Overview
ISO 8870:2006 (IDF 83:2006) - "Milk and milk‑based products - Detection of thermonuclease produced by coagulase‑positive staphylococci" specifies a microbiological method for detecting heat‑stable DNase (thermonuclease) produced by coagulase‑positive Staphylococcus spp. in milk and milk‑based products. The presence of thermonuclease is used as an indicator that staphylococcal growth has reached hazardous levels and may signal the potential presence of staphylococcal enterotoxins, a food‑safety risk.
Key topics and technical requirements
- Target enzyme: Thermonuclease (heat‑stable DNase) produced by coagulase‑positive staphylococci.
- Sample types: Raw milk, reconstituted milk powder, homogenized milk‑based products and certain cheese extracts.
- Sample preparation:
- Adjust samples to pH 3.8 (addition of skim milk powder for some products may be required).
- High‑speed centrifugation (27 000–34 000 g) and precipitation with trichloroacetic acid.
- Reconstitution in Tris buffer, adjust to pH 8.5, then heat in a boiling water bath for 15 minutes.
- Detection medium: Toluidine blue O‑DNA agar - DNA in the agar is cleaved by thermonuclease, producing a pink metachromatic halo around wells.
- Controls & interpretation:
- Use a thermonuclease‑positive Staphylococcus aureus strain as positive control (prepared in brain heart infusion broth).
- Incubate plates at 37 °C for 4 hours (extendable up to 24 hours). A pink halo ≥ 1 mm beyond the well indicates a positive result.
- A positive thermonuclease test correlates with coagulase‑positive staphylococci counts of ≈10 or more per gram.
- Equipment & reagents: incubator (37 °C), water baths (50 °C and boiling), centrifuges capable of >27 000 g, toluidine blue O, Tris buffer, TCA, skim milk powder free from thermonuclease.
Practical applications
- Food safety testing: Screening milk and dairy products for hazardous staphylococcal growth.
- Quality control: Routine monitoring in dairy processing and product release testing.
- Outbreak investigations: Rapid indication of products that may contain staphylococcal enterotoxins, prompting toxin testing.
- Method validation and laboratory protocols: Establishes a standardized detection technique for inter‑laboratory comparability.
Who should use this standard
- Dairy microbiology laboratories and QC teams
- Food safety regulators and inspection bodies
- Public health laboratories handling dairy‑related outbreaks
- Producers of milk and milk‑based products implementing HACCP and microbiological control
Related standards
- ISO 8261 | IDF 122 - preparation of test samples and dilutions for microbiological examination
- ISO 707 | IDF 50 - guidance on sampling of milk and milk products
Keywords: ISO 8870:2006, thermonuclease detection, coagulase‑positive staphylococci, milk testing, toluidine blue O, staphylococcal enterotoxins, food safety, dairy microbiology.
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