Quantification of Bacterial Infection in Host Cells by the Colony-Forming Unit Assay

Abstract

Source: Gayle, P. et al., Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses. J. Vis. Exp.  (2019).

This video demonstrates an in vitro quantification technique to determine Listeria monocytogenes infection of macrophages by the colony-forming unit assay. The bacterial colonies on the agar plate represent the intracellular bacterial population that escapes the host-defense mechanism and survives within the macrophages.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Preparing Cells for Infection

1. Propagate mouse macrophage-like cell line RAW 264.7 in DMEM tissue culture media supplemented with 10% fetal calf serum (henceforth referred to as DMEM/FCS) using a 125 mL flask and a tissue culture incubator maintained at 37 °C/5% CO₂.
2. The day before the infection, use a cell scraper to remove cells from the expansion flask and collect in a 15 mL screw cap conical tube. Centrifuge cells at 800 x g....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BHI (Brain Heart Infusion) broth	EMD Milipore	110493
DMEM media	Gibco	11965-092
FBS - Heat-Inactivated	Sigma-Aldrich	F4135-500ML
LB agar	Grow Cells MS	MBPE-4040
TPP Tissue Culture 48-Well Plates	MIDSCI	TP92048