Materials and Methods A mixed culture of two unknown bacteria was provided by the instructor. The methods used for identification of the two unknown bacteria were in the laboratory manual by Dry. Floyd and Dry. Kennedy (1) unless otherwise noted. The first step that was used was a three streak method, described in Lab 4, on a Blood Heart Infused/’Triplicate Soy Agar plate to isolate the two unknown bacteria. Once the two bacteria were incubated, grown, and isolated they were each individually streaked on a Triplicate Soy Agar plate to isolate individual loonies to be studied, tested and identified.
After incubation of the individual TTS plates, the morphologies were viewed and noted and a Gram stain was completed on each individual bacterium, which will be referred to as Bacteria #1 and Bacteria #2. After the Gram reaction was determined on Bacteria #1 and Bacteria #2, different biochemical tests were done according to the dichotomous keys provided in the lab manual. All the tests were performed by the methods outlined in the lab manual by Dry. Floyd and Dry. Kennedy (1). Table 1 and Table 2 list the tests performed, purpose, and results.
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Also Flow Chart 1 and Flow Chart 2 will show the results. Results The Unknown Bacteria 36/ Bacteria #1 on a TTS plate was examined by the naked eye and under a dissecting microscope. Bacteria # 1 was approximately 2 – mm in diameter. They were circular in form with an entire margin and convex elevation. The colonies were smooth, translucent, and had a white brownish color. The Gram stain resulted in Gram positive Cisco. After the Gram stain was completed, the bacteria were streaked on a Imitation-Salt Agar plate and a Catalane test was performed. After these test were completed a Phenol Red
Dextrose Fermentation tube was inoculated, and a SIMI Tube inoculated. The Unknown Bacteria 36/Bacteria # 2 on a TTS plate was examined by the naked eye and under a dissecting microscope. Bacteria # 2 was approximately 3 – 4 mm in diameter. They were circular in form with an entire margin and a flat elevation. The colonies were rough (granular), translucent, and white brownish color with black granules. The Gram stain resulted in a Gram negative rod. After the Gram stain was completed, the bacteria were streaked on an Eosin -Methyl Blue Agar plate and an Underwrote II was inoculated.
See Table 1 and Flow Chart 1 for results of Bacteria # 1 and Table 2 and Flow Chart 2 for results of Bacteria # 2. Table 1: Biochemical Test Results (Bacteria # TEST I PURPOSE I REAGENTS OBSERVATIONS RESULTS I Gram Stain I To determine the Gram reaction of Bacteria #1 | Crystal violet, Iodine, Alcohol, Seafaring I Purple Cisco I Gram positive Cisco I MASS Agar plate I To determine Gram positive Staphylococcus species I None I Yellow halo around streak I Staphylococcus erasures I Catalane Test I To verify bacteria identification I Hydrogen peroxide I No bubbling Negative I Phenol Red
Dextrose Fermentation Tube I To verify bacteria identification I None Reagent turned yellow ; no gas produced I Fermentation of Glucose ; Does not produce gas I SIMI Tube I To verify bacteria identification I Kava’s I Yellow color before and after reagent added.
Single streak at line of stab I Sulfide negative Indolent negotiation motile I Table 2: Biochemical Test Results (Bacteria # 2) Gram Stain I To determine the Gram reaction of Bacteria #21 Crystal violet, Iodine, Alcohol, Seafaring I Pink rods I Gram negative rods I EMBED Agar late I To determine the ability to ferment lactose I None I Dark purple streak I Fermentation of Lactose I Underwrote II I Identify bacteria I Kava’s I Interpreted results using the interpretation color chart.
Added reagent resulted in Indolent negative I Escherichia cold-blooded 34760 | FLOW CHART 1 BACTERIA # 1 FLOW CHART 2 BACTERIA # 2 It has been concluded that the Unknown 36/Bacteria # 1 is Staphylococcus erasures and Unknown 36/Bacteria # 2 is Escherichia coli. This was accomplished by first streaking a BI/TTS infused plate with the Unknown 36 broth to isolate he two bacteria. Once two different bacterial growths were observed, each different bacterium was streaked on individual TTS plates to isolate colonies for Gram staining and biochemical tests.
Gram staining resulted in Bacteria # 1 being Gram positive Cisco and Bacteria # 2 being Gram negative rods. Following the dichotomous key, Bacteria # 1 was streaked on a MASS plate. Fermentation was noted resulting in S. Erasures being identified. A Catalane test was performed to verify the identification of S. Erasures; however, the test was negative when it should have been positive. A false negative can result from the reagent not being fresh or not stored properly and/or the test run on cultures over 24 hours old.
Because of this, a Phenol Red Dextrose Fermentation tube and a SIMI tube were inoculated to confirm the bacteria were S. Erasures. Results from both test was consistent with S. Erasures characteristics. Staphylococcus erasures is a facultative anaerobic Gram positive Cisco that is salt tolerant. This bacterium is part of our normal flora found on human skin and in human nasal passages. S. Erasures is the cause of many minor infections such as follicular and pimples to more severe sissies such as pneumonia and toxic shock syndrome.
Following the dichotomous key Bacteria # 2 was inoculated into an Underwrote II. Also for verification an EMBED plate was streaked. Using the color interpretation guide for the Underwrote II a five digit code was reached. Once the code was recorded a code manual was used which identified Bacteria # 2 as Escherichia coli. The EMBED plate showed fermentation of lactose which is consistent with E. Coli characteristics. Escherichia coli are a member of the Interchangeable family and are part of human and warm blood animal’s normal intestinal flora.