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Indole Test Lab Report Essay

My partner and I were given unknown number 3 in the laboratory. After performing various tests over the course of a few weeks on our unknown, we came to the conclusion that our unknown organism is Klebsiella pneumoniae (K. pneumoniae). K. pneumoniae is a gram negative bacillus shaped microorganism. We observed that K. pneumoniae is a nonmotile organism. We performed multiple tests on our unknown culture, therefore we are very confident that it is correctly identified. We identified that K. pneumoniae is a facultative anaerobe.

When inoculated onto an agar slant, K. neumoniae took the shape of echinulate. When inoculated onto a nutrient agar plate, we identified that the growth of K. pneumoniae was round and smooth with a convex elevation. These are all characteristics of the K. pneumoniae bacterium. Through the use of resources such as the separation outlines provided in the lab manual and tables/charts provided by our lab instructor, we were able to deduce which organism we had based on the results of our tests. The tests that were crucial in our identification were gram staining, oxidase test, the motility, and the citrate utilization test.

The first step in narrowing down organisms was the gram stain. This test showed us the gram reaction as well as the general shape of the organism. Because the cell wall of a K. pneumoniae bacteria has a very thin layer of peptidoglycan, it does not retain the crystal violet stain in gram staining. The gram stain revealed that our unknown was a gram negative rod, narrowing down our choices of which microorganism it could be. The next crucial test performed was the oxidase test.

The oxidase test checks for the presence of the enzyme cytochrome oxidase. Our oxidase test was negative, showing K. neumoniae does not contain cytochrome oxidase in the electron transport chain. The next test we relied upon was the motility test. It is crucial to observe whether a bacteria is motile or not, since this can eliminate possibilities for the identity of the organism. By preparing a hanging-drop slide, we observed that our unknown was non-motile, limiting the options of what organism it could be. The final test we relied heavily on was the citrate utilization test. The citrate utilization test determines if the organism can use citrate as a source of carbon.

Our citrate test came out positive, showing that K. pneumoniae is able to utilize citrate. Through the use of these tests and the flow charts provided by our lab instructor, we were able to determine that our unknown organism was K. pneumoniae. The remaining tests performed allowed us to confirm our assumption that our unknown was K. pneumoniae. The Durham sugars test was done to determine if the organism ferments the sugars glucose, lactose, and mannitol. Our results were positive for each of the sugars, and gas was produced in each tube. This shows that K. pneumoniae ferments each of these sugars.

A catalase test determines if an organism produces the enzyme catalase, which is used to break down hydrogen peroxide into oxygen and water. The result of our catalase test was positive, demonstrating that K. pneumoniae produces the catalase enzyme. A nitrate reduction test is performed to determine whether an organism is able to reduce nitrate or not. Our test came out positive, showing that K. pneumoniae is able to reduce nitrate. The starch hydrolysis test is performed to determine whether the organism produces amylases, which are enzymes that break down starch molecules.

Our starch hydrolysis test was negative, showing that K. pneumoniae does not produce amylase and cannot break down starch. The casein hydrolysis test is performed to determine if the organism produces proteases, enzymes that break down protein molecules such as protein in milk. Our casein test was negative, demonstrating that K. pneumoniae does not produce proteases and cannot break down proteins via proteolysis. The fat hydrolysis test was performed to determine whether our bacteria produces lipase, an enzyme that breaks down fat.

The fat hydrolysis test was positive, showing K. pneumoniae produces lipase and can break down fats. The indole test was performed to determine if our bacteria can break down tryptophan via the enzyme tryptophanase. Our indole test came back negative, meaning K. pneumoniae does not produce tryptophanase and does not break down tryptophan into indole, ammonia, and pyruvic acid. The urea test was performed to determine if urea is hydrolyzed via the enzyme urease. The urea test was positive, meaning K. pneumoniae produces urease to break down urea.

An inoculation onto Kligler’s iron agar determines if an organism can ferment glucose and lactose, it also detects the production of hydrogen sulfide from the breakdown of cysteine. Our Kligler’s iron agar showed acid with gas production, meaning K. pneumoniae fermented both glucose and lactose. The hydrogen sulfide production was negative. A Litmus Milk test is done to determine whether the organism can ferment lactose, digest the milk proteins using proteases, cause the milk proteins to form clots, or reduce the litmus dye.

Our litmus milk test showed that our bacteria produces acid with clot. This means that K. pneumoniae ferments lactose and coagulates, producing a clot. The Voges-Proskauer test determines whether or not an organism produces butanedoil when fermenting glucose. Our VP test was positive, meaning K. pneumoniae ferments glucose to produce butanedoil. One test that did not match the tables or charts was the Methyl Red test. Our results for the MR test were false positive. This false positive can result from many different reasons.

A possible explanation for the false-positive result is cross contamination. There may have been cross contamination in the broth used for the MR test due to improper aseptic techniques used when inoculating the broth. A second possibility is cross contamination from the dropper used for the methyl red indicator. This dropper was placed in other students unknown tubes. If they did not observe proper aseptic techniques, they may have contaminated the dropper.

The chart given to us entitled “Percentage of Positive Reactions of Various Gram Negative Organisms” states that K. neumoniae has an 11. 3% chance of producing a positive reaction in a Methyl Red test. Despite our false positive, we were able to identify our unknown organism as being Klebsiella pneumoniae through the use of the different tests and the various flow charts provided for us. K. pneumoniae can be found in a wide variety of habitats, though it is most commonly found in the human gastrointestinal tract. Other habitats of K. pneumoniae include humans, soil, animals, and contaminated water. K. pneumoniae is an encapsulated organism, making it more resistant to the human immune system.

K. pneumoniae is an opportunistic pathogen in humans, meaning it is does not normally cause a disease in a healthy host, but when the host becomes sick and their immune system is compromised, K. pneumoniae can become a pathogen to the host. K. pneumoniae can cause pneumonia or bloodstream infections. Recently, there has been an increase in the resistance of K. pneumoniae to antibiotics. This is very dangerous because, if it continues to grow resistance, treating K. pneumoniae will be difficult. There has recently been an appearance of a strain of K. neumoniae that produces an enzyme called carbapenemase.

This enzyme works in conjunction with K. pneumoniae to create resistance aga iae to create resistance against the antibiotic carbapenem. The issue of K. pneumoniae carbapenemase (KPC) producing bacteria began in the United States, but has spread throughout the world. KCP producing bacteria pose a real dilemma for medical professionals in discovering how to properly treat these types of bacteria. Studies are currently being conducted to develop new ways to treat KCP producing bacteria in hopes to improve the quality of medicine world wide.

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