|Pyrazinamidase (Agar) Test
Pyrazinamidase is an enzyme that allows certain bacteria to resist pyrazinamide's activity by converting pyrazinamide to ammonia and
pyrazinoic acid. This attribute can be used to differentiate between the species, i.e. pyrazinamide is an anti-tuberculosis drug effective
on strains of Mycobacterium tuberculosis but not strains of M. bovis.
It is easier to detect the color by examining the tubes against a white background, using incident room light.
1. Lawrence G. Wayne 1973. Simple Pyrazinamidase and Urease Tests for Routine Identification of Mycobacteria. American Review of
Respiratory Disease, Volume 109, Issue 1, 147-151.
2. Sunil S.Trivedi, S.G.Desai. Pyrazinamidase activity of Mycobacterium tuberculosis - A test of sensitivity to pyrazinamide. Tubercle
Volume 68, Issue 3, September 1987, 221-224.
3. Hardy Diagnostics. PYRAZINAMIDASE AGAR Instructions for use. available online
https://catalog.hardydiagnostics.com/cp_prod/Content/hugo/PyrazinamidaseAgar.htm, accessed March 2020.
4. Paul G. Engelkirk, Janet L. Duben-Engelkirk 2008. Laboratory Diagnosis of Infectious Diseases: Essentials of Diagnostic
Microbiology, pg. 708.
(c) Costin Stoica
Dissolve 6.5 g of commercially available Dubos broth base in 1 liter of water. To this solution add 100 mg of pyrazinamide, 2 g of
sodium pyruvate, and 15 g of agar (other "Pyrazinamidase agar" formulations may also be commercially available).
Heat the mixture just long enough to melt the agar then dispense it in 5-ml amounts to screw-capped tubes. Autoclave the tubes and
then let the medium harden in an upright position (not slanted).
The surface of the agar butt is heavily inoculated from a fresh Lowenstein-Jensen stock culture, using enough inoculum so that it
could be readily seen with the naked eye. Inoculate at least two tubes. Incubate the tubes at 36 to 37° C for 4-7 days.
After incubation, approximately add 1 ml of freshly prepared 1% ferrous ammonium sulfate solution to culture tube. Then place the
tube in the refrigerator to prevent growth of contaminants from the non-sterile ferrous reagent solution. After 4 hours in the cold, the
tubes were examined for color change in the agar.
Appearance of a pink band on diffusion of the ferrous salt is indication of hydrolysis of pyrazinamidase to free pyrazinoic acid. If the 4
day test is negative, then is necessary to hold the second tube for the 7 day test.