Identifying Two Unknown Species of Bacteria

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 ...

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...indole, it is motile, there is no urease present and there is no coagulase activity. By deduction and logical reasoning Unknown 10a was determined to be Escherichia coli.

Unknown 10b is Staphylococcus epidermidis. According to Bergey’s Manual Staphylococcus bacteria are gram positive spherical cells that occur singly, in pairs or in irregular clusters. Unknown 10b was gram positive, spherical and occurred in clusters. Bergey’s Manual also says the bacteria grow well in high salt concentrations. Unknown 10b grew well on the mannitol salt agar. The optimum growing temperature is 30-37 degrees Celsius (Bergey’s Manual). Unknown 10b grew best at 37 degrees Celsius. The lab manual and past lab results confirmed all other test results. Unknown 10b was only able to use gamma lysis, it was unable to ferment mannitol and had no coagulase activity. When comparing to past labs it is confirmed that Unknown 10b is Staphylococcus epidermidis.

Unknown #10 contained two bacteria they are Escherichia coli and Staphylococcus epidermidis.

References

1.Holt, John G. et al Bergey’s Manual of Determinative Bacteriology, 1994.

2.Merkel, Brian Microbiology Laboratory, 2000.