Identification of an Unknown bacterium
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Identification of an Unknown
bacterium
Test Tube Number 23
Microbiology 2010C
March 31, 2004
Identification of an Unknown Bacterium
On February 25, 2004, we were given an unknown bacterium and asked to run two procedures that day to determine if we were dealing with a gram-negative or gram-positive bacterium and request an appropriate "shopping list" for proper identification. Our first two steps were (1) to prepare a slide utilizing the Gram stain process and (2) do a T-streak on TSA agar and incubating our petri plates for 24 to 72 hours at 37˚C. The Gram stain process entailed applying a "nickel-sized" amount of our pure culture to a sterilized glass slide and following the four-step procedure of placing a primary stain (crystal violet), a mordant treatment (Gram's iodine), a decolorizing agent (ethanol alcohol), and a counterstain (safranin) on our slides. These staining agents were to be applied in that order while utilizing aseptic procedures throughout each of our experiments. The crystal violet is applied for 60 seconds and lightly rinsed with H20 after the allotted time, next was the Gram's iodine for 30 seconds also rinsed with H20, then the ethanol alcohol which was rinsed immediately with H20, and finally the safranin for 30 seconds and rinsed with H20. After drying and heat-fixing our slides, we were able to determine if our bacterium was a Gram positive coccus (purple in color) or a Gram negative rod (pink/red in color) by viewing it under a compound light microscope using the oil-immersion lens. It was determined that mine was a gram-negative rod since it had short rods that were pink/red in color. Considering these findings, it was decided that the best nutrients to use would be Eosin-methylene blue (EMB), MacKoney plate (Mac), Phenol red-lactose, Phenol red-glucose, Phenol red-sucrose, and Gelatinase. All would have an incubation temperature of 37˚C with the exception of the Gelatinasewhich needs to be incubated at 27˚C for 24 to 72 hours.
On February 27, the experiments continued...