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.

9 Görüntüleme. Listeria monocytogenes Enfeksiyonunu Takiben Bir Fare Beyninde Bakteriyel Kolonizasyonun Değerlendirilmesi

Video: Listeria monocytogenes Enfeksiyonunu Takiben Bir Fare Beyninde Bakteriyel Kolonizasyonun Değerlendirilmesi - Deney

Experimental Infection with Listeria monocytogenes as a Model for Studying Host Interferon-&#947; Responses

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been highly informative on the role of innate and adaptive immune cells and specific cytokines in host immunity against intracellular pathogens. Production of IFN-&#947; by innate cells during sublethal infection with L. monocytogenes is important for activating macrophages and early control of the pathogen1-3. In addition, IFN-&#947; production by adaptive memory lymphocytes is important for priming the activation of innate cells upon reinfection4. The L. monocytogenes infection model thus serves as a great tool for investigating whether new therapies that are designed to increase IFN-&#947; production have an impact on IFN-&#947; responses in vivo and have productive biological effects such as increasing bacterial clearance or improving mouse survival from infection. Described here is a basic protocol for how to conduct intraperitoneal infections of C57BL/6J mice with the EGD strain of L. monocytogenes and to measure IFN-&#947; production by NK cells, NKT cells, and adaptive lymphocytes by flow cytometry. In addition, procedures are described to: (1) grow and prepare the bacteria for inoculation, (2) measure bacterial load in the spleen and liver, and (3) measure animal survival to endpoints. Representative data are also provided to illustrate how this infection model can be used to test the effect of specific agents on IFN-&#947; responses to L. monocytogenes and survival of mice from this infection.

This protocol describes how to inoculate C57BL/6J mice with the EGD strain of Listeria monocytogenes (L. monocytogenes) and to measure interferon-&#947; (IFN-&#947;) responses by natural killer (NK) cells, natural killer T (NKT) cells, and adaptive T lymphocytes post-infection. This protocol also describes how to conduct survival studies in mice after infection with a modified LD50 dose of the pathogen.

Deneysel Enfeksiyon<em&gt; Listeria monocytogenes</em&gt; Ana İnterferon-γ Yanıtları incelenmesi için Bir Model Olarak

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been ...

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Tuberculosis is caused by Mycobacterium tuberculosis (Mtb), a pathogen classified by the United Nations (UN) as a dangerous category B biological substance. For the sake of the workers&#8217; safety, handling of all samples presumed to carry Mtb must be conducted in a containment level (CL) 3 laboratory. The TB molecular bacterial load assay (TB-MBLA) test is a reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) test that quantifies Mtb bacillary load using primers and dual-labelled probes for 16S rRNA. We describe the use of heat inactivation to render TB samples noninfectious while preserving RNA for the TB-MBLA. A 1 mL aliquot of the sputum sample in tightly closed 15 mL centrifuge tubes is boiled for 20 min at either 80 &#176;C, 85 &#176;C, or 95 &#176;C to inactivate Mtb bacilli. Cultivation of the heat inactivated and control (live) samples for 42 days confirmed the death of TB. The inactivated sample is then spiked with 100 &#181;L of the extraction control and RNA is extracted following the standard RNA isolation procedure. No growth was observed in the cultures of heat treated samples. The isolated RNA is subjected to real-time RT-qPCR, which amplifies a specific target in the Mtb 16S rRNA gene, yielding results in the form of quantification cycles (Cq). A standard curve is used to translate Cq into bacterial load, or estimated colony forming units per mL (eCFU/mL). There is an inverse relationship between Cq and the bacterial load of a sample. The limitation is that heat inactivation lyses some cells, exposing the RNA to RNases that cause a loss of &#60;1 log10eCFU/mL (i.e., &#60;10 CFU/mL). Further studies will determine the proportion of very low burden patients that cause false negative results due to heat inactivation.

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Bacterial growth is a central concept in the development of modern microbial physiology, as well as in the investigation of cellular dynamics at the systems level. Recent studies have reported correlations between bacterial growth and genome-wide events, such as genome reduction and transcriptome reorganization. Correctly analyzing bacterial growth is crucial for understanding the growth-dependent coordination of gene functions and cellular components. Accordingly, the precise quantitative evaluation of bacterial growth in a high-throughput manner is required. Emerging technological developments offer new experimental tools that allow updates of the methods used for studying bacterial growth. The protocol introduced here employs a microplate reader with a highly optimized experimental procedure for the reproducible and precise evaluation of bacterial growth. This protocol was used to evaluate the growth of several previously described Escherichia coli strains. The main steps of the protocol are as follows: the preparation of a large number of cell stocks in small vials for repeated tests with reproducible results, the use of 96-well plates for high-throughput growth evaluation, and the manual calculation of two major parameters (i.e., maximal growth rate and population density) representing the growth dynamics. In comparison to the traditional colony-forming unit (CFU) assay, which counts the cells that are cultured in glass tubes over time on agar plates, the present method is more efficient and provides more detailed temporal records of growth changes, but has a stricter detection limit at low population densities. In summary, the described method is advantageous for the precise and reproducible high-throughput analysis of bacterial growth, which can be used to draw conceptual conclusions or to make theoretical observations.

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Assessing Bacterial Colonization in a Mouse Brain following Listeria monocytogenes Infection

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Assessing Bacterial Colonization in a Mouse Brain following Listeria monocytogenes Infection