Nitrate Reduction Test Mohammed Laqqan Mohammed Laqqan

Nitrate reduction test Chemolithoautotrophic bacteria (bacteria that obtain energy through chemical oxidation; they use inorganic compounds as electron donors and CO2 as their primary source), and many chemoorganoheterotrophs (bacteria that require organic compounds for growth; the organic compounds serve as sources of carbon and energy) can use nitrate (NO–3) as a terminal electron acceptor during anaerobic respiration. In this process, nitrate is reduced to nitrite (NO–2) by nitrate reductase. Some of these bacteria possess the enzymes to further reduce the nitrite to either the ammonium ion or molecular nitrogen as illustrated in figure 1. Mohammed Laqqan Mohammed Laqqan

Mohammed Laqqan Mohammed Laqqan

they possess other enzymes that reduce nitrite to ammonia; Organisms that possess the enzyme, nitrate reductase reduces nitrate to nitrite. The nitrite ions are detected by the addition of Sulfanilic acid and N,N-dimethyl-1-naphthylamine to the culture. Any nitrite in the medium will react with these reagents to produce red color. If a culture does not produce a color change, several possibilities exist: the bacteria possess nitrate reductase and also reduce nitrite further to ammonia or molecular nitrogen; they possess other enzymes that reduce nitrite to ammonia; nitrates were not reduced by the bacteria. To determine if nitrates were reduced past nitrite: a small amount of zinc powder is added to the culture containing the reagents. Since zinc reduces nitrates to nitrites, red color will appear and verifies the fact that nitrates were not reduced to nitrites by the bacteria (nitrate unreacted). If a red color does not appear, the nitrates in the medium were reduced past the nitrite stage to either ammonia or nitrogen gas (nitrate reacted). Mohammed Laqqan Mohammed Laqqan

Escherichia coli can reduce nitrate only to nitrite. Three different bacteria that give three different nitrate reduction results will be learned. Staphylococcus epidermidis is unable to use nitrate as a terminal electron acceptor; therefore, it cannot reduce nitrate. Escherichia coli can reduce nitrate only to nitrite. Pseudomonas fluorescens are characterized by excretion of diffusible yellow-green pigments that fluoresce in ultraviolet light) often reduces nitrate completely to molecular nitrogen. Mohammed Laqqan Mohammed Laqqan

Safety consideration Since N, N-dimethyl-1-naphthylamine might be carcinogenic (nitrite test reagent B), wear disposable gloves and avoid skin contact or aerosols. The acids in nitrite test reagent A are caustic. Avoid skin contact and do not breathe the vapors. Be careful when working with zinc. Do not inhale or allow contact with skin. No mouth pipetting. Mohammed Laqqan Mohammed Laqqan

Procedures How to Perform Test: Inoculate a nitrate broth with the test organism. Incubate at 37C for 24 hr. Add 5 drops of reagent A (Sulfanic acid) and 5 drops of reagent B (naphthylamine ) to the broth Property it tests for: The ability of some bacteria to reduce nitrate can be used in their identification and isolation. For example, E. coli can reduce nitrate only to nitrite, P. fluorescens reduces it completely to molecular nitrogen, and S. epidermidis is unable to use nitrate as a terminal electron acceptor. Media and Reagents: Nitrate broth containing 0.5% potassium nitrate (KNO3) Reagent A (Sulfanic acid) and reagent B (naphthylamine ) Mohammed Laqqan Mohammed Laqqan

Result Mohammed Laqqan Mohammed Laqqan

Mohammed Laqqan Mohammed Laqqan

Mohammed Laqqan Mohammed Laqqan

Mohammed Laqqan Mohammed Laqqan

Color after adding reagents Color after adding zinc NO3 to NO2 none REACTION N2 gas Color after adding reagents Color after adding zinc NO3 to NO2  none red -- (not added) NO3 to N2 yes no color NO3 to ammonia NO3 - no reaction pink-red Mohammed Laqqan Mohammed Laqqan

Significance of Nitrate Reduction test Most enteric bacteria are nitrate reducers. Pathogenic examples include Escherichia coli (opportunistic urinary tract infections), Klebsiella pneumoniae (bacterial pneumonia), Morganella morganii and Proteus mirabilis (nosocomial infections). Nonenteric nitrogen reducing pathogens include Staphylococcus aureus (staphylococcal food poisoning, bacteremia, various abscesses) and Bacillus anthracis (anthrax). Positive complete (full reduction— clear): Pseudomonas aeruginosa. Negative (pink): Acinetobacter. Mohammed Laqqan Mohammed Laqqan

The End Mohammed Laqqan Mohammed Laqqan