The Islamic University Of GazaFaculty Of ScienceDepartment Of Medical Technology

Detected By: Fatma M. EL-JamalSupervisor:

Dr. Abdelraouf Elmanama

PrinciplePrinciple SignificanceSignificanceMedium/ReagentMedium/ReagentProcedureProcedureResultsResults

Limitations of TestLimitations of Test QuestionQuestion

*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

*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

Colorless: - ve nitritetwo explanations for this observation:

*The nitrate may not have been reduced (nitrate – ve)

*The nitrate may have beenreduced to nitrite which hasthen been completely reduced to nitrogen oxide or nitrogen

(nitrite +ve )

After adding zinc dust:

*small amount ("knife point") of zinc dust is added to the incubated medium .

Red color : -ve nitriteColorless : +ve nitrite

* Metallic zinc catalyzes the reduction of NO3 to NO2 .

* 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 denitrificans nitrate +ve.

*Strains of M. catarrhalis and K. denitrificans have been misidentified as N.

gonorrhoeae nitrate –ve.

. *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 roomtemperature (15C to 30C)

*Nitrate reductase +ve control: Kingella denitrificans, CDC 10,236

*Nitrate reductase -ve control: Neisseria gonorrhoeae, ATCC 43069

*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

REACTION N2 gas Color after adding reagents

Color afteradding zinc

NO3 to NO2

 NO3 to N2

 NO3 to ammonia

NO3 ( no reaction)

none

none

none

yes

 no color

 no color

 no color

red

pink-red

 no color

 no color

( --not added)

* False -Ve, false +Ve .* Failure to detect pink color in the

uninoculated medium control tube after the addition of Zn dust may be due :

(1) medium not containing nitrate

(2)too much zinc dust NO3 NO2 (a false-positive )

N2

* uninoculated medium control Reagent A,B pink color

the media is contaminated with nitrite

* The reaction that occurs only under anaerobic conditions , in O2 no reaction .

* determine if oxygen medium a drop of oxidase reagent Purple O2 is present

Colorless O2 not present

*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.

*Is nitrate reduction an aerobic pathway or an anaerobic pathway ?

*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

anaerobic

*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