| Deoxyribonuclease |
DESCRIPTION
The DNA hydrolysis by DN-ase enzyme is revealed by toluidine blue incorporated in medium. Toluidine colors the DNA in blue and
the DNA degradation products in pink to red. The test is recommended for the detection of deoxyribonuclease activity of bacteria and
fungi, and especially for identification of pathogenic staphylococci. It is useful for differentiation between Serratia and Enterobacter,
Moraxella and Branhamella and other Gram-negative nonfermentative microorganisms.
The DNA hydrolysis by DN-ase enzyme is revealed by toluidine blue incorporated in medium. Toluidine colors the DNA in blue and
the DNA degradation products in pink to red. The test is recommended for the detection of deoxyribonuclease activity of bacteria and
fungi, and especially for identification of pathogenic staphylococci. It is useful for differentiation between Serratia and Enterobacter,
Moraxella and Branhamella and other Gram-negative nonfermentative microorganisms.
MEDIUM PREPARATION
DNase Agar
NaCl .............................................................................................................. 5.0 g
Casein enzymic hydrolysate .................................................................. 15.0 g
Papaic digest of soyabean meal ............................................................ 5.0 g
Deoxyribonucleic acid (DNA) ................................................................... 2.0 g
Agar ............................................................................................................ 15.0 g
Toluidine blue 0.25% solution ............................................................... 40 ml
Distilled water ......................................................................................... 960 ml
Dissolve ingredients in water by heating and mixing frequently; boil one
minute. Adjust to pH 7.3. Autoclave 15 min at 121 °C.
PROCEDURE
Harvest a well isolated colony and inoculate a DNase Agar plate. Incubate at 37 °C,
18-24 hours (or other required conditions).
READING
Reaction is positive if medium around colonies changes color to pink-red.
Reaction is negative if medium remains unchanged (blue). Plate should be reincubated for another 24h if negative.
NOTE
Toluidine blue may inhibit the growth of some bacterial strains.
For Campylobacter spp. testing: prepare anaerobic blood agar supplemented with DNA 2g/L. After incubation add HCl solution 1N in
plate. Reaction is positive if a clear zone appears around colonies and the rest of the medium become opaque.
REFERENCES:
1. Dumitru Buiuc, Mihai Zamfirescu: Teste de identificare a microorganismelor, in Dumitru Buiuc, Marian Negut, Tratat de
microbiologie clinica, 3-rd ed., 2009, 1103-1105.
2. DNase Test Agar Base M482. HiMedia Lab technical data. Revision : 1 / 2011.
DNase Agar
NaCl .............................................................................................................. 5.0 g
Casein enzymic hydrolysate .................................................................. 15.0 g
Papaic digest of soyabean meal ............................................................ 5.0 g
Deoxyribonucleic acid (DNA) ................................................................... 2.0 g
Agar ............................................................................................................ 15.0 g
Toluidine blue 0.25% solution ............................................................... 40 ml
Distilled water ......................................................................................... 960 ml
Dissolve ingredients in water by heating and mixing frequently; boil one
minute. Adjust to pH 7.3. Autoclave 15 min at 121 °C.
PROCEDURE
Harvest a well isolated colony and inoculate a DNase Agar plate. Incubate at 37 °C,
18-24 hours (or other required conditions).
READING
Reaction is positive if medium around colonies changes color to pink-red.
Reaction is negative if medium remains unchanged (blue). Plate should be reincubated for another 24h if negative.
NOTE
Toluidine blue may inhibit the growth of some bacterial strains.
For Campylobacter spp. testing: prepare anaerobic blood agar supplemented with DNA 2g/L. After incubation add HCl solution 1N in
plate. Reaction is positive if a clear zone appears around colonies and the rest of the medium become opaque.
REFERENCES:
1. Dumitru Buiuc, Mihai Zamfirescu: Teste de identificare a microorganismelor, in Dumitru Buiuc, Marian Negut, Tratat de
microbiologie clinica, 3-rd ed., 2009, 1103-1105.
2. DNase Test Agar Base M482. HiMedia Lab technical data. Revision : 1 / 2011.
(c) Costin Stoica
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