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The purpose of this laboratory experiment was to identify an unknown bacterium, designated as Unknown #19, using a series of microbiological tests. The tests performed included the citrate test, BCP lactose fermentation test, hydrogen sulfide test, indole test, methyl red test, motility test, and urease test. Through careful experimentation, Unknown #19 was identified as Klebsiella oxytoca, a bacterium commonly found in human intestines and healthcare settings. This report outlines the methods, results, and implications of this identification process.
The identification of unknown bacteria is a fundamental skill in microbiology.
In this experiment, we applied advanced thinking and previously learned skills to identify an unknown bacterium through a series of tests. These tests included Gram staining, citrate utilization, lactose fermentation, hydrogen sulfide production, indole production, methyl red test, motility test, and urease activity.
Klebsiella oxytoca is a bacterium commonly found in human intestines and healthcare settings. While it is not known to directly cause infections, it can become problematic if it enters the body through open wounds or other sources, leading to infections.
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Understanding the presence and characteristics of Klebsiella oxytoca is important, especially in healthcare settings, where it can be easily spread.
Unknown #19, suspected to be Klebsiella oxytoca, was isolated from a urine sample. To purify the culture, Unknown #19 was streaked onto an agar plate and incubated at 35°C for 48 hours. After incubation, colonies were observed, and a pure colony was selected for further testing, including Gram staining and a series of biochemical tests.
The citrate test was performed to determine the ability of the bacterium to utilize citrate.
A portion of the pure colony was streaked onto a citrate slant and incubated at 35°C for 48 hours. A color change from green to vibrant blue indicated a positive result, signifying citrate utilization.
The BCP lactose fermentation test assessed the bacterium's ability to ferment lactose. The pure colony was inoculated into a tube containing BCP lactose broth and incubated at 35°C for 48 hours. A yellow color change indicated a positive result, indicating lactose fermentation and a pH change.
The hydrogen sulfide test determined whether the bacterium could produce hydrogen sulfide and possess the enzyme cysteine desulfurase. The pure colony was stabbed into a PIA slant, incubated at 35°C for 48 hours, and refrigerated to prevent contamination. A black precipitate indicated a positive result, confirming hydrogen sulfide production and the presence of cysteine desulfurase.
The indole test assessed the bacterium's ability to utilize tryptophan to produce indole, pyruvic acid, and ammonia. The pure colony was inoculated into tryptone broth containing tryptophan and incubated at 35°C for 48 hours. Kovac's reagent was added, and a bright red ring layer indicated a positive result, demonstrating the production of indole.
The methyl red test determined whether the bacterium could ferment glucose to produce organic acids using formic hydrogenylase. The pure colony was inoculated into methyl red broth and incubated at 35°C for 48 hours. A red color change indicated a positive result, signifying the production of organic acid from glucose.
The motility test assessed the bacterium's ability to move using flagella. The pure colony was stabbed into a motility media and incubated at 35°C for 48 hours. Growth away from the original stab indicated a positive result, confirming motility.
The urease test determined whether the bacterium could utilize urease from urea to produce carbon dioxide and ammonia. The pure colony was streaked onto a urease slant and incubated at 35°C for 48 hours. A bright pink color indicated a positive result, signifying the hydrolysis of urea.
In the Gram staining process, Unknown #19 was observed as pink, indicating that it is gram-negative. Under the microscope, the bacterium appeared as rod-shaped bacilli arranged in chains with some individual cells. Colonies on the TSA plate exhibited a raised, smooth texture with small, yellowish-white colonies.
| Test | Media/Reagents Used | Enzyme Involved | Observations | Results | Interpretation |
|---|---|---|---|---|---|
| Citrate Test | Citric acid | Citritase | Vibrant blue throughout slant | POSITIVE | pH changed, unknown can utilize citric acid, grow, and metabolize ions |
| BCP Lactose Fermentation Test | Purple broth | Beta-galactosidase | Bright yellow | POSITIVE | Unknown can ferment lactose, pH change occurs |
| Hydrogen Sulfide Test | Peptone iron agar | Cysteine desulfurase | Solid purple | NEGATIVE | Unknown cannot produce the enzyme cysteine desulfurase |
| Indole Test | Tryptone broth | Tryptophanase | Bright pink ring on top of the culture | POSITIVE | Indole has been produced utilizing the enzyme tryptophanase |
| Methyl Red Test | MR broths | Formic hydrogenylase | Red color throughout tube | POSITIVE | Unknown can produce organic acid from glucose, pH drops below 5 |
The initial Gram stain test revealed that Unknown #19 is gram-negative, with rod-shaped bacilli arranged in chains. Subsequent biochemical tests helped identify the bacterium as Klebsiella oxytoca.
The positive citrate test result indicated that the bacterium could utilize citric acid, further narrowing down the potential candidates. Proteus vulgaris, Proteus mirabilis, Providencia alcalifacens, Providencia rettgeri, Providencia stuarti, Pseudomonas aeruginosa, Pseudomonas aureofaciens, Serratia marcescens, and Citrobacter farmeri were eliminated due to their inability to use citrate.
The BCP lactose fermentation test confirmed that the bacterium could ferment lactose, as evidenced by the yellow color change and pH alteration. This result eliminated several other potential candidates, including Proteus vulgaris, Proteus mirabilis, Providencia alcalifacens, Providencia rettgeri, Providencia stuarti, Pseudomonas aeruginosa, Pseudomonas aureofaciens, Serratia marcescens, and Citrobacter farmeri.
However, the hydrogen sulfide test yielded a negative result, indicating that the bacterium could not produce the enzyme cysteine desulfurase. This eliminated Citrobacter freundii, Enterobacter aerogenes, Klebsiella pneumoniae, Klebsiella ozaenae, and Pantoea agglomerans as potential candidates.
The positive indole test result, with the presence of a bright pink ring, confirmed the production of indole using the enzyme tryptophanase.
The methyl red test resulted in a positive outcome, with a red color indicating the production of organic acid from glucose and a decrease in pH below 5.
Based on the results of these tests, Unknown #19 was identified as Klebsiella oxytoca, a bacterium commonly found in human intestines and healthcare settings. Klebsiella oxytoca can be of concern in healthcare settings due to its developing resistance to antiseptics like chlorhexidine.
In conclusion, this laboratory experiment successfully identified Unknown #19 as Klebsiella oxytoca using a series of microbiological tests. Understanding the presence and characteristics of this bacterium is crucial, especially in healthcare settings, where it can be easily transmitted. Further research into the resistance of Klebsiella oxytoca to antiseptics like chlorhexidine is essential for maintaining patient safety in healthcare facilities.
Principles of Microbiology: Lab Report. (2024, Jan 02). Retrieved from https://studymoose.com/document/principles-of-microbiology-lab-report
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