Assessing Bacterial Colonization in a Mouse Brain following Listeria monocytogenes Infection

Abstract

Source: Ghosh, P., et al., Listeria monocytogenes Infection of the Brain. J. Vis. Exp. (2018)

This video demonstrates the quantification of the infective bacterial load from the perfused mouse brain infected with Listeria monocytogenes.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee. 

1. Organ Harvesting and Determination of Bacterial Burdens

1. Following perfusion, harvest organs (e.g., liver, spleen) by carefully removing any vasculature and ligaments attached and placing the organs separately into a sterile 15 mL conical tube containing 5 mL of sterile cold (4 °C) PBS. Store samples on ice until further processing occurs.
2. To harvest the brain, decapitate the head behind the ears using scissors and cut the scalp skin in between the animal's eyes down the midline. If required, trim excess tissue, keeping the scissors pressed against the skull.
   1. Gently insert one tip of the scissors into the foramen magnum (the hole in the base of the skull through which the spinal cord passes) and cut laterally into the skull on both sides.
   2. Gently cut the skull in between the rodent's eyes down the midline, applying lateral pressure. Avoid perturbing the brain and maintain minimum contact between the brain tissue and the scissors.
   3. Using fine-tip forceps, open the skull to expose the brain and place a spatula between the underside of the brain and the base of the skull to remove the brain.
      NOTE: This results in the tearing of brain nerve fibers.
   4. Carefully transfer the brain to a 15 mL conical tube containing 5 mL of sterile cold PBS.
   5. Discard the mouse carcass, and repeat these steps for the remaining animals.
      NOTE: Once all organs have been harvested, clean the procedure area and prepare for organ homogenization to determine bacterial burdens.
3. Clean and sterilize the tip of a tissue homogenizer placed in a biosafety cabinet by inserting the tip and running the homogenizer for 10 s sequentially in 5% bleach, sterile water, 75% ethanol, and sterile water, each contained in a separate 15 mL conical tube.
4. Homogenize the infected brain (~20 s on setting 6, maximum rpm) in the 15 mL conical tube until no visible tissue fragments remain.
   1. Clean the homogenizer as described in step 1.3 to prevent bacterial carry-over contamination to the next organ sample.
   2. Perform the homogenization process for all other organs (e.g., liver, spleen).
   3. Once the homogenization process is completed, clean the homogenizer as described in step 1.3 and store it until further use.
5. Prepare 10-fold serial dilutions of the organ homogenates in sterile PBS and plate the dilutions on BHI agar/streptomycin plates to determine the CFU per organ.
   NOTE: A similar method is performed to determine the CFU per mL of blood.
   1. After all dilutions are plated, transfer the plates to a 37 °C incubator overnight.
   2. The following day, count the number of colonies on each plate to determine the total number of bacteria per organ or mL of blood as follows:
      CFU/organ = (number of colonies x dilution factor) / mL of homogenate plated x 5
      CFU/mL of blood = (number of colonies x dilution factor) / mL of blood plated

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Brain Heart Infusion media	Becton Dickinson	237200
Streptomycin sulfate	Amresco	0382-50G
Petri dishes	VWR	25384-342
Glycerol	VWR	97062-832
IKA T18 ULTRA-TURRAX Basic Homogenizer	IKA	3352109	Model: T18BS1
Spectrophotometer	Beckman Coulter	DU 800 series
BALB/c mice	Jackson Laboratory	Model #000651
Ethylenediaminetetraacetic acid	Sigma-Aldrich	60004
15 ml conical tubes	VWR	21008-918
Phosphate-buffered saline	Corning	46-013-CM
Stainless steel spatula	VWR	82027-520
Stainless steel scissors (4.5 in)	VWR	82027-578
Stainless steel fine tip forceps (6 in)	VWR	82027-406