The purpose of this lab was to identify unknown bacteria cultures using various differential tests, and my unknown bacteria is #17. 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 Gram stain, Catalase, Mannitol Salt Agar (MSA), Blood Agar, Novobiocin, Coagulase, and DNAse (Alachi, 2007).
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.
Gram staining was used to determine whether the cells were gram positive or gram negative based on the color they appear at the end. The Gram stain utilizes four basic steps: apply a primary stain (crystal violet), fix it with Gram’s iodine (which fixes the primary dye inside the cell), decolorize with 95% ethyl alcohol to wash out the crystal violet-iodine (CVI) complex, and apply a counterstain, Safarin. Gram positive cells have a thick peptidoglycan layer that is external and has a higher degree of cross-thinking that traps the CVI complex better than gram negative cells. Therefore, they are less susceptible to decolorization by alcohol making them appear purple in color. Gram negative cells have a thinner peptidoglycan layer and a lipid membrane external to the cell wall which makes them susceptible to decolorization thus allowing them to be counterstained with Safarin making them appear red in color (Alachi, 2007).
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 (Alachi, 2007).
Manitol Salt Agar (MSA) is both a selective and differential media used for gram (+) cocci. A selective medium contains components that inhibits the growth of undesirable organisms and favors the desirable one. On the other hand, a differential medium seeks to show differences between different organisms growing in a medium. MSA 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. Therefore, staphylococci that metabolize mannitol (Staphylococcus aureus) turn the medium color yellow while those that do not ferment mannitol (Staphylococcus epidermidis) do not produce acid and the color indicator remains red. In some cases, as with Staphylococcus saprophyticus, MSA color may be redder due to the production of alkaline amines as by-products of protein degradation (Alachi, 2007). Blood Agar is a differential enriched-type medium that contains general nutrients and 5% sheep blood. It is useful for cultivating fastidious organisms and for determining the hemolytic capabilities of an organism. Some bacteria produce exoenzymes that lyse red...
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