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The medicinal need for new antimicrobial treatment options is undeniable, and all around the globe efforts are being made to discover and develop novel antimicrobial drugs. In this quest, one of the most chosen paths is unearthing natural substances of plant origin that would ideally replace the synthetic compounds widely used in todays’ practices. However, in order to discover new compounds with antimicrobial activities, excellent experimental methods are needed that will positively identify all the actives, and discard all the non-active compounds, that, don’t get your hopes up, will be so many of your potential samples. Practically, problems can be encountered on the path of achieving either one of these goals.

Let’s start by mentioning that there is no official standard for assessing the antibacterial activities of plant natural products or plant extracts. The lack of standardized and reliable testing methods causes a significant obstacle in the assessment of the antimicrobial activities of these products. Even though once you enter this field of research it’s easy to understand why, it still remains a complex issue that must be treated with an enormous amount of attention. The Clinical and Laboratory Standards Institute methods (CLSI 2015) are the official standards for the testing of conventional antimicrobial agents such as antibiotics, and are an excellent starting point. Modifications of these methods can be implemented in the examination of other types of molecules, yet very often the results between publications will vary due to the differences in the research methodologies. This makes the comparison of the data from different studies quite problematic.

The most popular techniques used for the assessment of the antimicrobial activities of plant compounds and extracts are the agar diffusion methods (disc diffusion or well diffusion) and the dilution methods (agar dilution and broth macro/microdilution). The agar diffusion technique is increasingly becoming the less popular choice, mostly because it can only generate preliminary, qualitative data for the activity of the antimicrobial agent being tested. Apart from this, it is limiting to the ability of the agent to diffuse in the agar medium, so it can often generate inaccurate results for agents with low-diffusion capabilities. In contrast to the diffusion methods, the dilution methods allow for the quantitative assessment of the antimicrobial susceptibility by determining the lowest concentration of the agent capable of inhibiting the growth of the tested organism, known as the minimum inhibitory concentration (MIC). The broth dilution methods can be performed on two scales, and both macro- and microdilution methods are similar and well established. Nonetheless, due to the use of smaller amounts of medium, reagents and tested agents the microdilution method is more economical and less laborious than the macrodilution method. To perform the tests, usually a series of tubes or plates is prepared with a broth or agar medium to which various concentrations of the antimicrobial agents are added. The tubes or plates are then inoculated with a standardized suspension of the test organism. After incubation at 35 ± 2°C for 16-20h, the tubes are examined and the MIC is determined. Even under the best of controlled conditions, a dilution test may not yield the same end point each time it is performed. Generally, the acceptable reproducibility of the test is within one twofold dilution of the end point.

The main problem with the use of the dilution methods for assessing the antimicrobial activities of plant compounds and extracts is the problem with dispersion of water insoluble compounds in the growth medium. Although, this goes mostly for the broth dilution method and liquid media, unequal distribution can also be encountered using the agar dilution method with very hydrophobic compounds. Furthermore, some of these compounds/extracts are strongly colored or opaque which makes the determination of the MIC value problematic due to concealment of the turbidity of bacterial growth and the inability to distinguish this growth in strongly colored media.

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So, what’s one to do?? Well.. read a lot and try different approaches. I am working with various plant extracts, and each extract has its particularities. Some are quite strongly colored, others are stubbornly insoluble in broth media, many precipitate at higher starting concentrations. In order to surpass these difficulties, I believe my most solid approach is using three different methodologies for each extract to conclude of its activity. Apart from that, I also use emulsifying agents in some cases. Although sometimes quite necessary, I am not particularly a fan of this solution, even more so of the more complex formulations such as the phospholipid complexes which completely engulf the hydrophobic agent. In these cases, the contact between the tested organism and the agent cannot be ensured. Varying the pH in order to improve the solubility of the compound/extract can also be a solution, as long as this change is still in the pH optimum of the tested organism. I would thread carefully with this approach because of the obvious effect a pH change will have on the growth and the metabolism of the tested microorganism. A good idea would be to construct a modified growth curve at the new pH point and if its’ slope and lag phase haven’t changed much from the one in neutral pH you can conclude that this pH change will not have a significant effect on the other parameters and the effect of your agent can be truthfully assessed. Lastly, using a low concentration of the extraction solvent (or any other solvent that dissolves the agent under examination) is the most common approach to go above this obstacle. I always use this approach as a last resort, since organic solvents have an effect of their own on the structures of the tested microorganisms. Therefore, I don’t like to be unsure that the effect I am observing is not coming from the solvent itself, or from a combination of the solvent/agent in which one augments the susceptibility to the effects of the other. Organic solvents very often have an effect on the permeability of the bacterial membrane and ease the access of some agents into the cytoplasm with the partial disruption of the integrity of the bacterial membrane. I have seen a lot of papers that use DMSO at quite high concentrations in their experiments and are very sure that the effect they have observed are due to their plant agent. Are they really? We’ll never know. Finally, thanks for dedicating your attention to read my thoughts on the antimicrobial test conundrum and I hope I was able to help! Good luck discovering a new antimicrobial!

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