Guest editorial

 

 

Agar diffusion: a reliable method?

The agar diffusion method, which is widely used in the assessments of the antimicrobial potential of various solutions, medicines or endodontic cements, is now being rejected for publication in indexed high-impact journals in Dentistry. Over the past ten years there was a considerable decrease in the number of articles that use this method in indexed journals, once studies with this experimental model are usually returned to authors before going through the peer review process. Although it appears radical, it is not a baseless decision, and this directs us to consider about this experimental model [1].

The agar diffusion methodology was originally developed to assess bacterial susceptibility to systemic antibiotics, in which the measurement of the antimicrobial effect on a given bacterial strain is based on the measurement of the zone of inhibition formed in the agar after contact with a disc containing antibiotic. Considering the differences in the pattern of diffusion of the materials being tested and the possibility of chemical interaction between the material and the chemical components of agar, decades were needed to develop the methodology in order to define and standardize the chemical substances involved in the test, thus making reliable results. To make the measurement of bacterial susceptibility a comparison curve was established so that the zone of inhibition of bacterial growth around the discs containing antibiotics was compared to the clinical concentrations of the antibiotic found in the human serum, thus helping to determine the clinical effectiveness of antibiotic in a given bacteria. Besides, reference strains of known origin and previously measured susceptibility to antibiotics are used as control in all tests. Thanks to this strict standardization, most aerobic and facultative bacteria can be reliably tested with regards to their susceptibility to antibiotics, but an internationally accepted standardization for assessing the anaerobic microorganisms in this aspect has not been reached so far [2].

However, when analyzing the antimicrobial potential of solutions or medicines with assumed disinfectant action, there is no standardization of the culture medium, neither of the substances to be tested. Moreover, the chemical reactions between agar and materials being tested are studied with low frequency, and therefore they are practically unknown. Nevertheless, the occurrence of any chemical interaction may lead to misinterpretation of the results on the antimicrobial potential of the material being tested. Materials with different chemical behaviors in relation to the culture medium may cause differences in the "zones of inhibition" that reflect these chemical interactions, so that the measurement of the antimicrobial action becomes inseparable from these reactions.

The culture medium sometimes presents, for example, buffering capacity. When testing materials which antimicrobial action is due to their high pH, their antimicrobial activity will certainly be underestimated, because in buffering medium the high PH of the material is neutralized, thus resulting in a small zone of inhibition.

It is also common to have a false positive result in the assessment of the antimicrobial action of given substances in agar. EDTA (ethylenediaminetetraacetic acid), a substance commonly used to remove smear layer during root canal biomechanical preparation, presents large zones of inhibition in agar tests. However, when the direct contact or minimal inhibitory concentration methodology is applied, EDTA has no antimicrobial power. That is, EDTA is capable of removing ions cations in agar, thus making it unsuitable for bacterial growth. Therefore, the zone of inhibition in these cases is not due to a real antimicrobial activity, but only to the chemical interaction between acid and agar.

It is imperative to emphasize that the objective of any in vitro experiment is to simulate and estimate within certain limitations the behavior of the material in vivo. That is, the objective of an antimicrobial test performed through the agar diffusion method shall be to determine the behavior of materials in a clinical situation. But the unknown chemical interactions between material and agar may cause a bias in interpreting the results, thus leading to conclusions that tend to overestimate or underestimate the antimicrobial potential of some substances. Considering that there is no evidence that the observation of a zone of inhibition on agar plates is capable of determining the clinical behavior of a material, we conclude that this methodology adds nothing to knowledge and, most important, may cause a skew in the clinical selection of different materials in several areas of Dentistry.

The assessment of the antimicrobial activity of substances used in Dentistry must therefore be performed through other methods, including the direct contact one. In this test, the bacterial suspension is placed in contact with other substances being tested for predetermined periods of time, and after that aliquots of the material are removed. If the objective is the assessment by the presence of turbidity, indicating bacterial growth, parts of the mixture of microorganisms with the tested solutions are incubated in liquid medium. If the analysis is quantitative, the material shall be diluted and aliquots of these dilutions are put on agar plates, incubated, and the CFU number is calculated [3].

When searching for "agar diffusion test" in PubMed with focus on Dentistry journals and periodicals from 2000 to 2010, 106 titles are found, of which 35 are by Brazilian authors. The high prevalence of Brazilian researchers using agar diffusion to asses the antimicrobial activity of antiseptics indicates that there is still lack of information on the limitation of this method and the risks of misleading conclusions when using it. Therefore, following the trend of high-impact journals in Dentistry, the editorial board of the South Brazilian Dentistry Journal agrees that studies developed with the objective to compare the antimicrobial capacity of materials or solutions in agar will not be accepted here, thus being immediately returned to authors.

 

Fernanda Geraldes Pappen
Professor, Doctor in Endodontics
Faculty of Dentistry
Federal University of Pelotas (UFPel)
Pelotas – RS

Erick Miranda Souza
Professor, Doctor in Endodontics
Faculty of Dentistry
University Center of Maranhão (Uniceuma)
São Luís – MA

 

References

1. Editorial Board of the Journal of Endodontics. Wanted: a base of evidence. J Endod. 2007;33(12):1401-2.

2. Haapasalo M, Qian W. Irrigants and intracanal medicaments. In: Ingle JI, Bakland LK, Baumgartner JC (ed). Ingle's endodontics. 6. ed. Hamilton: BC Decker; 2008. p. 992-1018.

3. Siqueira Jr JF, Uzeda M. Influence of different vehicles on the antibacterial effects of calcium hydroxide. J Endod. 1998;24:663-5.

1. Editorial Board of the Journal of Endodontics. Wanted: a base of evidence. J Endod. 2007;33(12):1401-2.         [ Links ]

2. Haapasalo M, Qian W. Irrigants and intracanal medicaments. In: Ingle JI, Bakland LK, Baumgartner JC (ed). Ingle's endodontics. 6. ed. Hamilton: BC Decker; 2008. p. 992-1018.         [ Links ]

3. Siqueira Jr JF, Uzeda M. Influence of different vehicles on the antibacterial effects of calcium hydroxide. J Endod. 1998;24:663-5.         [ Links ]