bacillus, Gram positive, swimming sporadically; 3) bacillus, Gram negative, swimming sporadically; 4) coccus, Gram positive, wave-like motion (all at 40x magnification)

It was concluded that the same species of bacteria that were found on tet(+) were also found in larger numbers on the tet(-). This is not true for the inverse. Therefore, Transect 3 does in fact harbor antibiotic resistant bacteria.

AP

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Thursday, January 21st, 2016

Taking a Microscopic Look at the Hay Infusion Culture

Purpose:

The purpose of this experiment was to identify the various kinds of organisms that grew on the top, middle, and bottom layers of the Hay Infusion Culture. In doing so, it is pertinent to notice how there are many different organisms that live in their own specific niches throughout the culture. It is expected that on the top layer, algae and other autotrophic organisms will reside because that is the best area to capture sunlight and photosynthesis, which would also mean there would also be some heterotrophs because the autotrophs provide a reliable food source. Within the culture in the middle and bottom layers, it is expected that we would find autotrophic organisms.

Materials and Methods:

Part 1: Hay Infusion Culture, microscope, glass slides and cover slips, transfer pipettes, and a dichotomous key in order to identify organisms

Part 2: 8 agar plates, P100 pipette and tips, samples from hay infusion, nutrient broth, 4 test tubes, test tube rack, glass spreader, bunsen burner

1. Observe the Hay Infusion Culture and note physical characteristics

2. Identify the different layers of the Hay Infusion Culture

3. Observe a sample from each niche using transfer pipettes, a microscope, and slides

4. Identify and measure each organism found using a dichotomous key.

5. Collect and label four tubes of 10mLs sterile broth with 10^-2, 10^-4,10^-6,10^-8 and four nutrient agar and four nutrient agar plus tetracycline plates. Label the plates with tetracycline with "tet" - and name one of each with 10^-3, 10^-5, 1^-7, and 10^-9

6. Add 100 micro Liters from the culture to the 10mLs of broth in the tube labeled 10^-2, swirl and pipette 100 micro liters of that to each 10^-3 plate.

7.Take 100 micro liters from tube 10^-2, put in 10^4, and repeat until you complete each agar plate with the serial dilutions.

8. Set the agar plates aside onto a rack. Leave them there at room temperature for a week.

The Hay Infusion Culture is exceptionally revolting, exuding the acrid scent of mold tinged with excrement akin to a sewer, resulting in a pungent mustiness that mildly stings the nose and brings tears of disgust to the eyes. It is very dark and murky, especially near the surface and looks very much like sewage. On the surface of the culture there is quite a bit of mold, forming a dense and sticky film that does not break very easily.

The organisms, from for example the surface versus those submerged, may differ because said organisms require certain environments in their niches to thrive. Some of the organisms on the surface may require direct oxygen from the air to survive, while those under the water prefer the more stable living conditions that water provides. The organisms living near plant life most likely do so because the decomposing plant matter provides them with last sustenance.

Most of the organisms observed were extremely motile, zipping along as if they had not a care in the world. The organisms were both protozoan and algal. It is most likely that those near the surface and with green coloration were in fact photosynthetic.

Types of Organisms found:

Raw Data: Top Layer Gonium, Pandorina, Colpidium (50-70µm)

Middle Layer Volvox (350-500µm), Paramecium bursaria (70-110µm), Spirostomum (1-3mm), Euglena (35-55µm)

Bottom Layer Blepharisma (400-600µm), Didinium cyst, Chilomonas (20-40µm), Spirostomum (1-3mm), Pandorina (20-250µm)

From left to right: Euglena, Spirostomum, Didium Cyst, Volvox, Eudorina, Colpidium

The Blepharisma can be categorized as a living organism because it possesses the five fundamental characteristics of all living things. They acquire energy by consuming other organisms such as bacteria and other ciliates by engulfing the organism and digesting it. It is a membrane-bound unicellular organism. It exchanges information between itself and the environment as well as passes its genes on after reproduction which is carried out by binary fission. These organisms have evolved from an ancestral unicellular organism that we also share.

If the Hay Infusion Culture “grew” for another two months what changes would you predict to occur? What selective pressures would affect the community of your samples? If the culture were allowed to grow for another two months, it is likely that the protozoans would consume many of the algal organisms leaving mostly protozoans. There would also be quite a bit more mold in the culture. The culture as a whole would likely be over run with all kinds of organisms due to the plethora of nutrients available and reproduction. Over a longer period of time, however, many of the organisms would probably die due to intense saturation and an inevitable decrease in oxygen levels.

Serial Dilution:

Conclusions:

It was observed that there were many different kinds of organisms with great diversity between the Top, Middle, and Bottom layers. This is indicative of the fact that certain organisms tend to thrive in their own specific niches.

-AP

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Thursday, January 14th, 2016

TRANSECT 3

The location of this particular transect is called the Butler Pavilion and is in the middle of Battelle-Tompkins Memorial Building, Hughes Hall, McDowell Hall, Woods-Brown Amphitheater, Mary Graydon Center, and Bender Arena. The topography of this location includes a multitude of different plant life such as flowers, bushes, and trees planted in a clay-like soil covered in a dark black mulch. There is a winding path of concrete that snakes through this garden-like place.

-AP