Overview:
Asparagine Broth is a minimal, chemically defined liquid medium used for the detection of nitrate reduction and denitrification ability in Mycobacterium species, particularly Mycobacterium tuberculosis and non-tuberculous mycobacteria (NTM). It is a key component of the classical Wayne method and is still widely used in reference laboratories for accurate identification of M. tuberculosis complex (negative reaction) versus most NTM (positive reaction). Asparagine serves as the sole carbon and nitrogen source, and nitrate reduction produces nitrite, which is detected by a red colour after adding sulfanilic acid and ?-naphthylamine reagents.
Applications:
?? Differentiation of Mycobacterium tuberculosis complex (negative) from NTM (usually positive)
?? Detection of nitrate reductase activity in mycobacteria
?? Part of the standard phenotypic identification panel (along with niacin, catalase, etc.)
?? Reference method in WHO, CLSI M24-A2, and national TB laboratory guidelines
Key Features:
?? Chemically defined � no interference from complex nutrients
?? Clear, colourless medium ? easy detection of red colour in positive reactions
?? Highly reproducible and specific for mycobacterial nitrate reductase
?? Essential test for confirming M. tuberculosis in culture
Composition (per liter):
L-Asparagine (anhydrous)   � 5.0 g
KH?PO?   � 1.0 g
Na?HPO?     � 2.5 g
MgSO?�7H?O     � 0.01 g
Sodium Citrate    � 0.1 g
Glycerol   � 5.0 mL
KNO?   � 1.0 g (nitrate source)
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 necessary.
Dispense 3�4 mL into small screw-cap tubes (e.g., 13 � 100 mm).
Autoclave at 121�C for 10 minutes only (do not over-sterilize).
Cool to room temperature. Tubes should remain clear and colourless.
Inoculation & Incubation:
Inoculate heavily with fresh growth from LJ or Middlebrook 7H11 (1�2 loopfuls).
Incubate at 36 � 1�C for up to 14 days (read at 3, 7, and 14 days).
Include an uninoculated control tube.
Reading (after incubation):
Add 0.5 mL Reagent A (0.2 % sulfanilic acid in 5 N acetic acid).
Add 0.5 mL Reagent B (0.1 % ?-naphthylamine in 5 N acetic acid).
Positive: red colour within 5�10 minutes (nitrite present)
Negative: no colour (M. tuberculosis complex)
If no colour, add a pinch of zinc dust: red colour now = true negative; no colour = false negative (nitrate reduced beyond nitrite).
Storage:
Prepared tubes: 2�8�C, protected from light, use within 6 months.
For laboratory use only. Reference method for mycobacterial identification.