This article provides guidance for laboratory users on selecting and using negative reference controls for Delvotest® validation, verification, and routine performance monitoring. Negative reference samples are used to establish Control Time (CT) and help confirm that the test system is performing as expected.
What Is a Negative Reference Control?
A negative reference control is a sample of the same matrix being tested that does not contain antibiotics or other inhibitory substances at concentrations that could affect the test.
For example, when testing raw cow milk, the negative control should be raw cow milk that does not contain antibiotic residues or other compounds capable of inhibiting bacterial growth.
Because the negative control is expected to produce a negative result, it serves as the reference sample for establishing Control Time.
Negative Controls for Validation and Verification
For validation and performance verification activities, the characteristics of suitable negative milk are outlined in ISO/TS 23758 🔗.
Additional guidance on local verification studies is provided in ✅ Local Verification Study 🔗.
Where possible, laboratories should use well-characterised reference materials supplied by accredited reference material producers. These materials provide a known and controlled starting point for performance verification.
Negative Controls for Routine Laboratory Use
For routine laboratory use, negative control milk can be obtained from a pool of milk from healthy animals that have not received antibiotic treatment.
To obtain a representative sample, the milk should be a commingled sample from at least 4 untreated animals. Its composition should be representative of the species and type of milk being tested.
The sample should be confirmed negative using a suitable quantitative reference method, such as LC-MS/MS. This helps confirm that the sample is representative of the milk being tested and suitable for use as a negative control.
Commercial Control Materials
Commercial milk controls may also be used for routine performance verification.
Potential advantages include:
- Standardised composition
- Improved consistency between verification runs
- Ease of storage and handling
- Ready availability
However, commercially prepared controls may not fully represent local milk characteristics, seasonal variation, or regional production conditions. For this reason, some laboratories supplement commercial controls with locally sourced milk as part of their quality assurance programme.
Next Steps
Once a suitable negative control has been selected, use it alongside the test samples to establish Control Time under the actual testing conditions.
⏭️ Next article: 🚧How to Establish Control Time Using Delvotest® Plates 🔗
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