An Assay to Determine Polymicrobial Biofilm Initiation on Virus-Infected Cells

In This Article

Abstract

Source: Plotkin, B. J. et al., Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi. J. Vis. Exp. (2016)

This video illustrates the initiation of polymicrobial biofilm formation on virus-infected cells by bacteria and fungi. Herpes simplex virus infection impedes bacterial attachment to the cells, preventing bacterial-fungal co-localization. In contrast, uninfected cells display bacterial-fungal co-localization and initiation of biofilm formation.

Protocol

1. HSV Strains and Handling

NOTE: Recombinant non-spreading HSV-1(KOS) gL86 and HSV-2 (KOS) 333gJ^- with beta-galactosidase reporter activity is used for the assay.

Use virus from a single lot and store at -80 °C at a 1:1 ratio of Dulbecco's modified Eagle's medium (DMEM) with 20% fetal bovine serum (FBS) and skim milk until use. Before viral lot storage, determine virus concentration by o-nitrophenyl-β-D-galactopyranoside (ONPG) and 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) assay.
Determine virus viability and multiplicity of infection ....

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Results

Virus infection assay; blue-stained cells; microscope image; varying MOI; cellular infection study.
Figure 1. X-gal Staining Pictures of Dosage Dependent HSV-1 Infection in HeLa Cells. HeLa cells infected with HSV-1 at various MOI and X-Gal stained. (A) HeLa cells in well of 96 well plate with X-Gal stained mock-infected HeLa cell cont.......