Basics

Detection of inhibitors and residual anti-infectives in milk

Microbiological inhibitor test systems are the most generally accepted world-wide for the detection of inhibitors or residual anti-infectives in milk. Microbiological inhibitor tests are especially suitable for routine inspections on account of their low cost, easy execution, relatively broad detection range and, in some cases, short test time (e.g. 2-3 hours for the most widely used test bacterium, Geobacillus stearothermophilus var. calidolactis C953).
 

Test principle of the microbiological inhibitor test

Microbiological inhibitor tests operate by making use of the sensitivity of specific micro-organisms - so-called test bacteria - to antibiotics, sulfonamides and other inhibitors. The inhibitors lessen or prevent metabolic activity and thereby also the growth of the test bacteria. The thickness of the bacteria or the opacity of the test medium can be used as a gauge to measure the amount of test bacterium growth. Acidification and reduction processes are used to measure metabolic activity with the help of coloured indicators. The colour of the indicator changes in the acidification process in response to the acidic metabolic products of the growing test culture. In the reduction process, such as the Brilliant Black Reduction Test, the growing test culture reduces specific indicators (e.g. brilliant black), which also leads to a colour change (e.g. from blue to yellow). Activity can however also be measured using other indicators, such as pH value, degree of acidity, amount of lactic acid present, curdling time, or conductivity.

One of the best-known and most widely-used procedures is the Brilliant Black Reduction Test (BRT).
 

Test principle of the Brilliant Black Reduction Test (BRT)

With the BRT, the test medium is contained in cavities, or wells, in microtiter test plates or ampoules. This test medium is a mixture of nutrients, test bacteria (Geobacillus stearothermophilus var. calidolactis C953), brilliant black, and other supplements which help improve detection sensitivity towards chosen inhibitors. The milk samples are pipetted into the wells. Any inhibitors present can then diffuse throughout the test medium. During incubation the growing test bacteria shift the redox indicator (brilliant black) to its yellow or colourless reduction stage through the division of double azocompounds, and the test medium changes from blue to yellow. If inhibitors are present in the sample, growth will be minimal or non-existent. There will then be no reduction of the colouring agent, or to only a very small degree, and the test medium will remain blue.