An in vitro study of the antimicrobial effect of Indigofera daleiodes plant tinctures using Disc Diffusion and Well Diffusion Assay

Title: An in vitro study of the antimicrobial effect of Indigofera daleiodes using Disc Diffusion and Well Diffusion Assay Background With the steady and consistent rise of antibiotic resistance (WHO 2014) the health care sector around the world is currently under much stress because of bacterial infectious diseases. Many pharmaceutical companies across the world are trying to develop new types of antibiotics, which is a very difficult and expensive process (Gould and Bal 2013). Hence alternative therapies like Phytotherapy, Homeopathy are being looked into more as possible alternative areas of treatment (van Vuuren 2009: 462-472). Aim of the study The purpose of this study was to determine the effect of various ethanolic extracts of Indigofera daleoides against Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecalis, Klebsiella pneumonia and Escherichia coli using the Disc Diffusion and Well Diffusion Assay microbiological methodologies. The efficacy of the tinctures was compared against the ciprofloxacin antibiotic and ethanol as positive & negative controls, measuring the minimum inhibitory concentration of each tincture according to the antibacterial potency they possess. Methodology Disc Diffusion and Well Diffusion Assay were used to investigate the antimicrobial effects of the Indigofera daleoides tinctures (derived from roots, leaves and whole plants). Ciprofloxacin was used as a positive control while 62% ethanol was used as the negative control. The tincture was prepared at a homeopathic dilution level (i.e. 1:10) according to the German Homeopathic Pharmacopoeia specifications (Bunyens 2005). Disc Diffusion Assay: Fifteen mueller-hinton agar plates were made. Each bacterium was then grown on three agar plates to make the experiments more valid i.e. 3 trials. Then six impregnated discs were placed at equidistance on the agar surface of each plate i.e. 3 discs were impregnated with the Indigofera daleoides tinctures (i.e. derived from the root, leaves and whole plant),1 disc was impregnated with 62% ethanol, another ciprofloxacin and the sixth one was plain. The 15 plates were then stacked and put in an incubator room at 37°C for 24hours. The results were then recorded by looking and measuring the zones of inhibition. Mixed factorial ANOVA was used to test the difference across the treatment groups. Oneway ANOVA was used to compare the differences between the whole plants, leaves and roots. Additionally, an independent t-test was further used to compare the differences between the two techniques employed. This was all done using SPSS version 25. Results The results of this study showed that all the Indigofera daleoides plant tinctures had no significant inhibitory effects on the selected panel of bacteria. Ciprofloxacin showed significant potency against all the bacteria, whilst ethanol was only slightly effective for some bacteria. Conclusion This study concluded that Indigofera daleoides plant tinctures in 62% ethanol are ineffective in inhibiting in vitro growth of the any of the selected panel of bacteria using Disc Diffusion and Well Diffusion Assay