Asparagine Broth

Asparagine Broth

Chemically Defined Medium for Mycobacterial Nitrate Reduction Testing
Catalog Number: AS-1125

Overview

Asparagine Broth is a minimal, chemically defined liquid medium used for the detection of nitrate reduction and denitrification activity in Mycobacterium species, including members of the Mycobacterium tuberculosis complex and non-tuberculous mycobacteria (NTM).

The medium is a key component of the classical Wayne method and remains in use in reference and research laboratories for phenotypic differentiation of M. tuberculosis complex (typically nitrate-negative) from most NTM (typically nitrate-positive). L-asparagine serves as the sole carbon and nitrogen source, ensuring high specificity and reproducibility.

Nitrate reduction results in the formation of nitrite, which is detected by the development of a red colour following the addition of sulfanilic acid and α-naphthylamine reagents.

Applications

• Differentiation of Mycobacterium tuberculosis complex (negative) from non-tuberculous mycobacteria (usually positive)

• Detection of nitrate reductase activity in mycobacteria

• Component of standard phenotypic identification panels (with niacin, catalase, etc.)

• Reference method described in WHO, CLSI M24-A2, and national tuberculosis laboratory guidelines

Key Features & Benefits

• Chemically defined formulation with no complex nutrients

• Clear, colourless medium allows easy visual detection of positive reactions

• High specificity for mycobacterial nitrate reductase activity

• Long-established and well-validated reference method

Principle of the Medium

Mycobacteria capable of nitrate reduction convert nitrate to nitrite during incubation. Following incubation, nitrite is detected by the addition of sulfanilic acid and α-naphthylamine, producing a red colour. Organisms lacking nitrate reductase activity do not produce nitrite, and no colour develops unless nitrate is reduced beyond nitrite.

Composition (per litre)

• L-Asparagine (anhydrous) – 5.0 g

• Potassium dihydrogen phosphate (KH₂PO₄) – 1.0 g

• Disodium hydrogen phosphate (Na₂HPO₄) – 2.5 g

• Magnesium sulfate heptahydrate (MgSO₄·7H₂O) – 0.01 g

• Sodium citrate – 0.1 g

• Glycerol – 5.0 mL

• Potassium nitrate (KNO₃) – 1.0 g

Final pH: 6.6 ± 0.2 at 25 °C

Preparation

• Dissolve all components in 995 mL distilled water

• Adjust pH to 6.6 with 1 N NaOH or 1 N HCl if required

• Make up to 1 L with distilled water

• Dispense 3–4 mL into small screw-cap tubes (e.g. 13 × 100 mm)

• Sterilise by autoclaving at 121 °C for 10 minutes only
  (do not over-sterilise)

• Cool to room temperature

• Prepared medium should remain clear and colourless

Inoculation & Incubation

• Inoculate heavily with fresh growth from Löwenstein–Jensen or Middlebrook 7H11 agar (1–2 loopfuls)

• Incubate at 36 ± 1 °C

• Examine at 3, 7, and 14 days

• Include an uninoculated control tube

Reading & Interpretation

After incubation:

1. Add 0.5 mL Reagent A
   (0.2 % sulfanilic acid in 5 N acetic acid)

2. Add 0.5 mL Reagent B
   (0.1 % α-naphthylamine in 5 N acetic acid)

Results:

• Red colour within 5–10 minutes: Positive (nitrite present)

• No colour: Negative

If no colour develops, add a small amount of zinc dust:

• Red colour after zinc: true negative

• No colour after zinc: false negative (nitrate reduced beyond nitrite)

Storage & Stability

• Prepared tubes: Store at 2–8 °C, protected from light

• Shelf life: Use within 6 months

Intended Use

For laboratory identification and research use only.
Not for direct clinical diagnostic use.

Quality & Compliance

• Classical reference medium for mycobacterial nitrate reduction testing

• Manufactured under controlled conditions for batch consistency

• Suitable for research, reference, and method-development laboratories

Customs & Trade Information

HS / AHECC Code: 3821.00.00
Prepared culture media for development or maintenance of microorganisms