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MicroOrganism
Unknown Experiment

Introduction: The purpose of this experiment is to distinguish and indentify an unknown bacterium. There are several tests that can help one eliminate and narrow down the options. The most useful test, and the very first one done, is a gram stain. This test will tell whether the bacterium is gram-positive or gram-negative. After the type of gram stain is identified, the tester has a wide array of differentiating tests at their disposal. Based on the results from these tests, and the numerous others that are available, one can accurately establish the identity of an unknown bacterium.

Materials: 1-Tube containing unknown bacteria, crystal violet, iodine, decolorizing agent, Safranin, Bunsen burner, sheep blood agar plate, TSA plate, and bacitracin antibiotic disks.

Procedures 1: 1.) Obtain a tube with an unknown bacteria and materials necessary to prepare a gram-stain. 2.) First place 5-8 loops of the bacteria onto the slide in a circular area and heat fix the bacteria to the slide. 3.) Place 3-5 drops, or enough to cover the bacteria, of crystal violet stain onto the slide and let sit for at least 1 min. 4.) Wash off crystal violet with water and then cover bacteria with 3-5 drops of iodine and let sit for at least 1 min. 5.) Wash off iodine with water and then add decolorizing agent until the runoff turns clear. 6.) Next add 3-5 drops of safranin to the slide and let sit for at least 1 min. 7.) Wash off safranin and then carefully blot the slide dry with a paper towel. 8.) Observe the bacteria through a microscope under the oil emersion lens. 9.) Be sure to note the morphology of the bacteria and whether it is gram-positive or negative. (Hold on to bacteria tube for future tests)

Procedures 2: (Tests performed based upon the results from gram-stain). 1.) Next obtain 1 sheep blood agar plate and inoculate it with the bacteria. 2.) Incubate blood agar plate, upside



Cited: A Photographic Atlas for the Microbiology Laboratory, 3rd ed., Leboffe and Pierce, Morton Publishing Co., 2005. Microbiology: Principles and Explorations, 7th ed., Jacquelyn Black, John Wiley & Sons, 2008.

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