2. General Microbiology Laboratory Biochemical Tests

3. Mohammed Laqqan NITRATE REDUCTION TEST

4. 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 as illustrated in figure1.  Some of these bacteria possess the enzymes to further reduce the nitrite to either the ammonium ion or molecular nitrogen as also illustrated in figure 1.

5. Mohammed Laqqan

6.  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 a pink or red color.  If a culture does not produce a color change, several possibilities exist: 1)the bacteria possess nitrate reductase and also reduce nitrite further to ammonia or molecular nitrogen; 2)they possess other enzymes that reduce nitrite to ammonia; 3)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, a pink or 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).

7. Mohammed Laqqan  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.

8. 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.

9. Mohammed LaqqanProcedures  How to Perform Test: 1)Inoculate a nitrate broth with the test organism. 2)Incubate at 37C for 24 hr. 3)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 )

10. Mohammed LaqqanResult 1)Observe the tubes for the presence of growth, and the absence of growth in the control tube. 2)Add 0.5 ml of nitrate test reagent A and 0.5 ml of test reagent B to each of the culture tubes and mix. (Alternatively, about 5 to 10 drops of each reagent works well.) A distinct pink or red color indicates a positive test, provided the uninoculated control medium is negative. 3)Negative tests should be confirmed by adding several grains of zinc powder and gently shaking the tube. If nitrate is present in the medium, it will turn red within 5 to 10 minutes; if it is absent, there will be no color change.

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13. Nitrate

14. Mohammed Laqqan REACTIONN2 gasColor after adding reagents Color after adding zinc NO3 to NO2 nonered-- (not added) NO3 to N2yesno color NO3 to ammonia noneno color NO3 - no reaction noneno colorpink-red

15. 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 calcoaceticus.  Neisseria and related species

16. Mohammed Laqqan End of lecture