VLD 421: Veterinary Clinical Biochemistry and Laboratory Diagnosis-II (0+1)

NITRATE REDUCTION TEST

Principle

• The ability of an organism to reduce nitrates to nitrites is an important characteristic used in the identification and species differentiation of many microorganisms.
• Organisms demonstrating nitrates reduction have the ability of extracting oxygen from nitrates to form nitrites and other reduction products.
• The presence of nitrites in the test medium is detected by the addition of sulfanilic acid and alpha naphthalamine . The sulfanilic acid and nitrite react to form a  diazonium salt. The diazonium salt then couples with the alpha - naphthalamine to produce a red, water soluble azo dye. 
• The development of a red colour within 30 seconds after adding the test reagents indicates the presence of nitrites and represents a positive reaction for nitrate reduction.
• If no colour develops after adding the test reagents, this may indicate either nitrates, have not been reduced (a true negative reaction) or that they have been reduced to products of nitrates such as ammonia, molecular nitrogen (N₂), nitric oxide (NO) or nitrous oxide (N₂O) and hydroxylamine. Because the test reagents detect only nitrites, the latter process would lead to a false negative reading.
• Thus it is necessary to add a small quantity of zinc dust to all negative reactions. Zinc ions reduce nitrates to nitrites and the development of a red colour after adding zinc dust indicates the presence of residual nitrates and confirms a true negative reaction.

Methodology

• Inoculate the Nitrate broth with test organism and incubate it at 37^ºC for 48 hours.
• At the end of incubation add 1 ml of each of sulfanilic acid and alpha naphthalamine reagent  to the tube.
• In case of negative reaction further confirm by adding Zinc dust.

Result

• Development of a red colour within 30 seconds indicates a positive reaction.

Nitrate reduction test

 

Controls

• Positive    : Escherichiacoli
• Negative  : Acinetobacter spp.