User:Charlotte E. Vogler/Notebook/Lab 3: Microbiology and Identifying Bacteria with DNA Sequences, Transect 2 on 7/8/14

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Lab 3: Microbiology and Identifying Bacteria with DNA Sequences on July 8, 2014

Introduction, Purpose, and Objectives

Today, we observed and identified bacteria species from Transect 2 that had previously been transferred onto four plates containing agar and four plates containing tetracycline. Our objective was to understand the bacteria species, observe their antibiotic resistance, and to understand how DNA sequences are used to identify a species.

Materials and Methods

Today, we identified species through a staining technique called Gram stain. Bacteria that are gram-positive have a thick layer of peptidoglycan in their cell walls and retain the dye. Those species that cannot retain the dye are gram-negative and have less peptidoglycan in their cell walls. Being gram-positive or –negative depends on the cell wall and different bacteria can be identified through this method. We used compound microscopes to view our plates individually to determine the characteristics of the bacteria species located in Transect 2. Later, we used the microscopes to view our gram stain slides and determine whether the bacteria were gram positive or gram negative. Finally, to prepare for the next weeks lab, we selected three samples to set up a PCR reaction. The purpose of the PCR reaction is to amplify the 16s rRNA gene to determine the species.

Data and Observations

Archaea species did not grow on the agar plates. This is because our environment at American University is not extreme enough. Archaea tend to grow in very extreme environments where other species are unable to survive, such as very hot or very cold niches.

Part 1: Quantifying and Observing Microorganisms

Table 1: 100-fold Serial Dilutions Results


Part 2: Antibiotic Resistance

The agar plates with tetracycline contained fewer colonies than the plates with just the nutrient. This is because tetracycline kills bacteria. The tetracycline prevented as much bacteria from growing in these plates as compared to the plates without the antibiotic. However, the plates with tetracycline still contained large amounts of an orange growth. Because this species did not seem to be hindered by the tetracycline, we believe one of two possibilities exist: a) the organism is not a bacteria or b) the organism is a bacteria, but has developed a resistance to the tetracycline. After testing the PCR reaction for the 16s rRNA gene, we will be able to conclude whether this organism is in fact a species of bacteria. We hypothesize that the orange organism is in fact a bacteria, but has become resistant to the effects of Tetracycline.

Tetracycline has an effect on the total number of bacteria because it is an antibiotic, which does not allow bacteria to grow or survive in the agar plate. It interferes with bacteria’s ability to produce the nutrients and proteins necessary for their survival. E. coli is a common type of bacteria that cannot survive in the presence of tetracycline. Conditions such pneumonia and other respiratory tract infections are treated with the tetracycline.

Part 3: Bacteria Cell Morphology Observations

Table 2: Bacteria Characterization


Conclusions and Future Directions

In next weeks lab, we will identify the bacteria species that we have been observing in previous labs by looking closely at the 16s rRNA gene in the species in order to determine their exact species. This is possible because the 16s rRNA gene is distinct in every species.

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