Disk-diffusion assay

Ampicillin residues in cultured fish muscle have been detected by a disk diffusion assay using Bacillus stearothermophilus (ATCC 10149) as the test organism (125). In this method, fish sample was homogenized with phosphate buffer, pH 6.0, and centrifuged. The supernate was heated at 82 C for 2 min, and analyzed according to the AOAC assay including the confirmatory -lactamase step. Recoveries of ampicillin ranged from 99 to 104%, whereas the limit of determination was 0.025-1.00 g/g. This method has also been successfully applied for the determination of amoxicillin residues in catfish muscle (126). 

Asperazine (168) showed significant leukemia-selective cytotoxicity in tests on different cell lines using the Corbett-Valeriote soft agar disk diffusion assay [187]. 

In summary, MIAs are the methods of choice when a cost-effective qualitative measure of the biological activity associated with unknown antimicrobial residues is required. When samples are received for analysis at a control laboratory or at a farm/abattoir site, there may be very limited (if any) information concerning what antimicrobial compounds the animal was treated with therefore, the use of multi-residue broad-spectrum MIAs can rapidly and efficiently identify the presence of many of the commonly used compounds. At the present time, a selection of commercial kits is available in the form of tube diffusion assays, disk diffusion assays, and swab devices. 

Cyclic disulfides have been shown to exhibit a variety of antimicrobial properties (85,88). The compounds 1,2,5,6-tetrathiacyclooctane (29a) and 1,2,5,6,9,10-hexathiacyclododecane (29b) were tested for antimicrobial activity, using a standard disk diffusion assay. The compound (29a) exhibited moderate antimicrobial activity toward Escherichia coli. 

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... 

Table 7. Inhibitory activity against Bacillus subtilis H17 and tec-assay (disk diffusion method) of isoprenoid-substituted phenols (75 pg/disk), their cytotoxic activities against HSC-2 and MT-4 cells, and other biological activities...

Toxicity. Initial assessment of the toxicity of these aryl dye molecules using a modification of the agar disk diffusion antibiotic sensitivity was inconclusive because of the limited diffusion of the compound and its intense binding to the cellulose disks. Liquid cultures supplemented with 1 mg of aryl dye dissolved in 1 ml of DMF were used to assay toxicity. Comparison of the growth of Candida lipolvtica (GSU 37-1) and Candida maltosa (GSU R-42) was made by observing the optical density at 595 nm in a Turner spectrophotometer model 380. Determination of the absorption by compound was made for uninoculated GYNB. The increase in absorbance in cultures with and without analogue was compared. 

For antimicrobial assays, there are several common methods employed. Due to its ease of operation, the most common method used is the disk diffusion method, which involves the application of a material onto a filter paper disk, and then the disk is placed onto solid medium previously seeded with the test microorganism of interest. Sometimes, the sample is dissolved in an appropriate solvent before application onto the paper disk. This method is very common in the evaluation of antibiotics and is the method adopted by the National Committee for Clinical Laboratory Standards (NCCLS). The method depends on the aqueous solubility of the antibiotics in order to facilitate diffusion through the solid medium. Essentials oils, however, are generally hydrophobic, do not readily diffuse through an aqueous medium and, therefore, the prevalence of false negatives or reduced activity might then be anticipated. 

Stapley et al. (1972) reported on the isolation of cephamycins A and B from Streptomyces griseus MA-2837. Antibiotic production was monitored using a disk plate diffusion assay with P. vulgaris MB-838 or V. percolans ATCC 2461 (Fig. 1). 

Fig. 1. Cephamycin A diffusion plate assay. This assay was performed versus P. vulgaris MB-838 in nutrient agar plus 0.2% yeast extract with 7-mm filter paper disks soaked in antibiotic solutions. Data from Stapley et al., 1972.

The standard E. coli pol Ai assay depends on the rate of diffusion of the test agent from a disk placed at the center of the plate. Results and their interpretation are then dependent on the differential inhibition of the pol A and pol Ai" strains by DNA-modifying agents. This effect is expressed as dif-... 

---