# Protocol for development, visualization, and quantification of mycobacterial biofilms on primary human airway epithelial cells

## TL;DR

This paper provides a detailed protocol for growing mycobacterial biofilms on human airway cells and measuring their structure and amount.

## Contribution

The novel contribution is a protocol for biofilm formation, visualization, and quantification on primary human bronchial epithelial cells.

## Key findings

- A protocol is presented for promoting mycobacterial biofilm formation on differentiated human bronchial epithelial cells.
- Methods for visualizing and quantifying biofilm matrix and biomass using microscopy and staining are detailed.

## Abstract

Tuberculosis (TB) displays several characteristics commonly linked to biofilm-associated infections, including recurrence of infection and resistance to antibiotics. Studying biofilm formation by mycobacteria on relevant mucosal surfaces advances our understanding of its role in TB pathogenesis and drug tolerance. Here, we present a protocol to promote biofilm formation by mycobacteria on differentiated primary pseudostratified human bronchial epithelial cell cultures. We then detail procedures to visualize and quantify biofilm matrix and biomass using electron and confocal microscopy and crystal violet staining.

For complete details on the use and execution of this protocol, please refer to Barclay et al.1

•Instructions for assessing mycobacterial biofilm formation on a biotic surface•Steps for visualizing biofilm matrix with electron and confocal microscopy•Guidance on quantifying biofilm biomass and matrix components

Instructions for assessing mycobacterial biofilm formation on a biotic surface

Steps for visualizing biofilm matrix with electron and confocal microscopy

Guidance on quantifying biofilm biomass and matrix components

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Tuberculosis (TB) displays several characteristics commonly linked to biofilm-associated infections, including recurrence of infection and resistance to antibiotics. Studying biofilm formation by mycobacteria on relevant mucosal surfaces advances our understanding of its role in TB pathogenesis and drug tolerance. Here, we present a protocol to promote biofilm formation by mycobacteria on differentiated primary pseudostratified human bronchial epithelial cell cultures. We then detail procedures to visualize and quantify biofilm matrix and biomass using electron and confocal microscopy and crystal violet staining.