Indole Nitrate Broth

Principle And Interpretation

Indole Nitrate Medium (Tryptone Nitrate Medium), due to the nutritive content, supports growth of aerobes, microaerophiles, and facultative as well as obligate anaerobes. It serves a dual purpose of detecting indole production and nitrate reduction in a wide range of microorganisms. Casein enzymic hydrolysate contains tryptophan, which is acted upon by certain microorganisms, resulting in the production of indole. Potassium nitrate acts as the substrate for determining nitrate reduction by microorganisms. Duplicate tubes of Indole Nitrate Medium may be inoculated and tested for the presence of nitrates or indole after incubation for various lengths of time. Nitrate test is performed by addition of 0.5 ml each of Sulphanilic Acid (R015) and alpha -Naphthylamine (R009). The development of pink colour indicates nitrate reduction. The colour develops due to presence of nitrite generated from reduction of nitrate. When nitrate is further reduced to ammonia, no colour develops. Add a pinch of zinc dust to the tube. The formation of pink colour after addition of zinc dust indicates that nitrate is not reduced. Indole production can be tested by the addition of Kovacs Reagent (R008) or Ehrlich reagent (R005) (1,2). The formation of a deep red colour in the reagent layer after gentle agitation indicates positive indole test. Indole Nitrate Medium is not recommended for indole test in coliform and other enteric bacteria, as they reduce nitrate to nitrite, which prevents the detection of indole (3). Indole Nitrite Medium should not be used for detecting indole production by members of the Enterobacteriaceae. The tubed medium should be boiled for 2 minutes and cooled, without agitation, before use.