Microbiological assays of antibiotics

The potencies of some antibiotics described in the European Pharmacopoeia are determined by microbiological assay (Microbiological Assay of Antibiotics 2.7.2... 

The procedure employed for the establishment of the chemical reference substances used in these assays has been previously published (Sandrin et al. 1997). The CRSs for the microbiological assays of antibiotics are first submitted to the chemical tests of the monograph. If the results are satisfactory, a collaborative microbiological assay is carried out, using the International Standard as calibrator. Thus, these reference substances are considered to be secondary reference substances since they are calibrated against existing standards. Potency is expressed in International Units. If an International Standard does not exist, European Pharmacopoeia Units are used. 

Sandrin J, Daas AGJ and Charton E (1997) Establishment of reference substances for the microbiological assay of antibiotics. Pharmeuropa 9 327-330. 

Kersey, Roger C., and Fink, Frederick C., Microbiological Assay of Antibiotics. 1... 

Standard Microbiology Assay of antibiotic susceptibility. Bacteria suspensions in saline ( 108 cell/mL) were assayed by Disk Method (DM) or Serial Dilutions Method (SDM) using automated analyzer Vitek (bio Meriuex, France). 

Microbiological Assay of Antibiotics. By Roger C. Kersey and Frederick C. 

A more precise assay of pancreatin can be made by an adaptation of the plate-diffusion method as used in the microbiological assay of antibiotics (see p. 814). The four point assay procedure is employed, that is, two dilutions of the standard preparation are compared with two equivalent dilutions of the test preparation, the stronger dilution in each case being four times that of the weaker. Large, flat-bottomed plates or trays are preferred, thus allowing an 8 X 8 Latin square design to be used, but petri dishes are also satisfactory. 

Microbiological Assays of Antibiotics (Antibacterial Agents) and OF Vitamins (Bacterial Growth Factors)... 

As is the case with antibiotics, the microbiological assay of vitamins is time consuming, and a considerable level of microbiological expertise is required. The... 

At the concentrations at which organic mercurials such as phenylmercuric nitrate, thiomersal, etc., are used as preservatives in pharmaceutical and cosmetic preparations microbiological methods of assay are frequently more reliable than are chemical ones. Either the plate-diffusion or the tube-dilution technique, as described on pp. 814 to 826 for the assay of antibiotics, can be used. The former is probably the method of choice although the latter is the more sensitive, being able to detect the organic... 

In the commonest form of microbiological assay used today, samples to be assayed are applied in some form of reservoir (porcelain cup, paper dise or well) to a thin layer of agar seeded with indicator organism. The drug diffuses into the medium and after incubation a zone of growth inhibition forms, in this case as a circle around the reservoir. All other factors being constant, the diameter of the zone of inhibition is, within limits, related to the concentration of antibiotic in the reservoir. 

Fig. 5-9 Decision tree used to accept or reject results from collaborative trials to establish the potencies of antibiotics to be used as chemical reference substances for microbiological assay standards.

United States Pharmacopoeia 28 [1] describes a microbiological method under antibiotics-microbial assays for the analysis of OTC and nystatin capsules, OTC and nystatin for oral suspension, OTC HC1 and hydrocortisone ointment, and OTC HC1 and polymyxin B sulfate ointment. The methods are relative rather than absolute, which are based on the determination of the level of oxytetracycline by a microbiological response to a series of standard oxytetracycline concentrations by a... 

The first study was performed by Venturini [97, 98] in both rats and dogs by using a microbiological assay (i.e. agar diffusion test and S. aureus 209 P FDA as test organism). Conversely from rifampicin, whose serum levels were already detectable 30 min after the administration and still measurable after 48 h, only trace amounts (i.e. 0.2 pg/ml) of rifaximin were detected in serum of fed rats 4 h later (fig. 6). The amount of detectable antibiotic was reduced by 50% in fasted animals. Similar results have been obtained in dogs after oral administration of 25 mg/ kg of both rifamycin derivatives [97, 98], No detectable amount of rifaximin was found in serum at any time. 

Microbiological assays are the primary assay method for antibiotics. They provide sensitive but non-selective methods. A variety of microbiological methods have been described for the assay of gramicidin. The official method in the United States is the turbidimetric method described in the Code of Federal Regulations127 12 . 

Overall, microbiological assay methods have been the most sensitive of all assay systems and the ability to measure residues in the ppb to ppm range is common and has been for over 20 years (1 ). However, most of the residue assay systems lack specificity and require confirmation by spectral systems for a proper identification of the individual antibiotic or the antibiotic family. 

The earliest methods for detecting antibacterial residues in food appeared in 1945-1948, soon after microbiological assays for the evaluation of antibiotics 687... 

Besides physicochemical methods, the use of microbiological growth-inhibition assays to test meat and milk for the presence of antibiotics residues is popular over a long period of time. These tests use antibiotic-sensitive bacterial reporter strains, such as Bacillus subtilis and Bacillus stearothermophilus var. calidolactis. These bacteria are inoculated under optimal conditions with and without sample. After culturing, results are read from visible inhibition zones or from the color change of the bacterial suspension in agar gels [6]. 

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