Processing of Infected Tissues for Bacterial Enumeration in a Mouse Co-Infection Model

Abstract

Source: Lenhard, A., et al. A Mouse Model for the Transition of Streptococcus pneumoniae from Colonizer to Pathogen upon Viral Co-Infection Recapitulates Age-Exacerbated Illness. J. Vis. Exp. (2022)

This video demonstrates the processing of lungs from a mouse co-infected with a pathogenic bacterium and a virus. Mincing, homogenizing, and plating the lung tissue allows quantification of bacterial load, indicating bacterial spread to the lungs and disease severity.

Protocol

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

1. Processing of infected tissues for bacterial enumeration

1. Lung collection
   1. Using dissection scissors, make a cut up the sides of the exposed rib cage and gently pull the ribs up toward the head of the mouse to expose the heart. Insert a 25 G needle attached to a 10 mL syringe prefilled with phosphate-buffered saline (PBS) into the right ventricle and begin slowly perfusing. Look for bleaching of the lungs as an indicator of successful perfusion. Flush slowly to avoid breaking the pulmonary tissue.
   2. Lift the heart with the forceps and make a cut to separate the lungs and heart. Once separated, pick up all lobes of the lung with the forceps and rinse in a dish with sterile PBS to remove any residual blood. In a Petri dish, mince the lungs into small pieces and mix well. Remove half of the lung mix for determination of the bacterial colony-forming units (CFU) or viral plaque-forming units (PFU) and place it in a round-bottom 15 mL tube prefilled with 0.5 mL of PBS for homogenization. 
      NOTE: It is important not to take different lobes of the same lung for the various assessments. Instead, all the lobes should be minced, mixed well together, and parsed out equally for the different assessments.
2. To homogenize the collected tissue, first clean the homogenizer probe by putting it in 70% ethanol and turning on the homogenizer at 60% power for 30 s. Repeat the step in sterile water for 10 s. Homogenize each tissue for 1 min. Clean the homogenizer probe in sterile water between each sample and in a fresh tube of 70% ethanol between each organ and sample group.
3. Enumeration of bacterial numbers
   1. Once all the organs have been harvested and homogenized, plate serial dilutions on blood agar plates. To calculate the total CFU, use 10 µL to plate and note down the final volume in mL for each sample. Incubate overnight at 37 °C/5% CO₂.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BD disposable syringes with Luer-Lok tips (1 mL)	Fisher	14-823-30
Fisherbrand delicate dissecting scissors	Fisher	08-951-5	Instruments used for harvest
Fisherbrand dissecting tissue forceps	Fisher	13-812-38	Forceps for harvest
Fisherbrand sterile syringes for single use (10 mL)	Fisher	14-955-459