Unknown Lab Report

Unknown Lab Report

April 25th, 2006

Introduction

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 observed.

Nitrate reduction was tested for by inoculating a nitrate broth with the unknown cultures, and allowing growth to take place. An inverted Durham tube was used again to test for the production of any gas. Adding 10 drops of both sulfanilic acid and б-napththylamine to the medium if 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 to 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. 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.

A gelatinase test was performed using nutrient gelatin to determine the ability of the bacteria to produce gelatinases. The gelatin was inoculated with the unknown cultures and incubated. The tubes were then placed on ice to reform the warm gelatin. If the gelatin remained solid after inoculation, the bacteria did not produce the gelatinase enzymes to liquefy the agar, constituting a negative test. However, if the gelatin was liquefied, the enzymes were secreted and the bacteria were positive.

A starch test was performed by plating the unknown cultures on starch agar to see if the bacteria could hydrolyze the starch in the medium. Iodine was the dropped on the cultures to react with the starch and form a dark brown color. The observance of a yellow halo around the culture indicates that the starch was hydrolyzed and the bacteria were positive for starch hydrolysis. No halo constitutes a negative test.

Manitol salt agar is both a selective and differential media used for gram (+) cocci. It is selective for salt tolerance and differential for maintol sugar fermentation. It also contains phenol red, which acts as a pH indicator, turning yellow under acidic conditions. The agar is most often used for the selection of S. auerus. Growth and a yellow color change are positive