Analysis of Data

Careful analyses of adhesion data can yield important insights into adhesion mechanisms. It is also useful to determine the numbers of combining sites (receptors) on a substratum, the affinity constant(s) describing the complex between an adhesin and its receptor, and the amount of an inhibitor required to cause a 50% reduction in adhesion. It is also important to determine the rate of an adhesion reaction and its corresponding desorption or dissociation. In a given system, the total (T) number of cells is equivalent to the combined bound (B) and unbound (U) or free cells. The knowledge of T and B data makes it possible to calculate U, or, conversely, knowledge of any two or the three entities provides a means of computing the third. In most adhesion experiments, T is known and B is determined.

The experimental protocol described in previous steps allowed us to obtain the following data:

3.4.1. Determination of the Number of Microorganisms (as CFU)

1. CFU/mL at the beginning of the experiment (Total [Bound + Free]).

2. CFU/mL adhering to the beads (Bound).

3. CFU nonadhering to the beads, after the adhesion steps (Free or Unbound).

4. Data from control tubes.

3.4.2. Plotting Procedure (see Note 3)

The general strategy in most properly designed adhesion experiments is to determine the extent of adhesion using several different bacterial cell densities but employing a constant amount of a receptor material (1). If the adhesion is directly proportional to the numbers of cells added, then Henry's law specifies that:

In actual practice, it is preferable to plot B vs log T (or U).

Another approach to the study of adhesion phenomena is through application of the Langmuir adsorption isotherm, one common derivative of the law of mass action. It has found wide usage in treating adhesion data:

Plots for U/B vs U are convenient for computing N (1/slope) and -1/K (the x-inter-cept). The N value represents the number of receptor sites, and K is the average association constant.

In the adhesion of microorganisms to surfaces, linearity may be observed only when reasonably narrow cells densities are employed.

The third approach to treat the adhesion data is through application of the Scatchard equation:

This method is probably of more value in microbial adhesion. A plot of B/U vs B yields a straight line with a slope of -K, and an ordinate intercepts yields N x K. As with the Langmuir equation, linearity is achieved only with a system that possesses site-site homogeneity and is free of cooperative phenomenum .

The extent of cooperativity (positive or negative) may be assessed by use of the Hill equation:

nH is the Hill coefficient, or index of cooperativity. When nH > 1, there is positive cooperativity. Similarly, negative cooperativity is indicated when nH < 1.

Finally, adhesion data should be subjected to statistical analyses, such as the parametric (unpaired I) test or unpaired (Mann-Whitney U) test specifically when comparing bacteria and/or substrata.

4. Notes

1. Once the suspension of microorganisms is obtained, they can be stored under refrigeration for no more than 1 h for the adhesion assay.

2. It is recommended to carry out the assay in a thermostatized shaker to favor the contact of the bacteria with the inert support.

3. After the plotting step, these data are used as the basis to determine the effect of different substances on the degree of adhesion. The value of 50% adhesion is taken from that plot, meaning to the number of bacteria able to bind to the beads in a value of 50%. This is the first reference point when studying the effects of the different substances that can increase or decrease this value by promoting or diminishing the potential adhesion of the bacteria under study. Many different substances can be studied in terms of their effect on the adhesion pattern of each strain:

a. Those related to the physical-chemical conditions of growth of the microorganisms i.e., culture media, pH, temperature.

b. Other substances which are present in the oral cavity, such as compounds of saliva, proteins, ions, solubles, and insoluble glucans, glucosyltransferase, fibronectin, and others that can affect or interfere in the adhesion phenomenon.

c. Some chemical products (such as proteolytic enzymes, saliva amylases, or other compounds) that can be tested to study the nature of the adhesion determinants are used to determine the chemical components of the bacterial surface.

Acknowledgments

This work was supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas de Argentina (CONICET), PIP 359.

References

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5 Ahumada, M. C., López, M. E., Colloca, M. E., and Nader Macias, M. E. (2001) Lactoba-cilli isolation from dental plaque and saliva of a group of patients with caries and characterization of their surface properties. Anaerobe 7, 71-77.

6. Marshall, K. C., Stout, R., and Mitchell, R. (1971) Mechanisms of initial events in the sorption of marine bacteria to surfaces. J. Gen. Microbiol. 68, 337-348.

7. Marshall, K. C., Savage, D. C., and Fletcher, M. (eds.) (1985) Mechanisms of bacterial adhesion at solid-water interfaces. In: Bacterial Adhesion. Mechanisms and Physiological Significance. Plenum, New York, pp. 133-161.

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