DNA hydrolysis test or Deoxyribonuclease (DNase) test is used to determine the ability of an organism to hydrolyze DNA and utilize it as a source of carbon and energy for growth. In this regard, Deoxyribonuclease test medium (DNase agar) prepared according to the formula of Jeffries, Holtman and Guse, is a differential medium used to test the ability of an organism to produce Deoxyribonuclease (DNase) enzyme.
This medium ispale green in colorbecause of the DNA-methyl green (indicator) complex(Note: Methyl green is a cation that binds to the negatively-charged DNA).It also contains nutrients for the bacteria.If the organism that grows in the medium produces Deoxyribonuclease, it breaks down DNA into smaller fragments. When the DNA is broken down, it no longer binds to the methyl green, andgreen color fades and the colony is surrounded by a colorless zone
Composition of DNase Test Agar
|3.||Deoxyribonucleic acid (DNA)||2.0|
|Final pH at 25°C: 7.3 ±0.2|
Objectives of Deoxyribonuclease (DNase)
- To determine the ability of an organism to produce the DNase enzyme.
- To differentiate and identifyS. aureusfrom other Staphylococcal species.
This test determines the ability of an organism that produce DNase. DNase are extracellular endonucleases that cleave DNA and release free nucleotides and phosphate. To detect these enzymes, DNase agar using no indicators or various indicators (toluidine blue or methyl green) are used to detect the hydrolysis of DNA.
In the DNase test, the test bacteria are grown on agar plates containing DNA. After colonies of the bacteria are visible, the plates are flooded with hydrochloric acid. If the bacteria have the ability to hydrolyse DNA, its colonies hydrolyse the DNA in the medium in the areas surrounding them, while the rest of the areas of the plates contain unhydrolysed DNA.
As a result, when flooded with hydrochloric acid, transparent clear zones are formed around the colonies, as the hydrolysed products, oligonucleotides, formed around them do not form precipitates with the hydrochloric acid.
Reagents And Material Used
- Petri dishes
- Conical flask
- Cotton plugs
- Inoculating loop
- Bunsen burner
- Laminar flow chamber
- Dispose jar
- DNase agar
- 1 N hydrochloric acid (or 0.1% toluidine blue)
- Isolated colonies or pure cultures of bacteria.
- Two petri dishes are cleaned, covered with craft paper and tied with thread or rubber band.
- The ingredients of DNase agar medium (containing DNA as the main component) or its ready-made powder required for 100 ml of the medium is weighed and dissolved in 100 ml of distilled water in a 250 ml conical flask by shaking and swirling.
- Its pH is determined using a pH paper or pH meter and adjusted to 7.2 using 0.1N HCI if it is more or using 0.1N NaOH if it is less.
- The flask is heated to dissolve the agar in the medium completely.
- The flask is cotton-plugged, covered with craft paper and tied with thread or rubber band.
- The two petri dishes and the conical flask containing DNase agar medium are sterilized at 121 °C (15 psi pressure) for 15 minutes in an autoclave.
- After sterilization, they are removed from the autoclave and allowed to cool for some time, without allowing the medium to solidify. Cooling of the medium prevents condensation and accumulation of water droplets inside the plates. If the medium has already been prepared and solidified during storage, it has to be liquefied by heating carefully till it melts completely.
- To prepare DNase agar plates, before the sterilized DNase agar medium cools and solidifies, in warm molten condition, it is poured aseptically, preferably inside a laminar flow chamber, into the two sterilized petri dishes (approximately 20 ml each), so that the molten medium covers the bottom of the petri dishes completely.Then, the plates are covered with their lids and allowed to cool, so as to solidify the medium in them. Water vapour that may condense on the inner surface of the plates and lids is evaporated by keeping the plates and lids in inverted position in an incubator at 37°C for about 1 hour.
- Each plate is marked on the bottom side into four quarters.
- “Spot inoculation” of the test bacteria is done aseptically, preferably inside a laminar flow chamber, on the center of each quarter by making a spot (or small smear) of the bacteria with the help of a flame-sterilized loop. The loop is sterilized after each inoculation.
- The inoculated plates are incubated in inverted position, top down, at 37°C for 24 to 48 hours in an incubator till colonies of the bacteria are visible.
- The plates are flooded with solution of 1 N hydrochloric acid (or 0.1% toluidine blue).
Observations And Results Interpretation
- DNase Positive:Transparent clear zones (or pink halo) formed around colonies.
- DNase Negative: Transparent clear zones (or pink halo) not formed around colonies.
With indicator (Toluidine Blue O)
- A positive test is demonstrated by the development of a pink or red halo around the colony or the well in the agar.
- A negative test is demonstrated by no change in the royal blue color of the medium.
Examples Of DNase Positive Bacteria
Staphylococcus aureus, Streptococcus pyogenes, Moraxella catarrhalis, Serratia species, Aeromonas, Vibrio.
Examples Of DNase Negative Bacteria
Staphylococcus epidermidis, Klebsiella, Enterobacter, E.coli, Serratia fonticola, Plesiomonas shigelloides.
Limitations Of DNase Test
- Agar must be inoculated with a suspension of a young broth culture (4 hours old) or an 18- to 24-hour colony in 1-2 mL of saline.
- Some MRSA strain do not give positive result and some strain of coagulase negative Staphylococci such as Staphylococcus capitis give weak positive reaction.
- For Moraxella and Gram-positive cocci with TBO testing, a low inoculum can result in a false-negative test, since these organisms may not grow well on the medium.
- 1N HCl is bactericidal for staphylococci. Once the HCl has been applied, the test must be read within 5 minutes and cannot be continued by re-incubation.
Uses Of DNase Test
- Used to determine the ability of an organism to hydrolyze deoxyribonucleic acid.
- Used to differentiateStaphylococcus aureuswhich produces the enzyme deoxyribonuclease from other Staphylococci which do not produce DNase.
- Particularly useful if plasma is not available to perform coagulase test or when the result of coagulase tests are difficult to interpret.
- It is also used to distinguishSerratia(positive) fromEnterobactersp.
- Moraxella catarrhalis(positive) fromNeisseria