Agar Diffusion Method for the Assay of Colicins

Abstract

An agar diffusion method for the assay of colicins A, B, D, E 2 , E 3 , and K is described. The assays were performed in large, square pyrex dishes that contained an agar layer seeded with an indicator organism sensitive to the colicin. The samples were applied to the agar in steatite beads positioned in a randomized sequence. The plates were stored at 4 C for 24 hr to allow the colicins to diffuse into the agar. After incubation at 37 C, the activity of each colicin preparation was estimated by measuring the diameter of the zone of inhibition of the growth of the indicator strain around each bead. The results of each assay were subjected to a statistical analysis, which included an analysis of variance and calculation of the theoretical regression and the confidence interval of the assay. The size of the inhibition zones, the form of the regression, and the slope of the regression of the responses were affected by the type and concentration of the agar, the depth of the agar layer, the indicator organism, the indicator inoculum density, and the time allowed for prediffusion of the colicins. Optimal conditions for the assay of each colicin were determined. Using a four-point assay design, the relative colicin concentration of unknown preparations was estimated in terms of a standard preparation of the same colicin. The experimental error of these assays (95% confidence interval) was about ± 10%.

A high throughput screen to test the biocompatibility of water-miscible ionic liquids

“…The Agar Diffusion method is an extremely well established test for the sensitivity of microorganisms to antibiotics (e.g.ref. [42][43][44][45][46] and other toxic compounds, 47 and is used very widely in clinical laboratories. The popularity of the method is due to its simplicity.…”

Microscale Assay for Screening of Inhibitory Activity of Lactobacillus

“…The inhibitory activity of Lactobacillus species has been well documented (1)(2)(3)(4)(5)7,(11)(12)(13)15) and is usually a result of the secretion of secondary metabolites including hydrogen peroxide, organic acids, and peptides (2-5, 11-13,15). Peptides that exhibit an inhibitory effect on any other microorganisms are classified as bacteriocins (5).…”

“…Peptides that exhibit an inhibitory effect on any other microorganisms are classified as bacteriocins (5). Bacteriocins have been shown to inhibit a wide range of microorganisms including Gram-positive bacteria (2,4,5,7,(11)(12)(13)(14)(15), Gram-negative bacteria (2,5,9,10), and even different bacteria within the same species (2,5,11). The traditional method for screening of bacteriocins is the agar diffusion method (7,11).…”

“…The corresponding zones of inhibition are measured in millimeters and are used to quantify the antimicrobial activity. However, the agar diffusion assay depends directly on zone of inhibition to quantify inhibitory activity, and the activity can be influenced by the rate of diffusion of the inhibitory compound through the agar medium (2,7,9,14,15). The agar diffusion assay is not cost effective; each assay requires the use of a large amount of specialized agar plates and sterilized well-forming rings.…”

“…The MODA also allows for the rapid examination of the effects of proteolytic enzymes, temperature, and pH on the bacteriocin. These are all common tests used in the screening for the inhibitory activity of Lactobacillus and are important tools to evaluate the antimicrobial activity of Lactobacillus (1)(2)(3)(4)(5)7,9,(11)(12)(13). To test the characteristics of the bacteriocin mentioned above, Lactobacillus supernatant can be treated with several proteases and catalase to rule out hydrogen peroxide.…”