The History and Basic Principles of Microbial Inhibition Assays

1 The History and Basic Principles of Microbial Inhibition Assays 

Shortly after their development in the 1940s, antibiotics were used in veterinary medicine, first to prevent or treat mastitis in cows and later for the treatment of other diseases. Initial concern about antibiotic residues in milk was not a public health issue but came from dairy processors who noticed inhibition of starter cultures used in the production of cheese and yogurt, thus generating a need for screening tests to examine milk for antibiotic residues. Since inhibition of starter cultures by penicillin in milk was the main problem, the earliest microbial inhibition assays were based on growth inhibition of lactic acid bacteria. Spores of Bacillus species were also utilized spores are easier to handle and far more stable than the vegetative cells. 

Another method to determine the presence of antibiotics in milk consisted in measuring coagulation. One of the earliest coagulation tests was described in 1952 by Lemoigne et al., who used four different strains of Streptococcus lactis. A defined number of cells of each culture were added to different milk samples and incubated overnight. If antimicrobial compounds were present in the milk at a certain concentration, the milk would not coagulate. This method was further optimized by Pien and co-workers ° using S. lactis strains with a defined resistance for certain concentrations of penicillin that enabled a semi-quantitative estimation of the antimicrobial concentration within the milk sample to be obtained. 

In 1944, Foster et al. were one of the first groups to develop an agar diffusion test for the determination of penicillin in liquid samples. Agar seeded with Bacillus subtilis spores was added to a petri dish. Sterile glass cups were then filled with liquid samples containing different concentrations of penicillin and placed on the 

In 1948, Welsh et al. developed a disk plate method using paper disks soaked in milk, which were placed on agar seeded with B. subtilis spores. Whey agar was first seeded with B. subtilis spores, and paper disks (7 mm) were soaked in the milk samples. The impregnated disks were placed on the agar surface, and the plates were incubated at 37°C for 4 h, after which time the zones of inhibition were determined. The sensitivity of this test for benzylpenicillin was found to be 5 xg/l. 

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