The purpose of this lab was to identify two unknown bacteria from a mixed culture. The reason for identification of unknown bacteria was to help students recognize different bacteria through different biochemical tests and characteristics. This is important in the medical field because identification of unknown bacteria can help treat a patient by knowing the contributing source of a disease. Also knowledge of different bacteria helped others make antibiotics used today. This lab was completed by using the methods learned thus far in identification of bacteria.
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 Dr. Floyd and Dr. 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/Trypticase 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 Trypticase Soy Agar plate to isolate individual colonies to be studied, tested and identified. After incubation of the individual TSA 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 Dr. Floyd and Dr. Kennedy (1). Table 1 and Table 2 list the tests performed, purpose, and results. Also Flow Chart 1 and Flow Chart 2 will show the results.
The Unknown Bacteria 36/ Bacteria #1 on a TSA plate was examined by the naked eye and under a dissecting microscope. Bacteria # 1 was approximately 2 - 3mm 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 cocci. After the Gram stain was completed, the bacteria were streaked on a Mannitol-Salt Agar plate and a Catalase test was performed. After these test were completed a Phenol Red Dextrose Fermentation tube was inoculated, and a SIM Tube inoculated.
The Unknown Bacteria 36/Bacteria # 2 on a TSA 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 -Methylene Blue Agar plate and an Enterotube 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 # 1)
TEST| PURPOSE| REAGENTS| OBSERVATIONS| RESULTS|
Gram Stain| To determine the Gram reaction of Bacteria #1| Crystal violet, Iodine, Alcohol, Safranin | Purple cocci| Gram positive cocci | MSA Agar plate| To determine Gram positive Staphylococcus species| None| Yellow halo around streak| Staphylococcus aureus| Catalase Test| To verify bacteria identification| Hydrogen peroxide| No bubbling| Negative| Phenol Red Dextrose Fermentation Tube| To verify bacteria identification| None| Reagent turned yellow & no gas produced| Fermentation of Glucose & Does not produce gas| SIM Tube| To verify bacteria identification| Kovac’s| Yellow color before and after reagent added. Single streak at line of stab| Sulfide negative Indole negativeNon motile |
Table 2: Biochemical Test...