The Activity of Deoxyribonuclease (Dnase)

Some microorganisms secrete an enzyme that attacks the deoxyribonucleic acid (DNA) molecule. This can be demonstrated by inoculating a plated agar medium containing the substrate DNA with a culture of the organism that produces the enzyme. The uninoculated medium is opaque and remains so after the culture has grown. If the plate is then flooded with weak hydrochloric acid, a zone of clearing appears around colonies that have produced DNase. This clearing occurs because the large DNA molecule has been degraded by the enzyme, and the end products dissolve in the added acid. Intact DNA does not dissolve in weak acid but rather is precipitated by it; therefore, the medium in the rest of the plate, or around colonies that do not produce DNase, becomes more opaque. Another way to demonstrate breakdown of DNA by DNase is to flood the plate with 0.1% toluidine blue. Intact DNA will stain blue, and DNase-producing colonies will be surrounded by a pink zone (see colorplate 21).

Purpose	To distinguish bacterial species that do and do not produce DNase
Materials	One DNA agar plate Dropping bottle containing 1 N HCl or 0.1% toluidine blue Slant cultures of Escherichia coli and Serratia marcescens

Procedures

1. Make a mark on the bottom of the DNA plate, dividing it in half.
2. Heavily inoculate one side of the plate with Escherichia coli by rubbing growth from the slant in a circular area about the size of a quarter.
3. Inoculate the other side of the plate with Serratia marcescens, in the same manner.
4. Incubate the plate at 35°C for 24 hours.
5. Examine the plate for growth.
6. Drop 1 N HCl or 0.1% toluidine blue onto the agar surface until it is thinly covered with fluid.
7. Examine the areas around the growth on both sides of the plate for evidence of clearing or opacity, or a pink color if toluidine blue was used.

Results
Make a diagram of your observations.