Nitrate Reduction Nitrate reduction test is used for the differentiation of members of Enterobacteriaceae on the basis of their ability to produce nitrate reductase enzyme that hydrolyze nitrate (NO3-) to nitrite (NO2) which may then again be degraded to various nitrogen products like nitrogen oxide, nitrous oxide and ammonia (NH3) depending on the enzyme system of the organism and the atmosphere in which it is growing. Nitrate (NO) I Nitrato reduktose Nitrite (NO) T Nitrito reduotas Nitric Oxide (NO) Nitric Oxide reductose Nitrous Oxide (NO) Nitrous Oxide reductose Nitrogen (N2) Requirements: 1. Media: Nitrate Broth with inverted Rutbams tube 2. Reagents: Sulpbalinis acid reagent, Alpha napthylamine reagent, zinc dust 3. Others: Inoculating loop, burner, dropper Procedure: 2 1. Inoculate nitrate broth with a heavy growth of test organism using aseptic technique. 2. Incubate at an appropriate temperature for 24 to 48 hours 3. Add one droppedfull of sulfanilic acid and one dconnecfull of a a-naphthylamine to each broth. 1. At this point, a color change to RED indicates a POSITIVE nitrate reduction test. If you get a red color, then you can stop at this point. Hitrate Broth This is a differential medium. It is used to determine if an organism is capable of reducing nitrate (NO3″) to nitrite (NO2) or other nitrogenous compounds via the action of the enzyme nitratase (also called nitrate reductase). This test is important in the identification of both Gram-positive and Gram-negative species After incubation, these tubes are first inspected for the presence of gas in the Durham tube. In the case of nonformenters, this is indicative of reduction of nitrate to nitrogen gas. However, in many cases gas is produced by fermentation and further testing is necessary to determine if reduction of nitrate has occurred. This further testing includes the addition of sulfanilic acid (often called nitrate I) and dimethyl-alpha-napthalamine (nitrate II). If nitrite is present in the media, then it will react with nitrate 1 and nitrate Il to form a red compound. This is considered a positive result. If no red color forms upon addition of nitrate I and II, this indicates that either the NO, has not been converted to NO2 (a negative result), or that NO, was converted to NO, and then immediately reduced to some other, undetectable form of nitrogen (also a positive result). In order to determine which of the preceding is the case, elemental zinc is added to the broth. Zinc will convert any remaining NO, to NO2 thus allowing nitrate and nitrate il to react with the NO2- and form the red pigment (a verified negative result). If no color change occurs upon addition of zinc then this means that the NO;’ was converted to NO, and then was converted to some other undetectable form of nitrogen (a positive result) of the nitrate broth turns red (tubes pictured in the center) after nitrate I and nitrate il are added, this color indicates a positive result. I instead, the tube turns red (tube pictured on the left) after the addition of Zn, this indicates a negative result. If there is no color change in the tube after the addition of nitrate I and nitrate II, the result is uncertain. If the tube is colorless (picture on the right) after the addition of Zn this indicates a positive test. Nitrate 1. What is the enzyme we are testing for? I 2. What is the inverted tube called? 3. What do you add if the initial result is negative?

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