User:Tunji Odunlami/Notebook/Biology 210 at AU

Lab 3. Microbiology and identifying Bacteria

Tunji Odunlami

Instructor: Alyssa Pederson

July 7, 2014

Introduction

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The purpose of this experiment was to understand the characteristics of bacteria and observe antibiotic resistance. Over the years, bacteria have grown resistant to antibiotics. This experiment analyzes this sensitivity, as well as the mechanism of action for the antibiotic tetracycline.

Materials and Method

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This lab required the observing of microorganisms. In a prior experiment, serial dilutions were made and used in conjunction with nutrient agar plates. Tetracycline was added to half of the agar plates, and they amount of bacteria growth was later recorded. The characteristics of the bacteria was also examined to measure the effect of tetracycline. Two organisms from the nutrient agar plate and tetracycline plate were then viewed using oil immersion.

The next procedure required gram staining. A sterile loop was used to scrape bacteria on the surface of the agar plates. The slides used were heat fixed and then while working with a staining tray, the slides were covered with crystal violet for one minute. This stain was rinsed and then covered in Grams Iodine for one minute. The slide was then decolorized using alcohol and smeared with safranin stain for 30 seconds. The slide was allowed to dry and then it was observed using 40x and the oil objective

PCR sequencing was the next step. A single colony of bacteria was transferred to 100 microliters of water. This was incubated at 100 degrees Celsius for 10 minutes. The centrifuge boiled samples for an additional five minutes. During this period 20 microliters of primer was added and the, 5 microliters of supernatant was transferred to the 16s PCR reaction.

Results

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The results of this lab showed an increase in bacteria growth for the nutrient agar plates in comparison to the tetracycline. The maximum amount of colonies counted on the tetracycline plate was 7. The maximum number on the agar plate was over 100. The results shown in table 1 display the amount of colonies per ml. Table two shows that most of the bacteria was gram negative and spherical in shape. The colonies appear to be punctiform and few were motile.

Tables and Graphs

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Table 1 ( Serial Dilutions Results)

https://drive.google.com/file/d/0B86Vlz1JSBUjcW9uSUVDb1BXY1E/edit?usp=sharing

Table 2 (Bacteria Count)

https://drive.google.com/file/d/0B86Vlz1JSBUjQ3J6Z05nY1lxQnc/edit?usp=sharing

Figure 1: (Nutrient agar 10^-3)

https://drive.google.com/file/d/0B86Vlz1JSBUjZjBPSjdPaUttOXJYdFNUbTc3Y2F1NHJGRUZv/edit?usp=sharing

Figure 2: (Nutrient agar 10^-9)

https://drive.google.com/file/d/0B86Vlz1JSBUjRlBidnh0M2M3cVBveHJVOVd3azlBby1PYmdJ/edit?usp=sharing

Figure 3: (Tetracycline 10^-3)

https://drive.google.com/file/d/0B86Vlz1JSBUjMERKN2plaEw3T205U3BNLXEtbjBZbjQ5LVlj/edit?usp=sharing

Discussion

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References

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Lab 2. Identifying Algae and Protists

Tunji Odunlami

Instructor: Alyssa Pederson

July 2, 2014

Introduction

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The purpose of this lab was to identify organisms using a dichotomous key, and understand the characteristics of algae and protists. A population is known as a group of organisms from the same species that occupy the same niche (lehman, 2009). The objective of this experiment expands on this idea, as it examines protists and algae from the same transect. Organisms such as plants are autotrophs, and synthesize organic compounds from light in a process called photosynthesis (Lehman, 2009). This mode of acquiring energy is different from that of heterotrophs that rely on engulfing their prey (Lehman, 2009). If energy can be obtained from light, then photosynthesizing organisms are more likely to use this method for obtaining organic materials.The experimental hypothesis for this lab states that photosynthetic organisms will only be found near the surface of the hay fusion culture.

Materials and Methods

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This experiment required the usage of a dichotomous key to identify unknown organisms. Four unknown organisms from two different niches were identified using the key. The microorganisms growing on both the surface of the liquid and at the bottom of the hay infusion culture represented the two different niches. The hay infusion culture was also observed for characteristics like color and visible plant matter. The next step involved preparing and plating serial dilutions. This was done by labeling four tubes with, 10^-2, 10^-4, 10^-6, and 10^-8 markers. 100 microliters from the culture was added to 10mls of broth in the tube. This step was repeated to make a 10^-8 dilutions. Further dilutions were done using four nutrient agar and four tetracycline plates. 100 microliters from each test tube was spread on the agar plates. This was repeated to make a 10^-9 dilution factor.

Results

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The hay infusion was observed and appeared to be opaque with a slight yellow and brown tint. There was plant matter and debris dispersed at the bottom of the culture. The culture was also odorless. The specimen found near the top of the culture was the Paramecium and Amoeba. The organisms at the bottom of the culture were believed to be Colpidium and Chlamydomnas.

Tables and Graphs

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Figure 1

https://drive.google.com/file/d/0B86Vlz1JSBUjZkNURUNKWVJSRy1sWHM1ZFZ3RmRrNEQ4T09r/edit?usp=sharing

(Serial dilution diagram)

Figure 2

https://drive.google.com/file/d/0B86Vlz1JSBUjNURfaHRFU1pSN0E/edit?usp=sharing

(Hay infusion culture)

Figure 3

https://drive.google.com/file/d/0B86Vlz1JSBUjV0o5MGprTkdwemxUM3Rqa3lDT2hYLUNMdlRV/edit?usp=sharing

(paramecium)

Figure 4

https://drive.google.com/file/d/0B86Vlz1JSBUjcG9WSHZPR0d0ZlRWbGFSZENlWlF0NDE1VVpF/edit?usp=sharing

(colpidium)

Figure 5

https://drive.google.com/a/student.american.edu/file/d/0B86Vlz1JSBUjNDQ1YmJXTzJWWjlzTmVOWi1pZ3J6RE8zZHUw/edit?usp=sharing

(Chlamydomonas)

Figure 6

https://drive.google.com/file/d/0B86Vlz1JSBUjc0NvWFhzcFgxTHVWMEJhVWY3Q2VPbmhiS01N/edit?usp=sharing

(amoeba)

Discussion

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The experimental hypothesis for this lab states that photosynthesizing organisms will only be found near the surface of the hay fusion culture. The results of this experiment led to this hypothesis being rejected. The results found that unicellular eukaryotes inhabited the surface. These organisms were the paramecium and amoeba. Unlike the algae, protists do not photosynthesize (Bentley, 2014). Amoeba are also protists, and they engulf bacteria (Bentley, 2014). The bottom of the culture also exhibited more free living protozoa such as Colpidium. Chlamydomonas was also observed at the bottom of the culture.

Organisms away from the plant matter versus being close may use the resources in its environment differently based on the organisms niche. The type of food, and how it consumes energy are all factors that shape an organisms niche (Lehman, 2009). So in theory, photosynthetic species may be found near the surface or closer to light than organisms that directly ingest prey. This idea was not substantiated in this experiment. Paramecium and Amoeba were observed at the top or near the surface of the culture. The amoeba appeared colorless and with out any distinct shape. They also were motile and approximately 25 micrometers in size. The paramecium had an oral shape and appeared ciliated. It was long, colorless, and about 50 micrometers in size. Both Paramecium and the Amoeba are motile and non-photosynthesizing. The unicellular and motile algae Chlamydomonas was observed and found at the bottom of the surface. It was 50 micrometers in size and looked to have some organelles. Chlamydomonas have chloroplasts, although this was not clearly observed, and have the ability to photosynthesize. The last organism found at the bottom of the surface was the colpidium, which had a smaller body in comparison to the other organisms, and was much faster. This protist does not have the ability to photosynthesize.

The Chlamydomonas meets all the needs of life described as, responding to stimuli, being made up of cells, evolution, reproduction, and taking and using energy. The Chlamydomonas is a unicellular, and uses a flagella to interact with its environment. It also has a chloroplast and is capable of photosynthesis. Along with being isogamous, Chlamydomonas is thought to be the origin of multicellular evolution in the volvacine line (Bentley, 2014).

Some sources of error included the dichotomous key. Using this to identify unknown organisms presented problems. It is likely that we incorrectly identified a certain organism because it was difficult to gage specific features in the organisms. Also, determining the size in micrometers of each organism was a source of error. Many of the organisms were motile and so it was problematic when trying to accurately measure using the ocular meter. This lab can be improved upon in the future by perhaps allowing for a longer incubation period. If the hay infusion culture was observed for another two months, it is likely that more organisms would appear, most notably bacteria. Once the plant matter and food from the culture is used up, the protists could die since they are heterotrophs. This would put protists at a disadvantage, and one would expect more photosynthetic organisms to dominate the remains of the infusion culture since they can obtain nutrients through photosynthesis.

References

1. Bentley, M. (2014). General biology 210 laboratory manual. Formally published manuscript, Department of Biology, American University, Washington, DC, United States.

2. Lehman, R. (2009). Biology. (2nd ed.). New York: Barrons Education.

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Lab 1. Biological Life at AU Campus

Tunji Odunlami

Instructor: Alyssa Pedersen

June 30, 2014

Introduction

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The purpose of this experiment was to make observations that would allow for a better understanding of natural selection and how it drives evolution. There are millions of different species that live on earth, some of which are unidentified, and these groups of species can coexist while inhabiting the same area at the same time (Bentley, 2014). The transect analyzed in this experiment was an open area with exposure to the sun. The topography of transect 5 led to the hypothesis stating that there will be more photosynthetic specimen in transect 5 than the other locations.

Materials and Methods

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This experiment required the observation of green algae in the Volvocine line. The samples of green algae examined were the Chlamydomonas, Volvox, and Gonium. Characteristics such as the number of cells, mechanisms of motility, and colony size were determined and later recorded. This was followed by observing a niche at American Universities campus. A 20 by 20 meter transect was marked and general characteristics such as topography, location, and abiotic components were identified and recorded. This experiment entailed the making of a Hay Fusion Culture. This was done by weighing 10 to 12 grams of soil from the transect and placing it in a jar with 500ml of water. About .1 grams of dried milk was mixed into the jar for 10 seconds. The top of the jar was then removed and placed on a flat surface.

Results

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Transect 5 appeared to have both abiotic and biotic components. Ants were seen covering several rocks along with a brown bird on a tree. The tree appeared to be approximately 20 to 25 feet in height. This tree stood directly in the middle of the transect and was visibly bigger that the other trees. The transect was filled with green patches of moss and occasional spots of long green grass. Soil was also seen on the ground in the midst of rocks and many different shaped stones. The largest stone was seen directly east of the transect and stood out in comparison to the other stones because of its apparent mass.

Tables and Graphs

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Table 1. https://drive.google.com/file/d/0B86Vlz1JSBUjcFVEdU5NLXlYWlk/edit?usp=sharing

(Abiotic and Biotic components Table)

Figure 1 https://drive.google.com/file/d/0B86Vlz1JSBUjbTVtMW50YkZkczg/edit?usp=sharing

(Aerial view of Transect 5)

Figure 2 https://drive.google.com/file/d/0B86Vlz1JSBUjS1hiUnRUUVVOQ3M/edit?usp=sharing

( Different rocks and stones in Transect 5, also displaying patches of moss and grass)

Figure 3 https://drive.google.com/file/d/0B86Vlz1JSBUjOHNhS05qMmVXRDA/edit?usp=sharing

(Tree centered directly in the middle of transect. A dry landscape relative to other areas of transect 5)

Discussion

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Populations of several different species are assembled in nature in what is known as a community (Lehman, 2009). This experiment exemplified this fact, as different species were observed cohabiting transect 5. Transect 5 appeared to be still developing as some areas were more cultivated than others. The ground and soil was warm to the touch, there were small plants, few trees, and different visible biotic organisms. Patches of moss were widespread along with intermittent weeds dispersed throughout the landscape. This experiment also observed samples of green algae from the volvacine line. The Chlamydomonas, Gonium, and Volvox samples displayed different characteristcs such as size and mechanisms of reproduction that may offer insight about the beginning of life and evolution.

In the future, the experimental design of this lab can be improved. One such improvement includes the dimensions of the transect. A bigger transect could be used in the future to observe a wider array of species in the environment. Also, only 10 to 12 grams of ground sample was used in this experiment. A larger sample could be used to analyze whether or not this affects the biodiversity of the ground sample. Lastly, more people could observe the transect instead of just two as with this experiment. There was only a limited amount of time to observe the transect and with more observers, we could better identify the abiotic and biotic aspects of the environment.

During this experiment we encountered some forms of error. One source of error was the sample itself. In theory, the 10 to 12 gram ground sample was representative of the ground and surface in our transect. This is highly unlikely due to the different landscape within transect 5. Some areas of the transect were muddy, while others appeared more dry. Some parts of the transect had lots of grass while others parts were barren. These factors definitely contributed to the type of ground vegetation sample we extracted. Another source of error involved the weighing of the .1gm dried milk. This amount was very small and difficult to transfer. Therefore, it is unlikely that exactly .1gms was successfully transferred into the Hay Infusion Culture.

The hypothesis of this experiment suggests that the most photosynthetic specimen would be found in transect 5. This conclusion was based on the landscape and design of the transect. Data has not been fully collected in this two part experiment, and we will look to future labs to conclude whether or not to accept this hypothesis.

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References

1. Bentley, M. (2014). General biology 210 laboratory manual. Formally published manuscript, Department of Biology, American University, Washington, DC, United States.

2. Lehman, R. (2009). Biology. (2nd ed.). New York: Barrons Education.