User:Hilary H. Strong

7/7/14

Lab 3: Microbiology and Identifying Bacteria with DNA Sequences

Introduction

Due to their small size, around 10 um, a microscope is necessary to study individual bacteria. In addition to comparing morphological differences notably, a stain can be applied to the sample to aid in identifying the bacterium. One defining characteristic of bacteria is the thickness of the layer of peptidoglycan,a component of the cell wall. The Gram stain is a good method for differentiating between thick peptidoglycan and thin ones, as thick layers hold onto the crystal violet dye used in the stain. Determining if the bacteria is gram-positive, has a thick layer of peptidoglycan, or gram-negative, has a thinner layer of peptidoglycan is useful picking the most effective antibiotic (Bentley et al. 2014).

Furthermore, a bacteria can be identified by performing a polymerase chain reactions (PCR) with specific primer sequences. Identifying species in this way is also useful for piecing together the phylogenetic relationships among species (Bentley et al. 2014).

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Methods/Materials

Procedure I: Quantifying and Observing Microorganisms

1. Examine the eight agar plates from the Hay Infusion Culture, looking for colonies

2. Count and record the number and type of colonies on each plate (Bentley et al. 2014)

Procedure II: Antibiotic Resistance

1. Compare the colonies on the agar nutrient plates to those on the agar + tetracycline plates (Bentley et al. 2014)

Procedure III: Bacteria Cell Morphology Observations

1. Add a sample from three chosen colonies to three sterile tubes filled with 100 uL of water

2. Incubate the samples for 10 minutes at 100 °C

3. Add the samples to the centrifuge for 5 minutes at 13,400 rpm

4.Prepare 3 PCR tubes by labeling them and adding 20 uL of primer and mix

5. Add 5 uL of the supernatant from the centrifuge sample to the PCR tubes

6. Move the tubes to the PCR machine (Bentley et al. 2014)

Procedure IV: Set up PCR for 16s Sequencing

1. Add a sample from three chosen colonies to three sterile tubes filled with 100 uL of water

2. Incubate the samples for 10 minutes at 100 °C

3. Add the samples to the centrifuge for 5 minutes at 13,400 rpm

4.Prepare 3 PCR tubes by labeling them and adding 20 uL of primer and mix

5. Add 5 uL of the supernatant from the centrifuge sample to the PCR tubes

6. Move the tubes to the PCR machine (Bentley et al. 2014)

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Data and Observations

Agar and Agar + Tetracycline Plates

Results of the colony count.

Drawings of Bacteria Cells

(Bonan 2014)

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Discussion/Conclusion

The appearance and smell of the Hay Infusion Culture might change from week to week because as the environment in the culture changes due to fluctuations in factors like temperature and light exposure, new organisms may grow while existing populations could die out or mutate in order to adapt.

There are significantly more colonies and varieties of bacteria as well on the agar nutrient plates than the agar plus tetracycline plates. This indicates that the bacteria is susceptible to tetracycline and thus the tetracycline inhibited bacteria from growing on the plates with it. Three species of bacteria grew on the tetracycline plates and thus were unaffected, or resistant to the antibiotic.

Tetracycline targets gram positive and gram-negative bacteria among many other microorganism. Tetracycline blocks aminoacyl-tRNA from to the acceptor site on the ribosome, thus preventing protein synthesis (Chopra & Roberts, 2001).

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References

Bentley, M., Zeller, N., & Walters-Conte, K. (2014). Bio210.

Bonan, N. (2014, July 7). Agar 10-9 wet mount [Drawing].

Chopra, I., & Roberts, M. (2001). Tetracycline Antibiotics: Mode of Action, Applications, Molecular Biology, and Epidemiology of Bacterial Resistance. Microbiology and Molecular Biology Reviews, 65(2), 232-260. doi: 10.1128/MMBR.65.2.232-260.2001

Freeman, S., Quillin, K., Taylor, E., Monroe, J., Allison, L., & Podgorski, G. (2014). Biological science (5th ed.). Glenview, IL: Pearson.

Rutgers University. (n.d.). Colony morphology: Describing bacterial colonies [Brochure]. Author. Retrieved July 9, 2014, from http://www.rci.rutgers.edu/~microlab/CLASSINFO/

HHS

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7/2/14

Lab 2: Identifying Algae and Protists

Introduction

If populations of organisms evolve as they adapt to the stresses of their environment by natural selection, then one would expect organisms to vary if the environment varies. In the Hay Infusion Culture, one would expect more photosynthetic organisms, like algae, near the top because of access to light and more protists towards the bottom.

If the bacteria are not resistant to antibiotics then there would be greater populations of bacteria in the agar than the tetracycline plates. If the bacteria are resistant to tetracycline, then they will thrive in the tetracycline plates, especially if other species they would normally compete for nutrients with are not resistant and are wiped out.

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Materials/Methods

Procedure I: How to Use a Dichotomous Key

1. Prepare wet mount of known organism

2. Use dichotomous key to confirm identify of the organism

3. Record observations of organism using compound microscope with 4x, 10x, and 40x objective lenses

Procedure II: Observing Hay Infusion Culture

1. Collect Hay Infusion Culture and make initial observations 2. Prepare two wet mounts from the top and bottom of the culture respectively 3. Observe the four samples under the compound microscope and use the dichotomous key to identify four organisms

Procedure III: Preparing and Plating Serial Dilutions 1. Ready materials by labeling four tubes with sterile broth with their final concentrations as well as two sets of four nutrient plates, one set of agar, and a second of agar plus tetracycline 2. Mix up the Hay Infusion Culture to ensure a representative sample 3. Add 100 uL to the first tube, mix well 4. Move 100 uL from the first tube to the second, mix well 5. Repeat twice more, until four tubes of decreasing concentration are prepared 6. Pipette 100 uL from each tube onto each pair of corresponding plates 7. Replace the covers of the plates and flip plates then upside down 8. Set aside the plates for incubation

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Data and Observations

Hay Culture Infusion: smells like mildew; appears murky with layer of mud at the bottom, some floating dirt, the opacity and brown color both increase as the depth of the sample increases

Top-down View of Hay Infusion Culture Straight-ahead View of Hay Infusion Culture

Diagram of Serial Dilution of Hay Infusion Culture

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Conclusion Paramecium is a protist which digests nutrients for energy, its cells are membrane bound with organelles to promote specialization, it has genes which encode hereditary information stored in its two nuclei, it is part of the alveolata lineage, and finally it can reproduce asexually or sexually, (Bentley et al. 2014) and (Freeman et al. 2014) If the Hay Infusion Culture is left over time it is expected that the population of protists would waste away from lack of nutrients, while the algae would thrive, assuming they have access to light, because of their ability to photosynthesize. Additional organisms could appear as the environment continues to change.

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References

Bentley, M., Zeller, N., & Walters-Conte, K. (2014). Bio210.

Freeman, S., Quillin, K., Taylor, E., Monroe, J., Allison, L., & Podgorski, G. (2014). Biological science (5th ed.). Glenview, IL: Pearson.

Ward's Natural Science Establishment, Inc. (2002). Ward's free-living protozoa [Brochure]. Rochester, NY: Author.

HHS

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7/1/14

Lab 1: Biological Life at AU

Introduction

In order to better understand the diversity of ecological life continually evolving via natural selection, compare samples of three of the 9,000 or so, species that comprise the Volvocine line. Observe the similarities and differences resulting from evolution in the volvocine line, especially related to motility, sexual reproduction, organization, and specialization. Then, examine a transect of the AU campus for a macro introduction to ecology in our surroundings. Differentiate between the biotic and abiotic elements in the transect to understand the stresses placed on the populations which reside there.

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Methods/Materials

Part I: The Volvocine Line 1. Prepare wet mounts of chlamydomonas and gonium 2. Observe using compound microscope 3. Prepare depression slide of volvox 4. Observe using compound microscope

Part II: Observing a Niche at AU

1. Analyze 20 by 20 meter transect

2. Observe general surroundings

3. Record notable abiotic and biotic components

4. Collect soil/surface plant sample in 50 mL conical tube

5. Prepare a Hay Infusion Culture by mixing 10-12 g of the sample, 500 mL of distilled water, 0.1 g dried milk, in plastic jar for 10 seconds

6. Remove top of Hay Infusion Culture and set aside

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Data and Observations

Differences among species of the Volvocine Line.

Transect #: 5

Location-outside Hughe's Hall

Aerial View of Transect

(Odunlami 2014)

Additional View of Transect

(Odunlami 2014)

Topography-slightly slanted

Abiotic Factors

Stone- rocks, pebbles Other- bench

Biotic Factors

Trees-ficus, other small trees Plants- day lily, modo grass, moss, daffodils, shrubs Animals- ants

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Conclusion

This transect is diverse in biotic and abiotic components and thus contains organisms of different types.

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References

Bentley, M., Zeller, N., & Walters-Conte, K. (2014). Bio210.

Environmental Protection Agency. (2012, June 25). Natural resources: Plankton. Retrieved from http://www.epa.gov/glnpo/image/viz_nat6.html

Freeman, S., Quillin, K., Taylor, E., Monroe, J., Allison, L., & Podgorski, G. (2014). Biological science (5th ed.). Glenview, IL: Pearson.

Office of University Architect. (2013, August 1). American University - Main campus [Map].

Protist Infomration Server. (n.d.). Protist images: Chlamydomonas reinhardtii. Retrieved July 7, 2014, from http://protist.i.hosei.ac.jp/pdb/images/chlorophyta/chlamydomonas/Euchlamydomonas/reinhardtii/sp_09.html

Odunlami, T. (2014, June 30). [Photograph].

University of New Hampshire, & Baker, A. L. (2012). Phytoplankton key - Phycokey - Volvox images. Retrieved from http://cfb.unh.edu/phycokey/Choices/Chlorophyceae/colonies/colonies_flagellated/VOLVOX/Volvox_Image_page.html

HHS

Contact Info

Hilary H. Strong (an artistic interpretation)

• Hilary H. Strong
• American University
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I work in the Your Lab at XYZ University. I learned about OpenWetWare from Bio 210, and I've joined because To participate in Biology 210..

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• 2013, B.A., Vanderbilt University

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