PYRAZINAMIDASE AGAR

Cat. no. K158 Pyrazinamidase Agar, 16x125mm Tube, 5ml Deep 20 or 100 tubes/box

INTENDED USE

Hardy Diagnostics Pyrazinamidase Agar is used to differentiate Mycobacterium species on the basis of pyrazinamidase activity.

SUMMARY AND PRINCIPLES

Pyrazinamide (PZA) is an anti-tuberculosis drug effective on strains of Mycobacterium tuberculosis but not strains of M. bovis . This attribute can be used to differentiate between the species, but susceptibility testing with the drug becomes complicated, as the drug works best at a low pH in vitro . The acidity of the media required for PZA activity prevents growth of some mycobacteria.

Pyrazinamidase Agar eliminates the problems of an acidic test medium by detecting the ability of certain Mycobacterium species to convert pyrazinamide (PZA) to pyrazinoic acid. The enzyme is characteristic of tuberculosis mycobacteria that are susceptible to PZA. Resistant strains, however, do not produce detectable amounts of the enzyme. (1)

All non-tuberculous mycobacteria are resistant to PZA, but some of them are pyrazinamidase positive ( M. marinum , M. avium , M. intracellulare , M. xenopi , M. malmoense , M. flavescens ), and some are negative ( M. kansasii , M. simiae , M. szulgai , M. gastri ). Therefore, this test can be used for differentiation between some of these species, for example, between M. kansasii and M. marinum . (1)

FORMULA

Ingredients per liter of deionized water:*

Disodium Phosphate 2.5gm
L-Asparagine 2.0gm
Pyruvic Acid, Sodium Salt 2.0gm
Monopotassium Phosphate 1.0gm
Casein Peptone 0.5gm
Ferric Ammonium Citrate 0.5gm
Tween ® 80 0.2gm
Pyrazinamide 0.1gm
Magnesium Sulfate 10.0mg
Calcium Chloride 0.5mg
Zinc Sulfate 0.1mg
Copper Sulfate 0.1mg
Agar 15.0gm

Final pH 6.6 +/- 0.2 at 25ºC.

* Adjusted and/or supplemented as needed to meet performance criteria.

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-8ºC. away from direct light. Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.

PRECAUTIONS

PROCEDURE

1. Place a heavy inoculum from an actively growing culture slant on the surface of each of 2 tubes of Pyrazinamide Agar. Use enough inoculum so that it may be readily seen with the naked eye. Do not stab the medium.

2. Incubate two (2) uninoculated tubes and two (2) uninoculated control tubes aerobically at 35-37ºC.

3. After 4 days incubation, remove one (1) uninoculated tube and one (1) control tube from the incubator and add 1ml of freshly prepared 1% ferrous ammonium sulfate solution (in deionized water) to the tubes.

4. Place the tubes at room temperature for 30 minutes, then examine for a pink band in the medium. Refrigerate negative tubes for 4 hours at 2-8ºC. to prevent growth of contaminants.

5. After 4 hours, examine for a pink color development by holding the tube against a white background using incandescent room light. If the 4 day test is positive, it is not necessary to hold the second tube for the 7 day test.

6. The control must be negative for the test to be valid. If the 4 day tube is negative or doubtful, perform test after 7 days using the second uninoculated and inoculated tube.

7. After 7 days incubation at 35-37ºC. remove the second inoculated tube and control tube from the incubator and proceed as described above.

INTERPRETATION OF RESULTS

Positive Test - a pink band in the agar

Negative Test - no color development.

LIMITATIONS

A small inoculum size may give false-negative reactions.

Incubation in a CO 2 atmosphere will interfere with the pyrazinamidase reaction.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, other culture media, swabs, applicator sticks, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Mycobacterium intracellulare
ATCC ® 13950
G 4 and 7 days 35°C Aerobic Positive: pink band develops within 4 hours after the addition of the ferrous solution
Mycobacterium kansasii
ATCC ® 12478
G 4 and 7 days 35°C Aerobic Negative: no color development

User Quality Control

PHYSICAL APPEARANCE

Pyrazinamidase Agar should appear slightly opalescent, and very light amber in color.

Pyrazinamidase Test

Showing positive (left tube) and negative (right tube) pyrazinamidase test
The left and right tubes of Pyrazinamidase Agar (Cat. no. K158) were incubated aerobically for four days with a heavy inoculum of Mycobacterium intracellulare (ATCC ® 13950) and Mycobacterium kansasii (ATCC ® 12478), respectively. Subsequent to incubation, 1mL of freshly prepared 1% ferrous ammonium sulfate solution (in deionized water) was added aseptically to each tube. The tubes were incubated aerobically at room temperature for 30 minutes and then at 2ºC. for four hours. Development of a pink color was indicative as positive for the pyrazinamidase test.

REFERENCES

1. Bloom, B.R., Ph.D. 1994. Tuberculosis: Pathogenesis, Protection, and Control. ASM Press, Washington, D.C.

2. Baron, E.J., et al. 1994. Bailey & Scott's Diagnostic Microbiology, 9th ed. Mosby, St. Louis, MO.

3. Konno, et al. 1967. Pyrazinamide Susceptibility and Amidase Activity of Tubercle Bacilli. Am. Rev. Respiratory Disease; 95:461-69.

4. Quality Assurance for Commercially Prepared Microbiological Culture Media, M22. Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS), Wayne, PA.


ATCC is a registered trademark of the American Type Culture Collection.
Tween is a registered trademark of ICI Americas, Inc.

040416gr