Agar plate diffusion assays

Due to the inherent variability of these assays either by agar-plate diffusion measurement or turbidimetry measurement, the fiducial hmits are calculated according... 

Waksman and Reilly have summarized the factors which have a bearing upon the choice of the method to be employed in measuring quantitatively the activity or potency of an antibiotic substance. They have cited literature references to the more commonly used methods. Loo and coworkers have described a suitable method for the quantitative determination of streptomycin by the filter paper disc, agar plate diffusion technique using Bacillus subtilis as the test organism. The procedure proved satisfactory in the assay of surface and submerged culture beers and of preparations obtained in isolation and purification... 

Microbiological assay by the agar plate diffusion method with a sensitive strain of an organism such as Sarcina lutea or Bacillus subtilis has been used to assay amoxicillin in biofluids [179,180,181], However, this lengthy and not very sensitive method has been largely superceded by chromatographic or chemical methods. 

Several factors affect the diffusion assay and must be controlled carefully. The depth of the agar in the cylinder-plate system must be minimal, as thin as possible and as uniform as possible to maximize diffusion of the analyte. 

For growth bioassays using solid media, modification of the agar diffusion assay used by Flores and Wolk [108] has been shown to provide reliable results [100]. Gross et al. [100] spotted methanol and ethanol extracts on 1% agar plates, dried the extracts in sterile air, and then overlaid the plate with a suspension of indicator cells in 1% agar. After several days of incubation, areas (zones) of no growth were measured and quantification of the inhibitory effects of the test extracts was estimated by comparison with/to the serial dilutions and controls. 

The United States Department of Agriculture (USDA), Eood Safety and Inspection Service (ESIS) developed a bioassay system which incorporates a seven-plate agar diffusion assay that can detect and quantify a range of antibiotic residues found in meat and poultty products. The USDA/FSIS system utilizes bacteria that are relatively sensitive or resistant to a particular class of antibiotic. The bacteria are used in combination with specific antibiotic test agars and four pH-specific, buffered sample extracts. If a detectable antibiotic residue is present in a sample, it produces a zone of clearing (inhibition) on one or more of the test plates. Certain antibiotic residues can be identified according to their characteristic patterns of inhibition. A chart called an antibiogram was developed that depicts expected patterns of inhibition that specific antibiotics are anticipated to produce on the seven plates. 

Antibiotic diffusion assays are based on the technique of allowing an antibiotic to diffuse throu an agar gel which has been previously seeded with a sensitive test organism. This diffusion may be of two types (a) linear diffusion, i. e., by bringing the antibiotic in contact with a column of seeded agar in a capillary or test tube and (b) radial diffusion around a suitable reservoir on a seeded agar plate. 

These methods are not generally applicable to the assay of supplemented feeds or biologic fluids because of the extremely low concentrations employed. Probably the first practical assay of an antibiotic was the plate diffusion method devised by Abraham et al. (1) in England. This assay made use of seeded agar plates the antibiotic was allowed to diffuse from holes cut in the agar. This method with slight modifications is the official method of the Food and Drug Administration and is described in detail below. 

The following antibiotics are also available commercially amphotericin B, framycetin sulphate, gramicidin, N.F., and kanamycin sulphate. Each of these can be assayed microbiologically for potency by the plate-diffusion method using a suitable medium, organism and pH value in the agar. 

By plate-diffusion The assay medium contains Oxoid peptone 10 g, Lab-Lemco 15 g and agar powder 15 g made up to 1 litre with water and adjusted to pH 7-2. For seeding the medium use about 0 4ml of a sensitive strain of Staphylococcus aureus (the Heat ley penicillin assay strain is suitable) grown for twenty-four hours in nutrient broth to each 100 ml of agar medium. 

Another reason for careful interpretation of the disk-diffusion assay is that it is subject to false-positive results that can be misinterpreted as antibiotic activity. For example, physical characteristics of the extract (viscosity, pH, etc.) can generate small zones of growth inhibition when bacteria are inoculated directly onto the surface of the agar plate. In addition, we have observed that some primary metabolites can inhibit growth when tested at high concentrations. It is also possible that simple molecules, or extract degradation products, can exhibit mild antibiotic properties. For these reasons, it is important that replicate extracts are tested and that small zones of inhibition are interpreted with caution. It is also important to clearly state the concentrations tested, even if naturally occurring concentrations are not known, so that activities can be reproduced and evaluated at a later time. 

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... 

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