In Vitro Biofilm Synthesis by Staphylococcus aureus

Abstract

Source: Rana, P. S. et al., Standardized In vitro Assays to Visualize and Quantify Interactions between Human Neutrophils and Staphylococcus aureus Biofilms. J. Vis. Exp. (2022)

In this video, we demonstrate the preparation of in vitro biofilm by S. aureus in a poly-L-Lysine-coated multi-well plate. The biofilm-formed plate can be used for downstream experiments.

Protocol

1. Preparation of in vitro biofilm Obtain isolated colonies of S. aureus from a cryopreserved stock using a streak-plate technique on a nutrient-rich agar plate, such as Tryptic Soy Agar (see Table of Materials). Coat individual wells of a 96-well plate with 100 µL of 0.001% (v/v) poly-L-Lysine (PLL) diluted in sterile H2O and incubate at room temperature for 30 min. Aseptically, aspirate the PLL solution using a vacuum-assisted asp…

Materials

BD Bacto Tryptic Soy Broth	BD	DF0370-07-5	Combine with 1.5% agar to make Tryptic Soy Agar
Clear bottom 96-well flat bottom polystyrene plates	Costar	3370
Hanks' balanced salt solution (HBSS) 1x	Corning cellgro	21-022-CV	Without calcium, magnesium, and phenol red
Poly-L-lysine solution	Sigma	P4707-50ML
White opaque 96-well plates	Falcon	353296	Tissue culture treated and flat-bottom plate
Minimal essential media (MEM) Alpha 1x	Gibco	41061-029