User:Ashlee Forbes/Notebook/0708 - Protists & Microbiology

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The purpose of the experiment described herein was to cultivate and observe prokaryotes present in Transect 5, a certified wildlife habitat in front of Hughs Hall on the campus of American University. More specifically, the organisms being observed were bacteria, as archaea (the other branch of the prokaryote group) were unlikely to be present on the AU campus, although it can be predicted that there will be a large amount of bacteria. The effects of tetracycline on these bacteria are also being tested. For the purposes of this experiment, the hypothesis will be: If bacteria are cultivated from a hay infusion of biotic material from Transect 5, half being treated with tetracycline, then the untreated bacteria will flourish more than the bacteria in the presence of tetracycline.

Materials and Methods The Hay infusion was agitated to mix up the organisms it contained. Then 100 μL were drawn from the middle of the jar, halfway to the bottom. These 100 μL were added to a plastic tube containing 10 ml of sterile broth. The tube was labeled 10<super>-2</super> (a 1:100 dilution). 100 μL from that tube were then added to another plastic tube containing 100 ml of sterile broth for a 1:10,000 dilution(10<super>-4</super>). This process was repeated to form a 1:1,000,000 dilution and a 1:100,000,000 dilution, labeled 10<super>-6</super> and 10<super>-8</super>, respectively. 100 μL of each of these dilutions were added to petri dishes containing nutrient agar. 100 μL of the 10<super>-2</super> solution were spread on the surface of nutrient agar in petri dishes labeled 10<super>-3</super>, one gel containing tetracycline, the other left plain. This was repeated for each of the other tubes, with 10<super>-4</super> going into dishes labeled 10<super>-5</super> and so on. A glass spreader was used for procedure, and sterilized in a flame between applications. The agar dishes were covered and left to incubate in sunlight for five days.

After five days, bacterial growth on the agar plates was observed and recorded using dissection and compound microscopes. To aid in viewing the bacteria, a Gram stain procedure was performed. A smear from four of the plates (two plain, two with tetracycline) was put onto a glass slide plate and mixed with a drop of water. The smear was heat fixed by passing the slide through a flame with the bacterial smear facing upward. Smears were then covered with crystal violet for one minute, then rinsed with water. The smears were then treated with Gram's iodine for one minute, then rinsed with water. The smears were then decolorized with a 95% alcohol solution for 10 seconds, then stained with safranin stain for 30 seconds. The slides were rinsed with water and blotted for excess water before being left to air dry for 15 minutes. Slides were observed using compound microscopes set for 40x and 100x.

Data and Observations No archaea were observed in the bacterial growth plates. Upon opening the petri dishes, there was a noticeable fecal smell from the bacterial growth. The bacterial growth was about twice as effective on the plain nutrient agar plates vs. the plates treated with tetracycline (Image 1, Table 1).

Ashlee Forbes - Lab3Image1.jpg Ashlee Forbes - Lab3Table1.jpg

On the highest dilutions, about 35 bacterial colonies were observed on tetracycline plates, while 61 were observed on the untreated plates. Each plate had a growth of yellow bacterial colonies, with the higher dilutions having more distinct colony formation. The untreated 10<super>-9</super> plate also had some white colonies in evidence (Image 1).

Four samples were selected for microscopic observation: the tetracycline treated 10<super>-3</super> and 10<super>-9</super> (T3 and T9) and the untreated 10<super>-5</super> and 0<super>-9</super> (U5 and U9). Tetracycline treated plates showed a growth of cocci bacteria, and were slightly larger, where the untreated plates showed a growth of bacilli, being slightly smaller. Full results are featured in Table 2 below.

Ashlee Forbes - Lab3Table2.jpg

Conclusions The data from the experiment supports the hypothesis. The bacteria on untreated plates did twice as well as the tetracycline treated plates. This is because tetracycline inhibits bacterial growth by inhibiting the synthesis of protein. Many forms of bacteria are susceptible to tetracycline because of this, but are only inhibited rather than killed. Because of its widespread use as an antibiotic, this has also resulted in several strains of resistant bacteria.

As expected, no archaea were observed. This is due to the mild environment, whereas archaea are usually found in climates that are extremely hot, saline and methane rich. The AU campus is none of those things.

In another run of this experiment, agar plates might be left longer to encourage more bacterial growth. The experimenters would also not rinse as heavily, as this washed away most of the bacteria on the plates. This would undoubtedly result in more bacteria as well as a stronger smell due to the proliferation of more bacteria.

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