Transcript Nitrate reduction test

General Microbiology Laboratory
Biochemical Tests
NITRATE REDUCTION TEST
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.
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Organisms that possess the enzyme, nitrate reductase reduces nitrate to
nitrite.
The nitrite ions are detected by the addition of Sulfanilic acid and N,Ndimethyl-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:
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, 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).
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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.
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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.
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Procedures
 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 )
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Result
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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Nitrate
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REACTION
N2 gas
Color after
adding reagents
Color after adding zinc
NO3 to NO2
none
red
-- (not added)
NO3 to N2
yes
no color
no color
NO3 to ammonia
none
no color
no color
NO3 - no reaction
none
no color
pink-red
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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
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End of lecture
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