ISO/TS 26844:2006 — Milk and Milk Products: Determination of Antimicrobial Residues by Tube Diffusion Test

Introduction to ISO/TS 26844:2006

ISO/TS 26844:2006 (IDF/RM 215:2006) specifies a microbiological inhibitor test for detecting a broad variety of antimicrobial substances in milk and milk products. Published jointly by ISO and the International Dairy Federation (IDF), this Technical Specification employs a two-tube diffusion method using Geobacillus stearothermophilus ATCC 10149 as the test organism. The method is applicable to raw milk, heat-treated milk, and reconstituted dried milk.

The principle is based on pH indicator colour change: normal growth of G. stearothermophilus causes the bromocresol purple indicator in the agar to turn from purple to yellow. When antimicrobial substances are present, growth is inhibited and the medium remains purple.

Dual-Tube Test System

The two-tube design provides complementary detection capabilities. Tube A operates at pH 7.0 with chloramphenicol as a synergist, showing enhanced sensitivity for tetracycline residues. Tube B operates at pH 8.0 with trimethoprim, targeting beta-lactams, macrolides, aminoglycosides, sulfonamides, and trimethoprim residues.

Procedure and Interpretation

The test procedure follows a carefully standardized workflow. Milk samples are first heat-treated at 80 C for 10 minutes to inactivate heat-labile natural inhibitory substances. After cooling, 0.3 ml of sample is pipetted into both Tube A and Tube B. The tubes are held at room temperature for 1 hour to allow diffusion, then the milk layer is poured off and the tubes are covered to prevent evaporation.

A heat shock step (70 C for 10 min) may optionally be applied to activate spores. Incubation proceeds at 63 C for 4 hours 15 minutes (plus or minus 30 minutes) until the negative control tubes have just turned from purple to completely yellow. A completely or partly purple colour in the test sample indicates a positive result — the presence of inhibitory substances.

Confirmation and Quality Assurance

Positive results can be confirmed through presumptive testing. Beta-lactam residues are confirmed using beta-lactamase enzymes — if enzyme treatment counteracts the inhibitory activity, beta-lactams are present. Sulfonamides are confirmed using p-aminobenzoic acid (PABA), which reverses sulfonamide inhibition. For other inhibitors, more sophisticated methods such as microbiological multiplate tests, immunoassays, or chromatographic methods (HPLC, LC-MS) are required per ISO 18330/IDF 188.

Quality control of the test organism suspension is critical. Each new batch must be checked against five standard solutions: benzylpenicillin (2 ug/kg), sulfadiazine (150 ug/kg), neomycin (30 ug/kg), erythromycin (10 ug/kg), and oxytetracycline (100 ug/kg). The test organism suspension should contain approximately 5,000,000 colony-forming units per millilitre and can be stored at below -20 C for up to one year.

Quality Assurance in Dairy Testing Laboratories

Successful implementation of the tube diffusion test requires rigorous quality assurance practices in the dairy testing laboratory. The test organism suspension preparation is critical — Geobacillus stearothermophilus ATCC 10149 must be maintained as a pure culture on slant agar and propagated under standardized conditions to ensure consistent spore viability and concentration. Each new batch of test organism suspension must be validated against the five standard antimicrobial solutions before use in routine testing. Laboratory personnel must be trained to recognize the subtle colour transition from purple to yellow and to standardize the interpretation endpoint. The optional heat shock step at 70 C for 10 minutes can help activate spores and improve test reproducibility. Sample handling procedures are equally important — milk samples should be tested without delay, and dried milk products must be properly reconstituted before analysis. The standard’s requirement for control samples in every test batch provides continuous quality monitoring, and any deviation in control performance triggers immediate investigation. For laboratories handling large sample volumes, the ability to use test samples that are expected to be negative as negative controls offers practical efficiency without compromising quality.

The two-tube approach with different pH values and synergistic agents represents a sophisticated design for broad-spectrum screening. Tube A’s inclusion of chloramphenicol at pH 7.0 selectively enhances tetracycline detection by interfering with bacterial protein synthesis pathways, making the organism more susceptible to tetracycline residues. Tube B’s use of trimethoprim at pH 8.0 exploits the pH-dependent activity of sulfonamides and optimizes detection of beta-lactams. This differential design allows a single test to cover multiple antimicrobial classes that would otherwise require separate analytical methods. The use of bromocresol purple as a pH indicator is also carefully chosen — its transition range (pH 5.2 to 6.8) from yellow to purple aligns well with the metabolic acid production of G. stearothermophilus during growth, providing a clear visual endpoint.

Precision Data and Collaborative Study Results

The informative annexes of ISO/TS 26844 provide valuable precision data from collaborative studies involving three experienced Dutch laboratories. These studies established the limits of detection for various antimicrobial substances in milk. For beta-lactams, benzylpenicillin is detected at 2 ug/kg in Tube A and 3 ug/kg in Tube B, while amoxicillin and cloxacillin are detected at 3 ug/kg and 20 ug/kg respectively. Tetracycline detection is notably better in Tube A (100 ug/kg for oxytetracycline and tetracycline) compared to Tube B (400 ug/kg and 300 ug/kg respectively), confirming the effectiveness of the chloramphenicol synergy approach. For sulfonamides, Tube B achieves detection limits as low as 2 ug/kg for dapsone and 150 ug/kg for sulfadiazine. These data allow laboratories to establish their own validation parameters and provide confidence in the method’s fitness for regulatory compliance testing.

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