April 25th, 2006
The purpose of this lab was to identify two unknown bacteria cultures using various differential tests. The identification of these unknown cultures was accomplished by separating and differentiating possible bacteria based on specific biochemical characteristics. Whether the tests performed identified specific enzymatic reactions or metabolic pathways, each was used in a way to help recognize those specifics and identify the unknown cultures. The differential tests used to identify the unknown cultures were oxidase, catalase, lactose and sucrose fermentation, Kugler/iron agar, nitrate reduction, gelatin hydrolysis, starch hydrolysis, manitol salt, MR-VP, citrate, bile esculin, indole, urease, DNase, and coagulase.
Material & Methods
The tests performed on the unknown bacteria cultures were all used to determine the identity of the bacteria. Each of the tests performed provided some key information about the bacteria in question and how it functions. Not all of the tests were performed on every culture, however, as some of the tests were used only for gram (+) or gram () bacteria, while others were even more specific and used only for cocci bacteria. The tests performed and what constitutes a positive and negative test are as follows.
The oxidase test was performed only on gram (-) bacteria and was used to test for the presence of cytochrome oxidase. Living bacteria were placed on a paper towel and saturated with a chromogenic reducing agent. Within seconds the reagent, acting as an artificial electron acceptor, will turn purple if oxidized cytochrome oxidase is present, indicating a positive test. If no color change is observed, no cytochrome oxidase is present and the test is negative.
The catalase test was performed only on gram (+) bacteria, as this test would not help in differentiating the gram (-) bacteria because all of the possible unknown gram (-) bacteria were catalase positive. This test is used to detect the presence of catalase, which helps to breakdown toxic hydrogen peroxide produced from the transport of high-energy electrons directly to oxygen. Catalase is tested for by adding hydrogen peroxide to the culture, and looking for the production of gas bubbles. If gas bubbles appear immediately, the culture is catalase positive. However, if no bubbles are observed, the culture is negative for catalase.
Lactose and sucrose sugar fermentation were tested using a broth containing the respective sugar compound, 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 originally 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 these sugars 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 would contain a bubble about 2/3 it's size.
The iron agar slant was used to test for the fermentation of glucose and lactose, as well as the production of H2S. Gas production was also monitored, looking for fissures produced by production of gas during fermentation. The conversion of the originally red slant and butt of the agar to yellow indicates that glucose fermentation took place. H2S could have been produced by the reduction of thiosulfate contained in the media. Production of this compound will cause a reaction to occur with ferrous sulfate and will result in the production of a black precipitate. Thus, the culture is positive for H2S production if a black precipitate is seen and negative if one is not...