Intended Use
Nitrate Broth is recommended as an aid in the identification of aerobic and facultative anaerobic gram-negative microorganisms by means of the nitrate reduction test.

Summary and Explanation
Microorganisms may be differentiated according to their metabolism of certain substrates. The ability to reduce nitrate to nitrite is characteristic of the family Enterobacteriaceae.
Nonfermenters and other miscellaneous gram-negative bacilli Vvry in their ability to reduce nitrates. Some members of this group are capable of denitrification, which is a reduction
of nitrate to nitrogen gas. The production of gas from nitrate is an important differential test for glucose-nonfermenting gram-negative bacilli. The end product of reduction dépends upon the bacterial species.

Nitrate Broth is a basal medium containing potassium nitrate.
The microorganism under evaluation is inoculated into the medium and after incubation, nitrate reduction may be determined. An inverted Durham fermentation tube in the prepared tubed medium serves to trap nitrogen gas produced through denitrification. The medium is evaluated for nitrate reduction by the addition of two reagents, Nitrate A Reagent (0.8% sulfanilic acid in 5N acetic acid) and Nitrate B Reagent (0.6% N, N-dimethyl-alpha-naphthylamine in 5N acetic acid), which detect the presence of a catabolic end product, and by the addition of Nitrate C Reagent, zinc dust, which detects the absence of remaining nitrate in the medium.

Identity Specifications
Difco™ Nitrate Broth
Dehydrated Appearance: Light to medium tan, free-flowing, homogeneous.
Solution: 0.9% solution, soluble in purified water. Solution is light to medium amber, clear.
Prepared Appearance: Light to medium amber, clear.
Reaction of 0.9% Solution at 25°C: pH 7.0 ± 0.2

Principles of the Procedure
Reduction of nitrate is generally an anaerobic respiration in which an organism derives its oxygen from nitrate. Depending upon environmental conditions, the end products of this metabolic process are usually not further oxidized or assimilated into cellular metabolism, but are excreted into the surrounding medium. Enterobacteriaceae characteristically reduce nitrate to nitrite which reacts with sulfanilic acid and N, N-dimethylalphanaphthylamine to produce a red color (Griess reaction).
The formation of other end products (ammonia, nitrous oxide, hydroxylamine, etc.) is also possible; therefore, the addition of zinc dust is used to detect unreduced nitrate. The
formation of nitrogen gas, an end product typical of certain organisms, is evidenced by displacement of the medium from the Durham tube by the gas produced.

Formula
Difco™ Nitrate Broth
Approximate Formula* Per Liter
Beef Extract : 3.0 g
Peptone : 4.0 g
Proteose Peptone No. 3 : 1.0 g
Potassium Nitrate : 1.0 g
*Adjusted and/or supplemented as required to meet performance criteria.