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DNase Test Agar with and without methyl green

Catalog Number: 1003417, 1003517
DNase Test Agar with and without methyl green


Description: For the detection of deoxyribonuclease production in microorganisms, especially suspected pathogenic staphylococci.

DNase Test Agar with methyl green as described by Smith, Hancock, and Rhoden1 is a modification of DNase Test Agar. It is also used for detecting DNase activity in microorganisms and for the isolation and differentiation of Staphylococcus aureus and Serratia marcescens from other organisms having similar characteristics except DNase production.

Formulation in g/L of DNase Test Agar:

Deoxyribonucleic Acid2.0 gCasein Peptone15.0 g
Soy Peptone5.0 gSodium Chloride5.0 g
Agar15.0 g

Final pH 7.3 ± 0.2

Formulation of DNase Test Agar with Methy Green:

Deoxyribonucleic Acid2.0 gTryptose20.0 g
Methyl Green0.05 gSodium Chloride5.0 g
Agar15.0 g
Final pH 7.3 ± 0.2

Preparation:

Suspend 42 g of the medium in one liter of distilled or deionized water. If required, add 10 g of mannitol and 0.025 g of bromothymol blue. Mix carefully to obtain a homogenous suspension. Heat with frequent agitation and boil for one minute. Sterilize in an autoclave at 118-121°C (12 to 15 lbs. psi) for 15 minutes. Cool to 45-50°C and pour into Petri dishes.

If desired, add 5% blood to the medium without mannitol to prepare a blood agar medium.

Uses:

Make a heavy band streak (2 cm in length) of the test organism (eg. staphylococci, Pseudomonas, Serratia, Bacillus, etc.) on the surface of the plate. You can simultaneously place in the same plate 4 to 5 different samples. Incubate for 18 to 24 hours at 35°C.

After good growth add a drop of 1 N hydrochloric acid or a few drops of 0.1% toluidine blue solution. With some strains it is necessary to increase the concentration of HCl to 2 N to obtain a good positive reaction, the appearance of a well defined bright clear halo around the bacterial streak.

In the presence of diluted hydrochloric acid, the reaction with deoxyribonucleic acid (DNA) in the culture medium forms a hazy precipitate.

On the other hand, the colonies producing deoxyribonuclease appear surrounded by a zone or a clear halo which contains fractions of soluble nucleotides from the degradation of DNA, which are not precipitated by the hydrochloric acid.

Kurnick5 showed that methyl green combines only with highly polymerized DNA and at pH 7.5. When combination does not take place, the color fades. This observation was applied to a test for determining DNase in body fluids and blood serum. Smith, Hancock and Rhoden1 applied this principle to modify DNase Test Agar for detecting staphylocci, streptococci, and Serratia. The method has the advantage that DNase activity can be detected by a clearing of the dye around DNase producing colonies. Acid does not have to be added and colonies can be picked directly from the plate for additional studies.

Mannitol fermentation can be determined simultaneously with DNase production by adding 10 g mannitol and 0.025 g phenol red to the DNase Test Agar before sterilization.

Results:

In the presence of hydrochloric acid:

In the presence of toluidine blue:

In the presence of methyl green:

The positive reaction takes approximately 5 minutes to form.

The medium is very rich in nutrients and most organisms grow very well on it. Nevertheless, for some fastidious organisms it may be necessary to add blood. In the presence of blood, the addition of diluted hydrochloric acid causes the formation of a well defined but opaque halo with DNase positive organisms. (The medium does not clear).

The DNase medium with blood should not be used in the study of hemolytic reactions and should only be added in absolutely necessary cases.

Weckman and Catting (1957), Disalvo (1959) and Fusillo and Weis (1959) proved that coagulase positive staphylococci degraded the DNA by hydrolysis and are considered DNase positive.

Typical Microbiology:

Typical cultural response on DNase Test Agar after 18-24 hours at 35°C.

Organism
Growth
Zone of Clearing
Serratia marcescens ATCC 8100
good to excellent
+
Staphylococcus aureus ATCC 25923
good to excellent
+
Staphylococcus epidermidis ATCC 12228
good to excellent
-
Streptococcus pyogenes ATCC 19615
good to excellent
+

Availability:

Catalog NumberDescriptionSize
1003417DNase Test Agar500 g
1003517DNase Test Agar with methyl green500 g

References:
  1. Smith, Hancock and Rhoden, Applied Microbiology, v. 18: 991 (1969).
  2. Blair, E.B., Emerson, J.S., and Tull, S.C., Am. J. Clin. Path., v. 47: 30-39 (1957).
  3. Disalvo Med. Tech. Bull., v. 9: 191 (1958).
  4. Weckman and Catting, J. Bact., v. 73: 747 (1957).
  5. Kurnick, Archives of Biochemistry, v. 9: 41 (1950).