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DNASE AGAR

Code: CM0321

For the detection of microbial deoxyribonuclease enzymes, particularly from staphylococci.

Directions
Suspend 39g in 1 litre of distilled water and bring to the boil to dissolve completely. Sterilise by autoclaving at 121°C for 15 minutes.

Description
Weckman & Catlin¹ suggested that DNase activity could be used to identify pathogenic staphylococci after they had established a close correlation with coagulase production. Jeffries et al.² incorporated DNA in the agar medium to provide a simple method of detecting DNase activity. Organisms are streaked on to the surface of the agar medium and incubated. The growth on the surface of the agar is then flooded with 1N hydrochloric acid. Polymerised DNA precipitates in the presence of 1N HCl and makes the medium opaque. If the organisms produce DNase enzymes, in sufficient quantity to hydrolyse the DNA, then clear zones are seen around the colonies.

Good correlation was shown between DNase production and coagulase activity when testing Staphylococcus aureus strains from clinical samples^2,3,4. Both Staphylococcus aureus and Staphylococcus epidermidis produce extracellular DNase^5,6,7 but Staphylococcus aureus produces greater quantities^1,7.

A modification of the medium is to add mannitol (1% w/v) and phenol red or bromothymol blue (0.0025% w/v) as an indicator of mannitol fermentation⁹. The pH reaction around the colonies must be read before the plate is flooded with acid.

The DNase reaction helps in the differentiation and identification of non-pigmented Serratia marcescens⁸ (positive DNase reaction) from Klebsiella-Enterobacter (negative DNase reaction).

Normal HCl is bactericidal and the organisms cannot be recovered from the surface of the agar after flooding. The incorporation of dyes into the medium which can distinguish hydrolysis of DNA is a further modification which avoids the use of acid. Toluidine blue⁸ and methyl green¹⁰ form coloured complexes with polymerised DNA; these colours change as the DNA is hydrolysed.

It should be noted that toluidine blue inhibits Gram positive organisms and it is used to detect DNase production by the Enterobacteriaceae. It has been used with ampicillin (30 mg/litre) to demonstrate DNase production by Aeromonas hydrophila from faeces¹¹.

Technique
Inoculate the plates by spotting the organism onto the surface of the agar so that a thick plaque of growth is evident after 18 hours incubation.
Examine plates for colour changes in or around the colonies if mannitol/indicator or dyes have been added to the medium. In the absence of dyes, flood the plates with 1N HCl and allow them to stand on the bench (lids uppermost) for a few minutes. Look for zones of clearing around the colonies.

Appearance of colonies with media modifications

Storage conditions and Shelf life
Store the dehydrated medium at 10-30°C and use before the expiry date on the label.
Store the prepared plates of medium at 2-8°C.

Appearance
Dehydrated medium: Straw coloured, free-flowing powder
Prepared medium: Straw coloured gel

Quality control

* This organism is available as a Culti-Loop®

Precautions
The DNase reaction for staphylococci is an indication of pathogenicity, it cannot be used as the sole criterion for identification.
Small zones of clearing may be caused by other enzymes or organic acid production⁷.
Other organisms than staphylococci, Serratia and aeromonads can produce DNases.
Once the hydrochloric acid has been applied to the medium the plate must be read within a few minutes and further testing cannot be carried out by re-incubation.
The methyl green must be purified by extraction with chloroform¹⁰.
Toluidine blue varies in performance according to source.
Merck Toluidine blue 1273 is satisfactory. Note that this dye cannot be used for Gram positive organisms.

References
1. Weckman B. G. and Catlin B. W. (1957) Journal of Bacteriology 73. 747-753.
2. Jeffries C. D., Holtman D. F. and Guse D. G. (1957) Journal of Bacteriology 73. 590-591.
3. DiSalvo J. W. (1958) Med. Techns. Suppl. to U.S. Armed Forces Medical Journal 9. 191-196.
4. Blair E. B., Emerson J. S. and Tull A. H. (1967) American Journal of Clin. Path. 47. 30-39.
5. Baird-Parker A. C. (1965) J. Gen. Microbiol. 38. 363-367.
6. Raymond E. A. and Traub W. H. (1970) Appl. Microbiol. 19. 919-921.
7. Zierdt C. H. and Gold D. W. (1970) Appl. Microbiol. 20. 54-57.
8. Schreir J. B. (1969) Amer. J. Clin. Path. 51. 711-716.
9. Coobe E.R. (1968) Ulster Med. J. 37. 146-149.
10. Smith P. B., Hancock G. A. and Rhoden D. L. (1969) Appl. Microbiol. 18. 991-994.
11. von Graevenitz A. and Zinterhofer L. (1970) Health Lab. Sci. T. 124-127.