Publisher Summary

This chapter discusses the screening methods for detecting bacteriocin activity. It also discusses the plating methods. The primary means for detecting bacteriocin activity of the lactic acid bacteria is the agar plate diffusion assay. Agar plate assays are popular for screening of antagonistic activity, monitoring expression, optimization, and inactivation of bacteriocins for their characterization. While these plating methods are straightforward, they are not without certain problems and limitations, as lactic acid bacteria can produce other antagonistic compounds and create conditions that mimic bacteriocin activity. Liquid methods are not as commonly used for the screening of bacteriocin activity. While plating methods are simpler and quicker to do, the use of liquid methods usually presents greater option for closer examination of bacteriocin activity. Assessment of the efficacy of a bacteriocin or bacteriocin-producing culture in a model food system is normally conducted by challenge with an indicator organism in the food and subsequent monitoring of the viability of the indicator using plate count methods.

I Historical Perspective

The demonstration of inhibitive effects between separate cultures of bacteria is well established. The earliest work includes a recorded observation by Antonie van Leeuwenhoek in 1676 that the product from one microorganism retarded the growth of another. Louis Pasteur, with J. F. Joubert in 1877, documented an antagonistic effect of bacteria from urine against Bacillus anthracis. As so often has been the case, such observations of antibiosis may not be elucidated to the point of determining the actual mechanism of action. Inhibition can be due to low molecular weight antibiotics, lytic agents, enzymes, defective bacteriophage, and other metabolic by-products such as ammonia, organic acids, free fatty acids, carbon dioxide, and hydrogen peroxide. In addition, nutrient depletion and lowering of the redox potential of the medium may antagonize growth of competing strains to some extent. Given the diversity of naturally occurring antagonistic compounds, it is often a challenge to quantify bacteriocin activity while eliminating or measuring other inhibitive effects, some of which may be synergistic with the activity of bacteriocins. Such factors in lactic acid bacteria have been earlier summarized by Klaenhammer (1988).

Total agreement does not exist for the definition of bacteriocin. For this chapter the broader and simpler description will be used, and that is, a bacteriocin is a peptide or protein produced by a bacterium that inhibits the growth of another bacterium.

In spite of numerous inhibitive factors, bacteriocins are ubiquitous in nature. As noted in the milestone review of bacteriocins from Gram-positive bacteria (Tagg et al., 1976), screening of a relatively large number of strains (100 or more) of any one bacterial species will usually offer some evidence of inhibition due to a bacteriocin. Tagg et al. (1976) exemplified this wide occurrence by summarizing 57 papers that surveyed different species of Gram-positive bacteria for strains that produced a bacteriocinlike effect. In eight studies that surveyed the production of bacteriocin activity from cultures of Staphylococcus aureus, the occurrence of bacteriocin activity ranged from 1 to 38% of the strains examined with an average of about 13%. The number of strains of staphylococci evaluated in these studies was from 65 to 2035. Five studies that screened for the presence of bacteriocin activity in group D streptococci found a range of 51 to 99% for bacteriocin-positive strains with an overall frequency of occurrence of about 74%. The number examined ranged from 77 to 108 strains.

II Agar Diffusion Techniques

A Introduction

The first antimicrobial susceptibility test that utilized diffusion of the antibiotic substance through agar media was done by Fleming in 1924 with penicillin against Staphylococcus aureus. Cutting wells into the agar to serve as reservoirs for liquid preparations of antimicrobial agents has been a popular modification (Reddish, 1929). Another approach has been to use sensitivity disks (sterile paper containing diffusible antibiotic) whereby the relationship between diameters of the zones of inhibition in lawns of indicator-seeded agar determines the minimum inhibitory concentration (MIC). Such MIC values are normally derived from serial dilutions of the antibiotic (usually twofold).

There have been many adaptations of the agar zone diffusion technique since it was first used in the testing of antimicrobial compounds (Abraham et al., 1941). Common to these techniques is the measurement of the zones of inhibition in indicator-seeded agar plates (Figure 1). The antimicrobial agent diffuses through the agar to inhibit growth of the indicator organism. With the agar diffusion method, Vesterdal (Cooper, 1964) found the gradient or zone of inhibition to be established when the charge or concentration at the source is well defined and a gradient is established by gradual exhaustion of the diffusing antibiotic (x or r;x = r − rd). There is a linear relationship between the response and the log10 dose. The zone size is a result of diffusion of the antimicrobial compound and the growth rate of the indicator organism (Linton, 1983). An antibiotic compound will diffuse through an agar at a constant rate depending upon its molecular weight, its ionic charge, and the composition of the gel. Temperature and solvent viscosity of the gel will affect this constant. Most microbiological assays are conducted isothermally, but should two different incubation temperatures be used to either prediffuse the antibiotic or to allow optimum growth temperatures to be used for the producing and indicator strains of a deferred antagonism assay, then the diffusion rate will change and the slope of an assay curve will be altered (Figure 2).

The amount of antibiotic compound will directly affect the zone of inhibition; the greater the concentration, the greater the zone diameter under standard conditions (Figure 2). The higher the amount of antibiotic substance present, the farther it diffuses out into the agar in a given time period. When there is a significant difference between the critical concentration of the antibiotic that inhibits the indicator organism under conditions of diffusion and the antibiotic concentration at the source (which is usually considered constant) the zone edge tends to be crisp. When they are similar, the zone edges may appear nondescript or diffuse (Linton, 1983).

Overnight incubation is the usual minimum length of time until measurement of zones of inhibition, but the actual location of the zone edge is fixed within a few hours of incubation, after which time the indicator organism grows outside the zone and becomes visible. This length of time for growth is determined by the type of microorganism tested for sensitivity, the nutritional status of the growth medium, the incubation temperature, and the inoculum size (Linton, 1983). Generally, the greater the inoculum, the shorter the required growth time; but too dense an inoculum results in no zones of inhibition because the initial high cell number will mask any subsequent zone formation (Figure 2). Large zones of inhibition are usually formed when the bacteria are slow growing (e.g., due to adverse temperature or minimal media), and small zones are usually formed when bacteria grow rapidly (Piddock, 1990).

In those systems in which the diffusion of the antibiotic and the growth of the indicator organism are simultaneous, all sensitive organisms within the forming zone of inhibition are initially inhibited by the critical inhibitory concentration of the antibiotic diffusing from the source. As incubation continues, cells beyond the zone grow to greater density. At the zone edge, an inhibitory concentration of antibiotic interacts with a cell density that is large enough to absorb antibiotic to an extent large enough to significantly lower the concentration of antibiotic to one that is no longer inhibitory. As the indicator grows in the log phase, the diffusing antibiotic is maintained at a subinhibitory level by the increased density of the indicator organism, and therefore the organism grows uninhibited and becomes visible.

Most antibiotics appear to be diffusible through agar gels, although in some cases this may occur slowly. In those assays in which prediffusion of the antibiotic compound is desirable because the agent diffuses slowly, (e.g., because of large molecular weight or polymeric forms), the initiation of indicator organism...