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

Published: March 29, 2024

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-&#946…

Representative Results

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 control; 20x initial magnification; (B) HeLa cells in well of 96 well plate with X-Gal stained HeLa cell infected with HSV-1 at…

Divulgations

The authors have nothing to disclose.

Materials

C.albicans
BBL Sabouraud Dextrose BD 211584
Fungisel Agar Dot Scientific 7205A
S.aureus
Mannitol Salt Agar Troy Biologicals 7143B
Sheep blood agar Troy Biologicals 221239
Hela cells
1xDMEM (Dubelcco's Modified Eagle Medium, with 4.5 g/L glucose and L-glutamine, without sodium pyruvate Corning 10-017-CM
Gentamicin 50mg/ml Sigma 1397 50µg/ml final concentration in the complete DMEM
Trypsin EDTA (0.05% Trypsin, 0.53M EDTA)Solution 1X Corning 25-052-CI
Fetal Bovine Serum Atlanta Biologicals S11150 10% final concentration in the complete DMEM
Other medium and reagents
ONPG Thermo Scientific 34055
Ultra-Pure X gal Invitrogen 15520-018
1x HBSS (Hanks' Balanced Salt Solution) Corning 20-021-CV
1XPBS Dot Scientific 30042-500
RIPA Lysis Life Technologies 89901
Supplies
96-Well plates Evergreen Scientific 222-8030-01F
Culture tubes 100×13 Thomas Scientific 9187L61

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An Assay to Determine Polymicrobial Biofilm Initiation on Virus-Infected Cells. J. Vis. Exp. (Pending Publication), e22008, doi: (2024).

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