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Escherichia coli bacterium is a facultative anaerobe that is gram negative microbe. This means that a cell wall is made up of a peptidoglycan layer as opposed to phospholid bi-layer found in gram positive bacteria. Again, the bacterium rarely requires presence of oxygen for its existence. However, it performs exemplary well in the presence of oxygen as it does in its absence. The bacterium is in most cases abbreviated as E. coli. The bacterium is rod-shaped and hence can be classified as Gram-negative bacillus. The word “bacillus” is usually a name given to any rod shaped bacterium whether positive or negative.
E. coli bacterium morphogenesis is in a cylindrical form and contains hemispherical caps. Furthermore, the cell is covered in fimbriae, which are tail-like projections that protrude from cell membrane and are meant to assist the bacterium to propel itself through a medium. Moreover, E. coli bacterium has got no nucleus and thus prokaryotic and its DNA is a continuous chromosome that is ring shaped. The bacterium is commonly found in the large intestine of warm blooded animals and performs well at a pH of about 7-8 and a 37o C body temperature. Unlike other disease causing bacteria, Escherichia coli can lead to infections even with small amounts of ingestion.
The bacterium can be contracted through drinking contaminated water or eating of already contaminated foods such as unpasteurized milk, ground beef where E. coli in the intestines gets to contact with the meat (Ishii S). An E. coli infections cause diarrhea as it damages the small intestine’s lining. Persistent infection or diarrhea can get worse causing a bloody diarrhea. The US Center for Disease Control & Prevention estimates that about 265,000 “Shiga toxin-producing” E. coli infections occur every year in the country (“E Coli Outbreaks”). The global infections are relatively high standing at around 2,801,000 cases annually (Leboffe M.J.). The main aim of performing these tests is to establish the unknown microbe that acts as a contributory agent to diarrhea and thus establishes a proper mechanism to treat the disease.
Materials and Methods Used
To perform the experiment, certain important equipments and chemicals were to be present. These chemicals included methyl red, Kovac’s reagent, oxidase-test reagents and ampoule of both Barritt’s A and B. Again, certain protective clothing, including lab coat, close-toed shoes, safety goggles and gloves, were required to ensure the safety of the individual. Inoculating loop was also in place to help move living bacteria and in some cases, such as testing for motility, an inoculating needle was brought to use. However, the equipment used to handle the bacteria should be allowed to cool down to avoid killing the bacteria.
The first step was to culture the ‘unknown J’ bacteria in Trypticase Soy Agar (TSA) plate through the performance of T-streak, thus producing isolated colonies of the bacterium from a mixture of cultures. T-streak procedure was done by picking a sample using sterilized loop, and then dragging it to and fro across agar’s surface beginning from the periphery, while ensuring that the loop is well flamed and sterilized after completion. Next, the streak plate would be incubated at a relatively hot room temperature for about 24 hours (Fotadar U). The singled colonies would be visible after the incubation period. Each test was important and had different incubation period as some required no incubation while others required up to seven days of incubation.
Aseptic technique was put to use any time the medium was sterilized with the unknown gram negative bacterium. This process was meant to avoid contamination of the culture or the surrounding. Every time inoculation was to be done, the instruments used had to be sterilized using non-luminous flame of the Bunsen burner. After carrying out all the required tests, the table was well cleaned using disinfectors to ensure that the environment was left uncontaminated in any way.
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The gram stain experiment showed pink rods that indicated that the bacteria that existed were gram negative bacilli. The bacteria were then grown under TSA and other test were performed. Oxidase test showed the paper color turn from purple to black which was a positive results indicating a number of bacterium including Escherichia coli. BCP lactose test showed color change from yellow to purple, another positive result that the bacteria caused fermentation of carbohydrates. Indole test conducted was positive forming a red ring on top of the broth. This depicted the ability of the bacteria to separate indole from tryptophane. Further, a citrate test was performed at a temperature of 35o C, causing the color of citrate to change from green to blue. Finally, a methyl red test carried out again at 35o C was negative indicated by the change of color from light yellow to dark yellow (Fotadar U).
After a number of depicting small disparities in test performance, it was evident that the ‘unknown J’ was Escherichia coli. The Gram stain had showed that the unknown was a gram negative rod. All tests were properly performed and depicted clear results on most occasions. However, in the case of indole test, there were challenges at first after it showed false results at the beginning. The mistake was realized after a clear inconsistency with the other results relating to tests performed earlier. There was a suggestion that the test be re-done to ensure clarity of the results. The repeated process gave positive results that were now consistent with the rest of the proved information about the unknown. Using the clear results from all the performed tests, a conclusion was made that the said unknown microbe J was Escherichia coli.
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