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UNKNOWN LAB REPORT
Kesan Mony Sisowath
The purpose of this lab was to identify two unknown bacteria from a mixed culture. The reason for the identification of unknown bacteria was to help students recognize different bacteria through different tests and characteristics. This is important in the medical field because the identification of unknown bacteria can help treat a patient by knowing the contributing source of a disease. Also, knowledge of different bacteria helped others make antibiotics used today.
Materials and Methods
The instructor provided a mixed culture, labeled #114, of two unknown bacteria. All these procedures were stated in the lab manual of general microbiology.
The first step that was used was a three-streak method on a nutrient agar plate to isolate the two unknown bacteria. Once the bacteria were incubated, grown, and isolated, a Gram stain was performed to identify the possible shape of one of the two bacteria in the mixed culture. Gram Stains is used to identify and classify bacteria as either gram-positive or gram-negative. Bacteria that lose color easily after decolorization are referred to as gram-negative, and bacteria that retain the color from the primary stain are called gram-positive. Bacteria stain differently because of chemical and physical differences in the cell wall. Under the microscope, if the gram stain is purple the bacterium is gram-positive, if the stain is red, it is gram-negative. Using the microscope, the unknown was determined to be gram-positive cocci.
Nitrate reduction was tested for by inoculating a nitrate broth with the unknown cultures, and allowing growth to take place. Adding drops of both reagents A and B to the medium is the first test to see if nitrite is present. If nitrite is present, the medium turns red, indicating a positive test. However, if the medium does not change, a second test is performed to see if nitrite was further reduced. In this second test, zinc powder is added to the broth to catalyze the reduction of any nitrate present in nitrite. If nitrate is present when the zinc is added the reduction of this compound will cause the medium to turn red, from the previously added reagents. The red medium on the second addition indicates nitrate was not reduced and a negative test result. However, if the medium does not change after the addition of the zinc, the unknown is positive for nitrate reduction, as the nitrite has just been further reduced, preventing its detection.
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Mannitol salt agar is both selective and differential media used for gram-positive cocci. It is selective for salt tolerance and differential for mannitol sugar fermentation. It also contains phenol red, which acts as a pH indicator, turning yellow under acidic conditions. Growth and a yellow color change are positive test results. No growth is a negative test result.
The urease test was performed only on the gram-negative bacteria, testing for the breakdown of urea. Urea hydrolysis to ammonia by urease-positive organisms will overcome the buffer in the medium and change it from yellow to pink. Rapid urease-positive bacteria will turn the broth pink within 24 hours. Urease-negative bacteria will produce no color change or turn the broth yellow.
Lactose and sucrose sugar fermentation were tested using a broth containing respective sugar compounds, phenol red, and inverted Durham tubes. The broths were inoculated with the unknown bacteria cultures and incubated for growth. If fermentation of the sugar molecules was carried out, the pH in the tube would be lowered, and the phenol red would be converted to yellow under the acidic conditions. Thus, the conversion of the original red broth to yellow signifies a positive test, indicating the bacteria can ferment using either lactose or sucrose. If the broth remains red, fermentation on theses sugar was not possible and the test is negative. The production of gas by the fermentation was monitored using the inverted Durham tubes. If gas was produced during the fermentation process, the Durham tube will contain a bubble.
EMB, eosin methylene blue, agar is selective for gram-negative bacteria. EMB agar will inhibit the growth of gram-positive bacteria. The gram-positive bacteria are inhibited by the dyes eosin and methylene blue present in the EMB medium. It is also differential because it distinguishes between gram-negative bacteria on whether they are able to ferment lactose.
MSA, mannitol salt agar, is a selective plate for the growth of Staphylococcus and differential for mannitol fermentation. It has a high concentration of salt. It is differential based on an organism’s ability to metabolize mannitol, a sugar. Organisms that metabolize mannitol and have acidic by-products will turn the medium yellow, as it contains phenol red, a pH indicator.
The catalase test was performed only on gram-positive bacteria, as this test would not help in differentiating the gram-negative bacteria because all of the possible unknown gram-negative bacteria were catalase positive. This test is used to detect the presence of catalase, which helps to break down toxic hydrogen peroxide produced from the transport of high-energy electrons directly to oxygen. Adding hydrogen peroxide to the culture, and looking for the production of gas bubbles test for catalase. If gas bubbles appear immediately, the culture is catalase positive. However, if no bubbles are observed, the culture is negative for catalase.
RESULTS – section was removed due to formatting problems with flowcharts
After isolation, a Gram Stain was conducted to identify one of the unknowns of gram-positive or gram-negative. Under the microscope, the bacteria stained purple and had shapes like circles concluding that one of the unknown is a gram-positive cocci. With the professor’s approval, the possible bacterium for gram-positive was staphylococcus aureus, staphylococcus epidermidis or enterococcus faecalis. After confirmation, a Nitrate test was conducted to further narrow down the bacteria possibilities. After incubation, the Nitrate test turned out negative, which differentiates the bacteria possibly be Enterococcus faecalis. To prove the statement accurate, a urea and mannitol test was conducted. The urea test, after incubation, has turned yellow, stating a negative test. The mannitol test turned yellow making the test positive. To narrow down to this single bacterium, two of each test were made for more accurate results. A catalase test was also performed because a negative result meant only for Enterococcucs faecalis. After the addition of hydrogen peroxide, the test resulted negative, which makes the unknown bacteria of the gram-positive, Enterococcus faecalis.
An EMB test was conducted from the mixed culture given by the professor to find the Gram-Negative in the unknown. After incubation, the EMB test turned metallic green proving that it is Escherichia coli. Informing the professor, the professor advises that the conclusion of the gram-negative was incorrect. Isolation from this medium was made to grow the bacteria all by itself. Performing another EMB test for better accurate results, after incubation the results were still the same. Using the isolated colony grown on the agar, two lactose tests were conducted to reduce the possible gram-negative. After incubation, the result from the lactose tests turned yellow meaning that the bacteria has fermented using either lactose or sucrose, narrowing the bacterium down to Escherichia coli, Klebsiella pneomoniae or Enterobacter aerogenes. Another EMB test was conducted for isolation of the grown colony. Since E. coli, was not one of the bacteria, 2 urea test was performed. The urea test turned pink, indicating a positive result, which meant the bacteria is possibly Klebsiella pneomoniae. Informing the professor of the results, the professor stated that this was also incorrect. Leaving the only last option of Enterobacter aerogenes, without conducting any test, this statement was also false. The professor identified that the Gram-negative bacteria is Proteus vulgaris.
Due to the lack of time, no further tests were performed to prove the Proteus vulgaris. Contamination must have happened running these tests because the results were never accurate. If time had permitted, an Indole and Simmon’s Citrate test would have been conducted to prove the bacteria of the unknown.
Enterococcus faecalis is gram-positive cocci. Its phylum classification is firmicutes and its kingdom is eubacteria. This bacterium is a non-motile facultative anaerobe. Some properties are that it ferments glucose without gas production and that is doesn’t produce catalase. This bacterium inhabits the gastrointestinal tract of humans and other mammals and in the right amount is considered normal flora. They actually give this as a main constituent of some probiotic food supplements. This bacterium, however, can cause life-threatening infections, especially in the nosocomial environment. The types of infections it can cause are local or systemic and include urinary tract infections, abdominal infections, wound infections, and endocarditis. Endocarditis is caused due to the bacteria’s surface pili, which leads to the formation of biofilms. These bacteria are usually arranged in pairs or chains and are non-encapsulated. These bacteria have a naturally high resistance to antibiotics, which is a factor in its pathogenicity. They also can live in extreme alkaline pH and high salt concentrations. Treatments for infections of this bacterium are antibiotics but only certain ones are effective due to its high resistance to antimicrobial treatments.