**5. Methodologies**

Medicinal use of plants is conducted through different ways, according to geographical availability, historic tradition and known efficacy and may include preparation of aqueous infusions and beverages using dried plants, tinctures (plants in alcoholic solutions), inhalation of steam from boiling preparations, ingestion of fresh or cooked parts as ingredients, preparation of topic ointments, essential oils, among others. However, the real scientific and therapeutic value of the plant properties, like antimicrobial potential, needs to be evaluated through proper techniques. The screening of antimicrobial activity may be pursued on two different pathways:


Antimicrobial Activity of Condiments 123

fail to grow in broth but are cultured when broth is plated onto agar and the bactericidal concentration is considered to be the lowest concentration at which bacteria fail to grow in

For susceptibility tests controls must be performed simultaneously, usually using the antibiotics most suited for the conventional treatment. Tetracycline was used for comparison with thirteen Thai condiments against *Vibrio parahaemolyticus* (Vuddhakul et al., 2007) and fifteen antiobiotics were used when testing the antimicrobial activity of condiments against multidrug resistant *Escherichia coli* isolated from water in Bangladesh (Rahman *et al*., 2011). Comparison with conventional antibiotics is necessary to categorize the sensibility of a

A rigorous evaluation of the individual MIC is sometimes difficult because of the ambiguity of results. Eugenol inhibited 9 out of 14 Gram negative and 12 out of 20 Gram positive bacteria, at a concentration of 493 ppm, by incorporating in Plate Count Agar. But the same paper reported a MIC of 32 ppm for *Candida glabrata* and *Aspergillus niger*, and a MIC of 63

The variation in concentration of antimicrobial substances is usual and expected. This, coupled with testing different preparations contributes for the difficulty of comparing results from study to study. For instance, allicin in garlic ranges from 0.3 to 0.5%, whereas

The problem is worsened by the fact each researcher may use different methods for preparing the samples: crude, aqueous extracts, ethanolic or methanolic extracts, chloroform or other solvents, as refered above. The results from testing the inhibitory effect of essential oils is not easy to ascertain, as the hydrophobic nature of such preparations may alter the inhibition areas because of the irregular diffusion, when compared with the more hydrophilic antibiotics. Some researchers add emulsifiers, as Tween 20 or Tween 80, to the oils, but the quantity and nature of the latter must not interfere with the results, like producing false-positive ones. Thus, standardisation of methods becomes difficult to achieve

There are other methodologies available to use in order to ascertain the effects of antibacterial activity of certain phytochemicals. The rate of inhibition and cell death can be observed through time-kill analysis and survival curves. The physical aspects of antibacterial activity concerning the structural modifications achieved can be observed by

Yeasts susceptibility can be evaluated by techniques similar to those used for bacteria, however the evaluation of antifungal ability of phytochemicals must be performed by other methods. One of these methods is the spore germination assay. Spores are put in contact with the testing compound for a period of time; afterwards they are observed microscopically in a slide (usually fixed with lactophenol cotton blue) and spore

In terms of antiviral ability of plant products, they can be determined by observation of cytopathic effects or plaque formation in cells infected and put in contact with the phytochemicals. Other option is, in the same conditions, to use molecular techniques for

detection of products resulting from viral replication, as nucleic acids.

broth and are not cultured when broth is plated onto agar (Burt, 2004).

specific microorganism to a plant, herb or condiment as sensible or resistant.

ppm for *Staphylococcus aureus* and *Escherichia coli* (Cowan, 1999).

eugenol in clove ranges from 16 to 18% (Shelef, 1984).

the use of scanning electron microscopy (Burt, 2004).

germination (or its absence) is observed.

(Nascimento *et al*., 2006).
