Identifying Two Unknown Species of Bacteria
Materials and Methods
Week 1, Day 1 (10 November 2000)
The first day an unknown sample was assigned to each group of students. The first test applied was a gram stain to test for gram positive or gram-negative bacteria. The morphology of the two types of bacteria was viewed under the microscope and recorded. Then the sample was put on agar plates using the quadrant streak method for isolation. There were three agar plates; one was incubated at room temperature, the second at 30 degrees Celsius, and the third at 37 degrees Celsius. By placing each plate at a different temperature optimal growth temperature can be predicted for both species of bacteria.
Week 1, Day 2 (12 November 2000)
After 48 hours of incubation the agar plates were viewed. Individual colonies were tested for successful isolation by gram staining and then viewing the stained bacteria under a microscope. Isolation was successful. One colony of each unknown bacteria was transferred to an agar slant for growth. The agar slants were stored at room temperature over the weekend so that they would not grow too much.
Week 2, Day 1 (17 November 2000)
After 5 days of growth each slant was tested using the gram staining technique to confirm the complete isolation of the bacteria. Both isolations were completely successful. Then each sample of bacteria was subjected to a series of tests for identification.
One bacterium was gram negative. It underwent four different tests. These tests were the EMB test (Eosin Mehylene Blue), the Sulfur Indole Motility (SIM) test, the Urease test, and the Simmon’s Citrate Utilization test. The EMB test checks for a bacteria’s ability to ferment lactose. This test is accomplished by placing the bacteria on Eosin Methylene Blue agar. The agar is selective for gram negative bacteria and those bacteria that can ferment lactose will have colored growth, usually a metallic green sheen.
The Sulfur Indole Motility agar tests for three separate characteristics; sulfur reduction, indole production, and motility. The SIM medium is a semisolid medium; this facilitates the motility test. The medium contains sulfur, if the bacterium has the ability to reduce sulfur the medium will turn black. The medium also contains tryptophan. If the bacterium has the enzyme tryptophanase, indole will be produced. A positive result will be a pink ring around the top of the medium after Kovac’s solution has been added.
The Urease test detects the presence of the enzyme urease. This medium contains urea and a pH indicator, phenol red. A bacterium containing urease will produce ammonia, which raises the pH and changes the color of the medium. A positive test shows a color change from a reddish orange to pink.
The fourth test used to identify gram negative bacteria is the Simmon’s Citrate Utilization test. This test measures a bacteria’s ability to use citrate as its sole...