Isolation, Animal_Science_&_Zoology, College Term Papers.com


		Data Bases
		• Custom Term Papers • Free Term Papers • Free Research Papers • Free Essays • Free Book Reports • Plagiarism?

		Links
		• Top 100 Term Paper Sites • Top 25 Essay Sites • Top 50 Essay Sites • Search 97,000 Papers @ DirectEssays.com • Search 101,000 Papers @ ExampleEssays.com • Search 90,000 Papers @ MegaEssays.com

		Free Essays
		• Term Paper Sites • Chuck III's Free Essays • Free College Essays • TermPaperSites.com • My Term Papers • Get Free Essays • Essay World • Planet Papers

Search Lots of Essays

Back to Subjects - Animal Science & Zoology

Isolation

Isolation Isolation and Identification of Unknown Bacteria Performed: 10 November 2000 through 19 November 2000 The purpose of this lab was to identify and isolate two unknown species of bacteria from one broth culture. Testing materials were supplied to help in the successful completion of this experiment. 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. 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. 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 carbon source for metabolic pathways. This solid medium contains Bromothymol Blue, which serves as a pH indicator. If the bacteria can break down citrate it produces carbon dioxide that reacts with sodium and water, also found in the medium, to produce sodium carbonate. Sodium carbonate elevates the pH causing a color change from green to blue (a positive result). The second bacterium was gram positive. There are three tests that are used to identify gram positive bacteria. The three tests are hemolyses on blood agar, Mannitol fermentation on mannitol salt agar, and the coagulase test using plasma-containing serum in coagulase tubes. The test on blood agar tests for a bacteria’s ability to hemolyze red blood cells to obtain the iron found in them. There are three types of hemolysis: gamma lysis, alpha lysis, and beta lysis. Gamma lysis is the absence of erythrocyte lysis showing no color change in the blood agar. Alpha lysis is a partial break down of hemoglobin and results in a color change from red to green. The third kind of hemolysis is beta lysis, which is the total degradation of hemoglobin and shows a color change from red to clear. The second test is on mannitol salt agar. This test is selective for those bacteria able to survive an environment containing high salt concentrations. The test also shows whether or not a bacterium has the ability to ferment mannitol. The medium contains a pH indicator, if mannitol is fermented then pyruvic acid is produced which drops the pH. If this happens a yellow halo will appear around the bacteria growth. The coagulase test checks for a bacteria’s ability to clot serum. If the serum clots then the bacteria possesses coagulase activity. The tests were performed and placed in incubators. The results were checked 48 hours later. All tests were viewed and the results were recorded. Analysis of the results was made and the bacteria were identified. After the initial gram test was complete the bacteria found in unknown #10 were determined to be as such: The first bacteria viewed will be referred to as Unknown 10a and the second will be Unknown 10b The optimal growing temperature for both unknown bacteria was found to be 37 degrees. The next series of gram stains showed that the isolation was successful. Both Unknown 10a and Unknown 10b were isolated. The isolated colonies were transferred to slants and stored over the weekend. The slants of Unknown 10a and Unknown 10b were again tested for total isolation using the gram staining technique. Both samples revealed successful isolation. Unknown 10a was then subjected to tests for gram negative bacteria. Unknown 10b was tested with tests for identifying gram positive bacteria. All test results were viewed and recorded. Lactose Fermentation Sulfur Reduction Indole Production Motility Urease Present Citrate Utilization Metallic Green Sheen No color change Pink ring formed Yes No color change No color change Unknown 10a was positive for lactose fermentation, negative for sulfur reduction, positive for motility, Negative for the presence of urease and negative for Citrate utilization. Hemolysis Mannitol Fermentation Coagulase Activity No Color Change No color change No clotting Unknown 10b participated in gamma lysis, could not ferment mannitol and did not have coagulase activity. By analyzing the results and using Bergey’s Manual of Determinative Bacteriology the identity of the unknown bacteria were found. Unknown 10a is Escherichia coli. Bergey’s Manual states that the genus Escherichia is made up of straight rods that occur singly or in pairs. The manual also states that the optimal growing temperature for this genus is 37 degrees Celsius. These two statements confirm the results found for Unknown 10a. Reviewing results from previous labs and consulting the lab manual confirmed all of the other test results. E. coli can ferment lactose, it can not reduce sulfur, it can produce 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. 1.Holt, John G. et al Bergey’s Manual of Determinative Bacteriology, 1994. 2.Merkel, Brian Microbiology Laboratory, 2000. Bibliography:

Word Count: 1379