Spirit Blue Agar (Fat Hydrolysis test): The testing use of the agar helps determine if the unknown organism has a lipid enzyme called lipase. The enzyme, in return, causes a dark blue precipitate to form, making it positive for lipase. Spirit blue is a pH indicator used for detecting lipase. Create a heavy, loop-streak in the middle of the plate. The positive control used is Staphylococcus aureus. Refer to Brown, Microbiological Applications p.255.
Fermentation (Sugar): The fermentation process allows determining the unknown organism to ferment several sugars. This is determining using Glucose, Lactose, Sucrose, Mannitol, and Trehalose broths. Reading the broths take place first, at 24 hour of incubation, and second, at 48 hours of incubation.…show more content… The chemical reaction that occurs for nitrate as follows: (NO3- + 2e- + 2H+) Nitrate reductase (NO2- + H2O). The process in which the unknown organism uses is called nitrate respiration. It acts as a terminal electron acceptor in anaerobic respiration. The NO3- can be reduced to a gaseous product of N2O or N2, which is said to be denitrification. The reagents used to test for nitrate reductase are Sulfanilic acid (reagent A) and dimethyl α–naphthylamine (reagent B). If a sudden, dark red color occurs after adding 5 drops of each reagent into the media indicates a positive for nitrate reductase. However, if no sudden change of color occurs, it indicates a negative for nitrate reductase. The explanation for this occurring is one of two variable factors. The first variable factor, nitrate was not reduced and the second variable factor was some form of reduction occurred via denitrification (e.g. Ammonia, hydroxylamine). When this occurs, an addition reagent, Zinc powder, is used to check for possible nitrate reductase. If a dark red appears after zinc powder is added into the media, then nitrate reductase occurred and is a negative result. Shake the media well after adding reagents (A & B) and again if zinc must be added. The positive control used is Escherichia coli. Refer to Brown, Microbiological Applications
