assessing bacterial colonization in a mouse brain following listeria monocytogenes infection

Assessing Bacterial Colonization in a Mouse Brain following Listeria monocytogenes Infection

Using 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 carefully transfer the brain into a 15 milliliter tube containing 5 milliliters of 4 degrees Celsius PBS.
In a biosafety cabinet, insert the tip and run a tissue homogenizer for 10 seconds in sequential 5% bleach, sterile water, 75% ethanol, and sterile water solutions in separate 15 milliliter conical tubes. When the tip has been thoroughly sterilized, homogenize the infected brain in its tube.
Prepare tenfold serial dilutions of each organ homogenate in sterile PBS and plate the dilutions on brain heart infusion agar plates. Then, incubate the homogenate dilution cultures at 37 degrees Celsius overnight, and count the number of colonies on each plate to determine the total number of bacteria per organ or milliliter of blood.

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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.&#160;
1. Organ Harvesting and Determination of Bacterial Burdens
<ol>
	<li>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 &#176;C) PBS. Store samples on ice until further processing occurs.</li>
	<li>To harvest the brain, decapitate the head behind the ears using scissors and cut the scalp skin in between the animal&#39;s eyes down the midline. If required, trim excess tissue, keeping the scissors pressed against the skull.
	<ol>
		<li>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.</li>
		<li>Gently cut the skull in between the rodent&#39;s eyes down the midline, applying lateral pressure. Avoid perturbing the brain and maintain minimum contact between the brain tissue and the scissors.</li>
		<li>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.</li>
		<li>Carefully transfer the brain to a 15 mL conical tube containing 5 mL of sterile cold PBS.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>Homogenize the infected brain (~20 s on setting 6, maximum rpm) in the 15 mL conical tube until no visible tissue fragments remain.
	<ol>
		<li>Clean the homogenizer as described in step 1.3 to prevent bacterial carry-over contamination to the next organ sample.</li>
		<li>Perform the homogenization process for all other organs (e.g., liver, spleen).</li>
		<li>Once the homogenization process is completed, clean the homogenizer as described in step 1.3 and store it until further use.</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>After all dilutions are plated, transfer the plates to a 37 &#176;C incubator overnight.</li>
		<li>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</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Brain Heart Infusion media</td>
			<td>Becton Dickinson</td>
			<td>237200</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptomycin sulfate</td>
			<td>Amresco</td>
			<td>0382-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>VWR</td>
			<td>25384-342</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>VWR</td>
			<td>97062-832</td>
			<td></td>
		</tr>
		<tr>
			<td>IKA T18 ULTRA-TURRAX Basic Homogenizer</td>
			<td>IKA</td>
			<td>3352109</td>
			<td>Model: T18BS1</td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Beckman Coulter</td>
			<td>DU 800 series</td>
			<td></td>
		</tr>
		<tr>
			<td>BALB/c mice</td>
			<td>Jackson Laboratory</td>
			<td>Model #000651</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>60004</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml conical tubes</td>
			<td>VWR</td>
			<td>21008-918</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline</td>
			<td>Corning</td>
			<td>46-013-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless steel spatula</td>
			<td>VWR</td>
			<td>82027-520</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless steel scissors (4.5 in)</td>
			<td>VWR</td>
			<td>82027-578</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless steel fine tip forceps (6 in)</td>
			<td>VWR</td>
			<td>82027-406</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Ghosh, P., et al., <a href="https://app.jove.com/v/58723">Listeria monocytogenes Infection of the Brain.</a> J. Vis. Exp. (2018)
This video demonstrates the quantification of the infective bacterial load from the perfused mouse brain infected with Listeria monocytogenes.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.&#160;
1. Organ Harvesting and ...

Take a perfused mouse brain in buffer.
The brain is infected with streptomycin-resistant Listeria monocytogenes, a pathogenic bacterium.
The perfusion step removes blood from the brain, ensuring that bacterial burden measurements reflect the load of Listeria monocytogenes in the tissue.
Next, homogenize the brain using a sterilized homogenizer to break down the tissue and release bacteria.
Now, prepare serial dilutions of the brain homogenate using buffer.
Plate these dilutions on brain heart infusion agar plates containing streptomycin, an antibiotic.
Incubate the plates. The brain heart infusion agar provides nutrients for Listeria monocytogenes growth, while streptomycin selects for resistant bacteria, allowing only streptomycin-resistant Listeria colonies to form.
Count the number of colonies on the plate to determine the colony-forming units, a measure of viable infective bacterial load in the brain.

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Assessing Bacterial Colonization in a Mouse Brain following Listeria monocytogenes Infection

Transcrição

Assessing Bacterial Colonization in a Mouse Brain following Listeria monocytogenes Infection