Assessing the Survival of a Bacterial Pathogen in Mouse Peritoneal Macrophages

Abstract

Source: Zhang, C. et al. Development and Assessment of Intracellular Infection Models for Staphylococcus aureus. J. Vis. Exp. (2025)

This video demonstrates the quantification of intracellular survival of a methicillin-resistant bacterial pathogen in mouse peritoneal macrophages. It outlines the steps involved in infection, extracellular bacterial clearance, host cell lysis, and colony enumeration to assess bacterial survival within host macrophages.

Protocol

1. Preparation of intracellular bacterial
   1. Incubate the peritoneal macrophages with methicillin-resistant Staphylococcus aureus (MRSA) 252 for 2 h. Remove the supernatant and wash cells 2x with phosphate-buffered saline (PBS). Add Dulbecco's modified Eagle medium (DMEM) complete medium with 100 µg/mL gentamycin (1 mL per well) and incubate for 2 h at 37 °C in 5% CO₂.
   2. Remove the supernatant and wash cells 3x with PBS. Use a cell scraper to collect mouse peritoneal macrophages in 50 mL centrifuge tubes, centrifuge at 300 x g for 5 min. The intracellular bacteria of MRSA252 were successfully prepared in peritoneal macrophages.
   3. For the calculation of intracellular bacteria, to the peritoneal macrophages infected with intracellular MRSA252, add lysozyme (10 µg/mL, see Table of Materials) at 37 °C for 10 min and then wash with PBS 2x. Then, add 1 mL of PBS to resuspend the cells. Lyse the cells by adding 0.1% Triton X-100 for 5 min.
   4. Dilute the lysis with a series of dilutions and drop it onto a tryptic soy agar (TSA) plate. Culture overnight at 37 °C. Calculate the intracellular bacteria of MRSA252 in peritoneal macrophages, which were 1.8 x 10⁶ colony-forming units (CFU) per mouse, by counting the bacterial colonies.
      NOTE: When the cell scraper collects cells, the action should be gentle. The cells are scraped down in one direction to avoid cell breakage, which could affect the subsequent experiment.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
6-well plate	Corning Incorporated, USA	3516
Biohazard safety equipment	Heal force, China	VS-1300L-u
Cell incubator	ESCO, Singapore	CCL-170B-8
Cell scraper	Nest	710001
Centrifuge M1416R	RWD, China	M1416R
Centrifuge tube	Guanghou Labselect, China	CT-002-50A
Dulbecco’s Modified Eagle Medium	Thermo Gibco, USA	C11995500BT
Fetal Bovine Serum	Hyclone	SV30208.02
Gentamycin	Shanghai Sangon, China	B540724-0010
Lysozyme	Beijing Solarbio, China	L9070
MRSA252	Third Military Medical University, China	null
Penicillin and Streptomycin	Shanghai Beyotime, China	C0222
Phosphate Buffer Solution	Shanghai Beyotime, China	ST476
Saline	Sichuan Kelun, China	null
Triton X-100	Shanghai Beyotime, China	P0096-100ml
Tryptic Soy Agar (TSA) plates	Beijing AOBOX Biotechnology Co., LTD,China	02-130
RAW264.7 cell	USA, ATCC	null