THERMONUCLEASE AGAR

Technical Data #1669a / 2003.05.29

The Thermonuclease Test Agar (TTA) is used for identification of heat stable nuclease produced by most strains of Staphylococcus aureus, Staphylococcus schleiferi, Staphylococcus intermedius and Staphylococcus hyicus.

 

FORMULA
in grams per liter purified filtered water

 

Tris Buffer (pH 9.0) 0,05M

1000 ml
DNA

0,3 g
CaCl2

1,1 mg
Sodium Chloride

10,0 g
Toluidine Blue

0,05 g
Agar

10,0

pH 9.0 +/- 0,2 at 25º C

This approximate formula may be adjusted and/or enriched to obtain best results.


PRECAUTIONS
This medium is for IN VITRO diagnostic use only.


STORAGE
Store prepared media at 2-8º C protected from direct light.


SIGN OF DETERIORATION
Media should not be used if the expiry date has passed. Prepared media should not be used if there are signs of contamination or deterioration (shrinking, cracking, evaporation or discoloration).


PROCEDURE
Prior to inoculate, the prepared media should be brought to room temperature.

  1. Inoculate BHI Broth with 2-3 colonies at 35º C for 24 hours.
  2. Boil tubes for 15 minutes and cool down to room temperature.
  3. With a Pasteur Pipet cut no more than 12 wells per plate.
  4. Fill each well with 2 drops of inactivated BHI broth. A maximum of 12 strains may be tested per plate.
  5. Incubate at 35º C from 1-4 hours, upside down. Negative results should be incubate for an additionnal 20 hours.

Note: from a blood culture broth, start test by boiling blood culture broth.


QUALITY CONTROL
Results after 4-24 hrs at 35ºC

 

Organisms

ATCC

Clair zone

Blue ring
Staphylococcus aureus

25923

+ pink

+
Staphylococcus aureus MRSA

clinical

+

+
Staphylococcus epidermidis

12228

-

-
Staphylococcus epidermidis

clinical

-

-

INTERPRETATION
A positive control is observed by a clair zone around the ring and a dark blue ring around the zone. A pale pink halo may appear around the well.


LIMITATIONS OF METHOD
This medium is only a part of the identification. Other tests may be required.


REFERENCES

  1. Balows, A., W.J. Hausler Jr., K. L. Herrmann, H. D. Isenberg and H.J. Shadomy, Lennette, Manual of Clinical Microbiology. 5th ed. 1991 Washington D.C. American society for Microbiology.
  2. Mac Faddin, Jean F., 1985 Media for Isolation-Cultivation-Identification-Maintenance of Medical Bacteria. Vol.1. Baltimore, MD.: Williams & Wilkins.
  3. Victor, R., F. Lachica, S.S. Jang, and P.D. Hoeprich Thermonuclease Seroinhibition Test for Distinguishing Staphylococcus aureus from other Coagulase-positive Staphylococci. 1979. J. Clin. Microbiol. 9:141-143.
  4. Zarzour, J.Y., and E.A. Belle. Evaluation of three Test Procedures for Identification of Staphylococcus aureus from Clinical Sources. 1978. J. Clin. Microbiol. 7:133-136.


CATALOGUE NUMBER

 

Prepared media    
Mono-Plates, 100x15 mm 10/pkg 1669