Effect of various solvents on bacterial growth in context of determining MIC of various antimicrobials

摘要

Dimethyl sulfoxide (DMSO) and ethanol are frequently used as solvent for natural as well as synthetic antibacterial compounds, in order to determine their MICs. Effect of these solvents on bacterial growth is an important factor to be considered, while considering reproducibility of experiments for MIC determination. Present study aimed at determining the effect of different concentrations (1% to 6%) of DMSO, ethanol, and methanol on the growth of five different bacteria. DMSO scored better followed by methanol and ethanol, in terms of their compatibility with MIC determination. Lower concentrations of solvents, which apparently do not affect the bacterial growth significantly, may still potentiate the effect of antibacterial compound under test. Introduction Due to rising incidence of drug resistance among pathogenic bacteria, new antibacterial compounds are constantly being searched5. Potency of any newly reported antibacterial preparation can be quantified and compared with those already known by determining its MIC value. Minimum Inhibitory Concentration (MIC) is defined as the lowest concentration of antimicrobial agent required to inhibit growth of a test organism over a defined interval related to the organism’s growth rate, most commonly 18 to 24 h. MIC has been accepted as the most basic laboratory measurement of the activity of an antimicrobial agent against an organism7.Conventionally broth dilution method is applied for measuring the MIC. It requires preparing various dilutions of the compound under test in a suitable solvent. In case of natural products, generally extraction is carried out using solvents of varying polarity, ethanol and methanol are most commonly applied. To quantify antimicrobial activities, extracts have to be dried. Frequently it is difficult to resolubilize extracts even in the solvent originally used. In serial dilution assay the solvent has to be miscible with water. Water frequently doesn’t dissolve the intermediate polarity or non-polar components of a dried extract. An alternative is to use solvents such as methanol, ethanol or DMSO. Selection of appropriate solvent is one of the most significant factors which can influence MIC measurements in vitro. Ethanol3 and DMSO6 are preferred since they are miscible with water. DMSO is a highly polar, stable substance with exceptional solvent property6. However, DMSO1, ethanol4 and other solvents used in various bioassays have been reported for their antimicrobial effect2. Thus it becomes essential to ensure that the final concentration of the organic solvent is not likely to interfere with the bioassay (MIC determination). It should also be noted that each organism may exert varying susceptibility to these solvents.Present study aimed at determining the effect of different concentrations (1% to 6%) of DMSO, ethanol, and methanol on the growth of five common bacterial pathogens. Materials and Methods Microorganisms Staphylococcus epidermidis MTCC 435, Pseudomonas oleovorans MTCC 617, Vibrio cholerae MTCC 3906, Shigella flexneri MTCC 1457 and Salmonella paratyphi A were used as test organisms. All the bacterial strains except Salmonella paratyphi A were obtained from the Microbial Type Culture Collection (MTCC), Chandigarh, India. S. paratyphi A was obtained from the Microbiology Dept., Gujarat University, Ahmedabad.Growth MediumMueller Hinton (MH) Broth (HiMedia, Mumbai, India) Solvents Dimethyl sulfoxide (DMSO) (sd fine chemicals Ltd., Mumbai, India), methanol (Merck, Mumbai, India), and ethanol (Baroda chemical industries Ltd, Vadodara, India). ProcedureVarying volumes of MH broth and respective solvent were mixed in different test tubes (table 1) so that concentration of the solvent ranged from 1-6 % v/v. To this was added 500 μL of inoculum, which was prepared from overnight growth of the test organism, so as to match the turbidity of 0.5 McFarland standard. Total volume in each of the tube was thus made 2 mL. A tube containing growth medium (1