Question

The plate method used for detection of thermonuclease enzyme employs ..............................

• A. Toluidine blue DNA agar
• B. Malachite green DNA agar
• C. Hematoxyline DNA agar
• D. Desoxycholate DNA agar

Hint:

Remember the diagnostic indicators:
- Target: Thermonuclease (TDNase) from Staphylococcus aureus.
- Agar: Toluidine Blue DNA Agar (TDA).
- Color shift: Blue to pinkish-red (metachromasy due to DNA hydrolysis).

Answer 1

The Correct Option is A

Step 1: Understanding the Concept:
Thermonuclease (TDNase) is a highly heat-stable extracellular deoxyribonuclease produced by enterotoxigenic strains of Staphylococcus aureus.
Since the enterotoxin and the thermonuclease enzyme are produced under similar conditions, detecting this heat-stable enzyme serves as a rapid and reliable indicator of S. aureus growth and potential enterotoxin contamination in dairy products.

Step 2: Detailed Explanation:
The standard plate method used to detect thermonuclease activity is performed using a specialized agar medium:
-Medium Composition:
The assay uses Toluidine Blue DNA Agar (TDA). This agar contains calf thymus DNA as the substrate and Toluidine Blue O as a metachromatic dye.
-Reaction Mechanism:
Toluidine blue complexes with intact, double-stranded DNA, producing a royal blue color in the agar.
Before testing, milk or cheese extracts are boiled to denature any heat-labile nucleases produced by other organisms, leaving only the heat-stable staphylococcal thermonuclease active.
The boiled sample is then added to wells cut into the TDA agar plate.
If active thermonuclease is present, it diffuses into the agar and hydrolyzes the DNA.
The degradation of DNA releases the dye, causing a metachromatic shift from the original royal blue to a bright pinkish-red halo around the sample well.
The diameter of this pink zone is proportional to the concentration of active thermonuclease in the sample.

Step 3: Final Answer:
The plate method used for the detection of the thermonuclease enzyme uses Toluidine blue DNA agar.