Nitrate Reduction Test: Principles, Significance, and Results

1. The Islamic University Of Gaza Faculty Of Science Department Of Medical Technology Nitrate Reduction Test Detected By : Fatma M. EL-Jamal Supervisor : Dr. Abdelraouf Elmanama

2. Agenda : Principle Significance Medium/Reagent Procedure Results Problems & Solutions Limitations of Test Question

3. Principle : * Bacterial species may be differentiated on the basis of their ability to reduce nitrate to nitrite or nitrogenous gases . * The biochemical pathway involved in nitrate reduction is shown .in the Figure

4. * The nitrate reduction test is based on the detection of nitrite in the medium after incubation with an organism . If nitrite present in the medium: Red color

5. Colorless: - ve nitrite two explanations for this observation: * The nitrate may not have been reduced (nitrate – ve) * The nitrate may have been reduced to nitrite which has then been completely reduced tonitrogen oxide or nitrogen (nitrite +ve)

6. * small amount ("knife point") of zinc dust is added to the incubated medium. * Metallic zinc catalyzes the reduction of NO3 to NO2 . After adding zinc dust : Red color : -ve nitrite Colorless : +ve nitrite

7. Significance : * Most G-ve bacilli +ve nitrate , and –ve nitrite * Use to identification of only few G-ve bacilli such as :Moraxella , Acinetobacter , Pseudomonas ,Vibrio , and Flavobacterium . * strains of Neisseria mucosa, Moraxella catarrhalis, and Kingella denitrificansnitrate +ve.

8. * Strains of M. catarrhalisand K. denitrificans have been misidentified as N. gonorrhoeaenitrate –ve .

9. Medium/Reagent: .* Medium: Nitrate broth * Reagents: (1)Sulfanilic acid solution (Nitrate reagent A): 0.8% in 5N acetic acid . (2)Alpha-Naphthylamine solution (Nitrate reagent B): 0.6% in 5N acetic acid . (3)Zinc powder : Store at room temperature (15C to 30C)

10. Quality Control: * Nitrate reductase +ve control: Kingella denitrificans, CDC 10,236 * Nitrate reductase -ve control: Neisseria gonorrhoeae, ATCC 43069

11. Procedure : * Inoculate a nitrate broth with the test organism * Incubate at 37C for 24hr * Add 5drops of reagent A (Sulfanilic acid) And 5drops of reagent B (naphthylamine ) *If it’s colorless add a pinch of zinc dust to the tube *Read and record results

12. Results : Color afteradding reagents Color after adding zinc N2 gas NO3 to NO2 -- (not added) none red  NO3 to N2 yes  no color  no color none NO3 to ammonia  no color  no color NO3 ( no reaction)  no color pink-red none

13. *Reaction Observed with Uninoculated Nitrate Medium.

14. *Reaction Observed with Nitrate-Negative Species

15. * Reactions Observed with Nitrate-Positive Species

16. Problems & Solutions : * False -Ve,false +Ve . * Failure to detect pink color in the uninoculated medium control tube after the addition of Zn dust may be due : • medium not containing nitrate • too much zinc dust NO3 NO2 (a false-positive ) N2

17. * uninoculated medium control Reagent A,Bpink color the media iscontaminated with nitrite * Thereaction that occurs only under anaerobic conditions, in O2 no reaction . * determine if oxygen mediuma drop of oxidase reagent Purple O2 is present Colorless O2 not present

18. Limitations of Test : *If the test is performed properly and quality control strains give appropriate results, there should be no limitations to this test. Care must be taken to ensure that all components of the test are performed properly . *No identification of genus or species can be made on the basis of the nitrate reduction test alone.

19. Question : * Is nitrate reduction an aerobic pathway or an anaerobic pathway? anaerobic *Why add zinc powder? Metallic zinc catalyzes the reduction of NO3 to NO2 When do you add it? After incubation and adding reagent A,B and give colorless

20. References : *http://www.cdc.gov/std/Gonorrhea/lab/tests/nitrate.htm *http://www.rlc.dcccd.edu/mathsci/reynolds/micro/lab_manual/nitrate.html *http://web.clark.edu/tkibota/240/Unknowns/Nitrate.htm *http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=378807