Diffusion test, agar-plate

The Sarcina lutea test is the official US Food and Drug Administration (FDA) test for detecting penicillin residues in milk and dairy products (41). In this test, milk samples are placed in stainless steel cylinders on an agar plate seeded with Sarcina lutea ATCC 9341. As milk diffuses into the agar, inhibitors prevent the growth of the organism, causing a zone the width of which is a measure of the antibiotic concentration. The test is sensitive to about 0.006 g/ ml penicillin G, and confirmation of positive results can be performed by the addition of penicillinase. 

That both components were antibodies was established by agar diffusion tests with these preparations and solutions of the glycan performed in the usual manner. The results of the agar diffusion tests are shown in Figure 3, right plate. The two preparations of antibodies have been designated as anti-galactose (anti-gal) antibodies and anti-lactose (anti-lac) antibodies and some of the properties of the two sets of antibodies are described in a later section. 

The fate of tubercle bacilli or E. coli in human or bovine serum was determined not only by the use of the agar-plate diffusion test but also by experiments performed in serum-containing test tubes. In tube experiments, serum is inoculated with bacterial suspensions adjusted to contain a known number of bacteria in the inoculum. Infected sera are incubated at 37 °C and, after various periods of time, bacterial numbers are determined by plating the samples on the growth-supporting IPAM. The number of developed colonies on agar medium shows the number of bacteria in the experimental tubes. 

Diffusion in agar performed by the conventional method shows a strong reaction between the antibody and Man-BSA, but not with BSA (Fig. 12, plates A). However, both compounds give precipitin complex with anti-BSA serum. Oxidation of the antigens with periodate no longer gives a precipitin reaction with the anti-mannose antibodies, but has no effect on the anti-BSA antibodies. Hapten-inhibition results are also shown in Fig. 12, plates C and D. These tests are conducted with the purified antibody... 

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... 

The historical gradient plates, ditch-plate and cup-plate techniques (see Hugo 8c Russell, 1998) have been replaced by more quantitative techniques such as disc diffusion (Fig. 11.4), broth and agar dilution, and E-tests (Fig. 11.5). All employ chemically defined media (e.g. Mueller-Hinton or Iso-Sensitest) at a pH of 7.1—1 A, and in the case of solid media, agar plates of defined thickness. 

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. 

Okerman et al. compared the performance of a Tetrasensor with three microbial inhibition assays for the analysis of tetracycline antibiotics in tissue. The group concluded that when large numbers of samples have to be analyzed without the requirement for immediate results, classical agar diffusion tests with thin plates and performed as prescribed for the EPT still seem the most economical choice. However, the receptor-based test Tetrasensor was recommended for use in official surveys and also in cases when immediate results are required. Unlike the inhibition tests, this receptor test does not require a well-equipped laboratory for use and is more suited for the meat industry. ... 

The agar diffusion method (Kirby—Bauer) is also sometimes used for the evaluation of antibacterial activity of textiles. This is a relatively quick and easily executed semiquantitative method to determine antibacterial activity of diffusible antimicrobial agents on treated textile material. The bacteria are grown in nutrient broth medium and after appropriate dilution (e.g., lOOx) from the culture, test organisms are swabbed over the surface of agar plates. Ten-millimetre-diameter disks of the test fabric and control fabric are then gently pressed onto the surface of the plate. The plates are then incubated at 37 °C for 18—24 h. The antibacterial activity of the fabrics is demonstrated by the diameter of the zone of inhibition in comparison to the control textile sample. 

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