User:Abigail M. Fogg/Notebook/Biology 210 at AU
2/6/14, lab 1 notes
Great job! Some notes:
-Make sure you sign each entry with your initials
-Make sure you include an intro and conclusion section (refer to email with outline attachment)
-Start working on building a map of your transect to detail your land and where your samples are taken from. We will talk about this more Wednesday
Plantae and Fungi: Returning to Transect
Hay Infusion Observation II
One week prior to this lab, plates a serial dilution of a sample from the hay infusion culture was prepared so that it could be observed. In lab today the various plates were observed in order to determine the characteristics of bacteria. The plates that contained an antibiotic (tetracycline) were also compared to plates that had no antibiotics in order to observe antibiotic resistance. A PCR preparation was also started in order to illustrate the significance of DNA sequencing in the identification of species.
1. Take qualitative observations of Hay Infusion Culture
2. Qualitative observations of the Agar plates.
1. Label the slides. Use a sterilized loop to obtain a small portion of bacteria. Transfer it to its respective slide.
2. Heat fix the sample of the bacteria to the slide by passing it through a flame three times with the bacterial smear side up
3. Working with a staining tray, cover the smear with crystal violet for 1 minute.
4. Rinse the stain off using a wash bottle filled with water.
5. Cover the smear with Gram's iodine mordant for 1 minute. Rinse gently.
6. Decolorize by flooding the smear with 95% alcohol for 10-20 seconds. Rinse gently. Decolorization has occurred when the solvent flows colorlessly from the slide
7. Cover the smear with safranin stain for 20-30 seconds. Rinse gently.
8. Blot excess water carefully with a paper towel and air dry.
9. After the gram stain is done, focus in on the sample at a low magnification, then observe the slide under 40X and the oil objective. (coverslips are not used with gram stains).
Hay Infusion Culture
-Water Level Decreased -Decreased amount of material in jar -Increased transparency of water -No film coating the top of the water -Less potent Smell
I would not anticipate to find any Archea species growing on the agar plates because these specimen have a preference for extreme environments (ex. extreme halophiles, thermophiles, methanogens etc.). When water has a foul odor this is indicative of the presence of bacteria. Because the smell of the water was particularly potent the first week, and far more fain the second this likely indicates a larger population of bacteria the first week in comparison to the second. A smaller bacteria population would also account for why there was less organic matter seen in the jar the second week. Perhaps the material was consumed and many of the bacteria died due to lack of resources (food). When observing the agar plates it was very noticeable that the presence of tetracycline altered the growth patterns of the bacteria. Plates that only contained nutrient agar had large colonies in comparison to those growing in the presence of tetracycline. Also there seemed to be more bacteria of different color and texture on the strictly agar plates in comparison to the tetracycline containing plates. This indicates that the antibiotic does work to kill bacteria. There were two fungi groups found on the plates. Antibiotics can be used to kill bacteria that will grow fungus but not kill fungus specifically. Antifungals can be used for this purpose. (U.S National Library of Medicine) Tetracycline inhibits protein synthesis of bacteria which causes the death of the organism. A bacteria that is resistant to this antibiotic will develop genes that code for an energy-dependent efflux of tetracyclines, or alternatively be able to create a protein that protects the bacterial ribosomes from the antibiotic. (Chopra 2001)
"Fungal Infections: MedlinePlus." U.S National Library of Medicine. U.S. National Library of Medicine, n.d. Web. 16 Feb. 2014. <http://www.nlm.nih.gov/medlineplus/fungalinfections.html>.
Chopra, Ian. "Tetracycline Antibiotics: Mode of Action, Applications, Molecular Biology, and Epidemiology of Bacterial Resistance." NCBI. N.p., n.d. Web. 16 Feb. 2014. <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC99026/>.
Hay Infusion Observation
In lab today the hay infusion that was created last week was observed. Organisms from the Hay Infusion were observed in order to grasp an understanding of the characteristics of Algae and Protists. A dichotomous key was used to identify/characterize/classify these organisms.
1. Qualitative observations were taken (appearance,smell of jar etc.)
2. A micropipette was used to extract a sample of the hay infusion from two different environments (the top and the bottom)
3. The liquid sample was placed on a slide and covered with a cover slip
4. The sample was observed. Observations were recorded.
Procedure II: Serial Dilution (For net weeks lab)
1. Obtain four 10mL test tubes filled with sterile broth
2. Obtain four nutrient agar, and four nutrient agar plus tetracycline plates (make sure they are labelled accordingly)
3. Swirl the Hay Infusion Mixture. Using a micropipette draw 100 microliters of the Hay infusion culture so that it can be transferred to the first 10mL test tube. Swirl the test tube well.
4. Draw 100 microliters from the test tube and place it in the next test tube. Mix well. Continue to do this for the remaining test tubes.
5. Transfer 100 microliters of the solution from test tube one to agar plate. Make sure the mixture is spread well. Do the same for the Agar + Tetracycline plate.
6.Repeat this process for the remaining test tubes.
7. Leave the plates to incubate for one week
The contents of the jar had a very potent odor. Inside the jar the water was fairly clear with an exception of a brown and green film that coated the top of the water (likely some mold). In the water there were two plants, one that had a thin leafy stalk and one that had a thick white stalk. Organisms near these plants would differ from other organisms because they would likely use the plant as a source of food, oxygen or shelter. There was also a thin layer of sand in the bottom of the jar. A sample was selected from two regions of the jar; the brown film on the top layer of the jar and the bottom of the jar near the leaved plant. Sketches of the organisms observed are located in the third image.
Slide 1-Top Environment
Specimen 1 - A Large brown individual was observed. Did not appear to be mobile. Occupied approximately 10 spaces at 10x (100 um). This cell did not fit the description of any other cell discussed in class.
Specimen 2- An individual observed in very close proximity to the brown individual. Small, mobile, black, vibrating and moving in a circular pattern. Occupied approximately 2 spaces at magnification 40X (5um). This could have possibly been a chlamydomonos cell
Specimen 3- Mobile individual, clear in color. Appeared to have some sort of inner membrane. Occupied 12 ocular spaces at 40x ( 30um)
Slide 2 - Bottom Environment
Specimen 1 - Spherical individual moving quite quickly with flagella. Occupying approximately 62um
Specimen 2 - Very long stick like figure. Occupying approximately 2mm in length
Specimen 3 - Small vibrating spherical organism.
If this hay infusion was observed for another two months I believe that many more organisms would be seen in this environment. I also believe though that with the very limited resources (food, shelter, mates etc.) that natural selection would be very prevalent. The most fit organisms would be those that could find shelter, nutrition and if necessary a mate within their lifetime. It is these individuals that have the greatest probability of reproducing and passing on their genetic information to offspring.
A future experiment could make a hay infusion that incorporates more organic matter (plants, decaying leaves, etc.) with hopes that this would provide organisms with more means for growth. While observing this hay infusion it was often quite difficult to find organisms. The organisms that were observed were identified/characterized by the dichotomous key.
Wednesday, January, 15
1. A transect (20 x 20 space of land on the American University Campus) was assigned to each group.
2. Each group was taken to their transect where they observed the topography, the biotic and the abiotic components of the area.
3. A sample of the transect was then taken using a trowel. A sample that contained some biological life (weeds) was selected.
4. Between 10 and 12 grams of the sample was then placed in a large jar.
5. 500 mL of Deerpark water and 0.1 grams of dried milk were added to the jar
6. The jar was labeled for identification and then shaken for 10 seconds
7. The jar was then left for 1 week to culture
Transect location: AU Community Garden
Biotic Components: Plants, weeds, grass, bugs, grubs. Many larger animals will be in this area during the spring and summer months such as deer and squirrels
Abiotic Components: Wooden planks for boxed gardens, plastic labels, soul, rocks, wood chips. Area not sheltered by trees or buildings so it is exposed to sun, snow, wind and rain.
Topography: Fairly flat. The garden rests at the bottom of one hill and at the top of a large crevasse.