Nitrate Broth

Technique

Inoculate 2-3 tubes of broth with one loop of pure culture and incubate at 37 ± 1 °C, reading after 18-24 h until 72 h. Reading is performed after the incubation (18-24 h), adding a few drops of each reagent (Nitrate A and B) or, better, the mixture of both in equal parts. Cherry red is taken as a positive signal. Absence of colour is a presumptive signal of negative. Verification is carried out adding a little bit of zinc powder: if a red colour appears, test is negative, otherwise (there is no colour change) test is positive.

Instability of red colour, that easily and quickly becomes brown red, is due to nitrite excess, and it has no other effect on the test.

Negative cultures after 24 hours must be repeated after 48 and 72 hours in other tubes before discarding them as negative.

Gas production in non-fermentative strains is usually a signal of denitrification. With fermentative strains it may be just CO₂. Anyway, it is advisable to inoculate with Durham tubes.

Nitrate reduction assays must always be performed with control tubes without inocula, since the pollution of the material with nitrous oxide or nitrite is enough to provide false positive reactions.

An inverted Durham fermentation tube in the prepared tube medium serves to trap nitrogen gas produced through denitrification.

It is possible that air bubbles are generated in the Durham tube during the transport. If air bubbles are present in the Durham tube prior to inoculation, the tube should be inverted until the air is released from the Durham tube. Failure to remove air bubbles prior to inoculation may result in reading the result as a false-positive reaction in gas production.

Physical/chemical control

Microbiological control

Inoculate a pure culture.

Aerobiosis. Incubation at 35 ± 2 °C, reading after 18-24 hours.

Sterility control

Incubation 48 hours at 30-35 °C and 48 hours at 20-25 °C: NO GROWTH.

Check at 7 days after incubation in same conditions.

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