Microbiological antibiotic assays

Microbiologically based assay systems invariably measure the active antibiotic(s) or forms of the antibiotic that can be inhibitory to microorganisms. Immunological assays can measure both the active antibiotic as well as microbiologically inactive species. 

Drug/Lab test interactions Methotrexate, pyrimethamine, and most antibiotics invalidate folic acid and vitamin Bi2diagnostic microbiological blood assays. 

Antibiotics may be assayed by a variety of methods (see Chapter 8, pages 166-188, in Pharmaceutical Microbiology, 5th edition 1992). Only microbiological and radio-enzymatic assays will be considered briefly here see Figs 25.5 and 25.6 and sections 4.1.1 and4.1.2. 

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. 

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. 

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.

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

For assaying oxytetracycline content in injections, tablets, capsules, ointments, and oral suspensions, the United States Pharmacopoeia 28 [1] uses a liquid chromatography method described in the assay under oxytetracycline. For oxytetracycline and Nystatin capsules and for oral suspension, United States Pharmacopoeia 28 [1] uses a microbiological method listed under antibiotics-microbial assays <81>. 

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. 

KATZ Microbiological Assay Procedures for Antibiotic Residues... 

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]. 

The fermentation batches are incubated aerobically under stirring at 28°-30°C. At intervals the antibiotic activity is assayed microbiologically by the agar diffusion method using Sarcina lutea as the test organism. The maximum activity is reached after 96-120 h of fermentation. 

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

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