Quantifying inhibitors in milk  

Whilst the microbial inhibition test detects the presence of inhibitors in milk based on a certain cut-off level, it does not actually provide the antibiotic level present. This newsletter discussed how to quantify inhibitors in milk.


Quantifying the levels of milk inhibitors present in a positive milk samples not only confirms the initial results, but also provides valuable data in terms of the extent of the contamination.  

Ranking samples in terms of how much they exceed MRL’s helps identify milk suppliers that may need some assistance to improve the quality of milk supplies. 

Positive rapid milk antibiotic residue tests are further examined in New Zealand using antibiotic disk assays to help pinpoint problem farm suppliers. 

An easy test to do use is the paper disc assay method. This test is based on the same organism used in the microbial inhibition test (Bacillus stearothermophilus), but is greatly expanded to include a range in which milk inhibitors (expressed as equivalence of ug/mL of penicillin G) can be measured.


  Antibiotic disks assay 

This test has stood the test of time, having been published by Edwin J. De Beer and Marion B. Sherwood in 1945 (click here for the original paper). 

Test method

The principle of the test is agar is seeded with the spores of the bacterium Bacillus stearothermophilus var. calidolactis (sensitive to penicillin and other inhibitory substances).

Details of the method are as follows: 

Prepare some PM Indicator agar and seed with microbes. 

Heat test sample to 82°C for >2 min and cool promptly to room temperature. 

After heat treatment, the milk sample to be tested is absorbed onto sterile paper discs. There are 2 ways to do this:

1.       with clean, dry forceps, touch paper disk to surface of well-mixed milk, so that capillary action moistens the filter paper discs; or

2.       using micropipettor, add 90µL of sample directly on to the paper disc. 

Using tweezers, place the paper discs onto the seeded agar and the plates incubated.  


Incubate at 64 ± 2°C until well-defined zones of inhibition (16-20 mm) are obtained with the 0.008 unit/ml control (about 4-6 hours).  

If growth inhibitors are present in the milk they will diffuse out from the disc and inhibit the growth of bacteria. 


Examine plate for a clear zone of inhibition (>16 mm) surrounding disk against the background lawn of bacterial growth, indicating presence of inhibitory substance.


  Microbial disk inhibition zone  

The diameter of the zone (measured in mm) can be related to a concentration of penicillin or inhibitory substances by using a standard response graph.

The larger the zones, the greater the concentration of the inhibitor. 

An inhibitory substance in a milk samples may be further identified as “penicillin” or “not penicillin” by pre-treating part of the milk sample with the enzyme penicillinase - add 0.05 ml penicillinase to a 5 mL sample. 


Assay of test milk in screening and confirmatory test may produce the following results:

1.       No zone around disk containing untreated milk in screening test is a negative test for inhibitory substances.

2.       Zone around disk containing untreated milk but no zone around disk containing penicillinase-treated milk in the confirmatory test is a positive test for -lactam residue.

3.       Clear zone of equal size around both disks in confirmatory test indicates presence of inhibitors other than -lactam residues.

4.       Clear zone of 4 mm around penicillinase-treated milk smaller than that around untreated milk disk in confirmatory test indicates presence of -lactam residues as well as another inhibitor(s).  

The penicillin-positive control solution at 0.008 unit/mL should produce clear, well-defined zones of inhibition (16-20 mm).

If no zone of inhibition is produced by penicillin-positive control, test sensitivity is not adequate and test should be repeated. 


Also, the normal whole milk that is to serve as the control diluent should be tested before use to ensure that it exhibits no antibacterial activity against the test organism. 

Be Careful

Although the test is simple to perform, the preparation of the test ingredients requires some technical skills to prepare, in particular:

  • the viability of the culture,
  • the seeding onto the agar plates to ensure it grows correctly, and
  • the preparation of the standards to the correct concentration.

If you need help with this method, let me know.


 Charm Bacillus Sterothermophilus Disc Assay (BDSA)  

If all of the above sounds too hard to prepare, which it can be, there is a simpler method. It’s called the Charm Bacillus Sterothermophilus Disc Assay (BSDA) which contains everything you need to get started – the spores, the controls, the discs, the enzyme and the standards. 

Unlike the standard disc assay method, the BSDA also includes a colour indicator within the agar. his indicator changes colour from purple to yellow when the bacteria produce acid making easier to distinguish the zones. 

The BSDA is not only easy to prepare and use, it’s also officially approved in the US. 

BSDA antibiotic disk assay   

Stay tuned for my next issue. 


Arrow Scientific Pty Ltd

Unit 1, 332 Burns Bay Road, Lane Cove NSW 2066

Ph: 02 9427 7455  -  Fax: 02 9427 7456  -   www.arrowscientific.com.au


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