User:Mark Skaggs/Notebook/Lab Three: Microbiology and Identifying Bacteria with DNA Sequences

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Objective: To observe and identify bacteria species present within transect and agar plates through visual inspection and DNA sequencing. Also, determine antibiotic resistance within bacteria.

Methods:

1. Agar Petri Dishes. Observe agar plates prepared in Lab Two and differentiate bacteria colonies, species, and quantify each from the eight petri dishes (four with agar and four with agar and tetracycline).

2. Antibiotic Resistance. Note the specimen that have grown on the dishes with tetracycline.

3. Gram Stains. Prepare gram stains from agar plates by extracting bacterial colonies and rinsing sterilized bacterial slides in crystal violet, iodine, 95% alcohol, and safranin stain. After allowing to dry, view using micrscopy and note cell & colony characteristics.

4. PCR Reaction. Conduct the beginning of a PCR (polymerase chain reaction) by transferring bacterial colony to 100 µl water in sterile test tubes. Incubate at 100°C for 10 minutes. Centrifuge at 13,400 rpm for 5 minutes. Add 20 µl of primer to PCR tube. Transfer 5 µl of supernatant liquid from centrifuged samples into 16s PCR reaction tubes. Place three tubes in PCR machine.


Observations, Results, and Conclusion:

Archaea thrive in extreme conditions such as volcanoes and the Marianas trench. Due to the nature of their preferred habitat, it is improbable that archaea would be present on the agar plates. While archaea may thrive within the petri dishes and in the presence of tetracycline, they will unlikely be within the initial sample from transect 2.

The bacteria present on each of the agar plates will change over time and in different concentrations. Also, the tetracycline plates will yield different bacteria species. As time passes, some bacteria will flourish and others will diminish. This will result in varying smells and appearances.

Dilution-------Agar Type---------Colonies Counted------Conversion Factor------Colonies/ml

10^-3-----------nutrient--------------lawn----------------------x10^3------------------lawn

10^-5-----------nutrient--------------lawn----------------------x10^5------------------lawn

10^-7-----------nutrient--------------lawn----------------------x10^7------------------lawn

10^-9-----------nutrient--------------163-----------------------x10^9------------------1.6x10^11

10^-3-----------nutrient + tet-------lawn-----------------------x10^3------------------lawn

10^-5-----------nutrient + tet-------lawn-----------------------x10^5------------------lawn

10^-7-----------nutrient + tet-------189------------------------x10^7------------------1.89x10^9

10^-9-----------nutrient + tet-------18-------------------------x10^9------------------1.8x10^10

Quite noticeably, the agar plates that contain tetracycline grow different bacteria than plates that do not contain it. White and orange bacteria grow on the normal agar plates, but only orange bacteria grew on the tetracycline plates.