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Nitrate Reduction Test
Mohammed Laqqan
Nitrate reduction test
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Chemolithoautotrophic bacteria (bacteria that obtain energy
through chemical oxidation; they use inorganic compounds as
electron donors and CO2 as their primary source),
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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
•
•
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 red color.
If a culture does not produce a color change, several possibilities exist:
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the bacteria possess nitrate reductase and also reduce nitrite further to
ammonia or molecular nitrogen;
•
they possess other enzymes that reduce nitrite to ammonia;
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nitrates were not reduced by the bacteria.
To determine if nitrates were reduced past nitrite:
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a small amount of zinc powder is added to the culture containing the
reagents.
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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).
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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.
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.
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Procedures
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1.
2.
3.
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 )
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Result
Mohammed Laqqan
Mohammed Laqqan
Mohammed Laqqan
Mohammed Laqqan
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
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):
aeruginosa. Negative (pink): Acinetobacter.
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Pseudomonas
The End
Mohammed Laqqan