Microbial growth inhibition assays

Assays for detection of antimicrobial residues in foods are based on the microbial growth inhibition, microbial receptor, and enzymatic colorimetric formats. 

More versatile than the growth-inhibition assays and potentially applicable to determining the presence of different antibiotic residues in different matrices are the microbial receptor CHARM I and II test assays (19, 20). The Charm I test, developed exclusively for -lactams in milk, constitutes the first rapid test recognized by The Association of Official Analytical Chemists (AOAC) with a test time of 15 min (19). The speed and sensitivity of this test permitted testing of milk tankers before they unloaded at the processing plant (21). In 1984-1985, the CHARM I test was further developed to test for antibiotics beyond -lactams to include tetracyclines, sulfonamides, aminoglycosides, chloramphenicol, novobiocin, and macrolides. The extended version has been referred to as CHARM II test. 

In addition to the agar plate disk-diffusion assay, spectrophotometric methods have frequently been used to measure microbial growth inhibition. Spectrophotometric methods generally require that the test microorganism be grown in liquid... 

A preservative is a substance that extends the shelf-life of drug products by preventing oxidation or inhibiting microbial growth.14 Preservatives must be monitored in the products since they are considered to be active components. Generic HPLC assays are typically developed for preservatives such as buty-lated hydroxytoluene (BHT), an antioxidant for solid dosage forms, and antimicrobials such as parabens, sodium benzoate, or sorbic acid in liquid formulations. For these additive components, typical assay specifications are 85-115% of label claim. 

It is probable that the largest number of medicinally directed semi-synthetic agents ever produced containing a specific pharmacophore, the -lactam ring, are the penicillins, cephalosporins and mono-bactams. To these must also be added the synthetic cephems and cephams and the many variations upon those basic themes. Although current figures are not available, it was estimated in 1980 that over 10000 variations on the basic penicillin structure had been made and assayed for their ability to inhibit microbial growth (Hoover, J.R.E., personal communication). 

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. 

Figure 1.1. Representation of a typical disk susceptibility assay. A paper disk impregnated with a suspected antibiotic compound is applied to an agar plate seeded with a test microorganism and the area of inhibited microbial growth is indicated by a cleared zone surrounding the disk.

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