Journal of Bioscience and Biotechnology Discovery Volume 3. Page 59-64. Published 17th April, 2018 ISSN: 2536-7064: Article Number: JBBD-01.02.18-063 www.integrityresjournals.org/jbbd/index.html Full Length Research Preparation, characterization, and well diffusion assay of Silver and Selenium nanoparticles against Biofilm forming Staphylococcus aureus Poonam Verma 1 * and Sanjiv Kumar Maheshwari 2 1 School of Biotechnology, IFTM University, Moradabad, India. 2 Institute of Bio-science and Technology, Shri Ramswaroop Memorial University, Lucknow-Deva Road, India. *Corresponding author. Email: poonam.phdbiotech@gmail.com Copyright © 2018 Verma and Maheshwari. This article remains permanently open access under the terms of the Creative Commons Attribution License 4.0, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Received 1st February, 2018; Accepted 15th March, 2018 ABSTRACT: In this study, biofilm-forming isolates from 36 clinical samples (Urine, Blood and Pus) were analyzed phenotypically via well-diffusion of nanoparticles, using Congo Red Agar, Tube and Tissue Culture Plate methods. Silver (Ag) and Selenium (Se) nanoparticles were synthesized by chemical reduction method. Antimicrobial activity was performed by well diffusion test of Ag and Se nanoparticles against biofilm forming Staphylococcus aureus strains. Distribution pattern showed the highest isolation rate from pus 30 (83.33%) followed by blood 4 (11.11%), and urine 2 (5.56%). All strains 36 (100%) were found to be non-biofilm forming S. aureus using CRA method. Another method for biofilm formation was performed by TM, 5 (13.89%) was strongly positive while 20 (55.56%) isolates unable to produced biofilm producer, whereas TCP method showed 12 (33.33%) high biofilm producer while 16 (44.44%) were non biofilm producer. Ag-NPs showed morphology average size and shape with scanning electron microscopy (SEM) reveals spherical particles with the size of 80.32 nm whereas, Se-NPs showed the size of 74.29 nm with SEM. In well diffusion assay, 50 μl of the nanoparticles (silver and selenium) and 50 μl antibiotic (amoxicillin as a positive control) were used. In this study, silver nanoparticles were found to have the active inhibitory effects against biofilm forming S. aureus strains (SA12, SA15, SA32), whereas selenium nanoparticles demonstrated low inhibitory effects (SA23). Key words: Biofilm-forming, nanoparticles, Selenium, Silver, Staphylococcus aureus, Well-diffusion. INTRODUCTION Staphylococcus aureus (S. aureus) is a gram positive cocci bacterium. It is an either commensal that colonizes healthy nasal mucosa (Williams, 1963) or pathogen of humans. The coagulase-positive species S. aureus is well known as a human pathogen, which can produce serious and sometimes life-threatening infections. Species of the S. aureus is identified on the basis of a variety of conventional physiological or biochemical tests. The key characters for S. aureus are colony pigment, heat-stable nuclease, clumping factor, free coagulase, and acid production from mannitol (Murray et al., 2003). Clinical isolates of S. aureus species have different capacity to form biofilm. This may be due to the differences in the expression of biofilm related genes, genetic make- up and physiological conditions (Verma and Maheshwari, 2017). S. aureus biofilm mode of growth is tightly regulated by complex genetic factors. However, in recent decades some latest studies have taken biofilm development into account in terms of elucidating host immunity toward infection and may lead to the development of efficacious anti-biofilm S. aureus therapies (Verma and Maheshwari, 2017). A biofilm can be defined as a microbial derivative sessile community, typified by cells that are attached to a substratum, interface, and embedded in a matrix of extracellular polymeric substance. Biofilm exhibit an altered phenotype with regard to growth, gene expression and protein production (Donlan and Costerton, 2002). Biofilm thickness can range from a single cell layer to a