Bladder infections are usually caused by gram-negative bacteria. These bacteria reduce nitrates derived from food to nitrites when urine remains in the bladder three to four hours, sufficient time for this reaction to occur.

Nitrite is not present in urine under normal circumstances. When present, nitrites react with p-arsanilic acid to produce a diazonium compound. The diazonium compound in turn couples with 3-hydroxy-1,2,3,4 tetrahydrobenzo-(h)-quinolin to produce a pink color.

A first morning, clean, voided midstream specimen is optimal for detecting nitrites in urine.