eoe sub articles

EoE Sub Articles

1. Bacterial growth and staining
<ol>
	<li>Perform all bacterial work in a Biosafety Cabinet, Biosafety Level 2 laboratory.</li>
	<li>Inoculate all bacterial cultures, including Pseudomonas aeruginosa&#160;(PAO1),&#160;Escherichia coli,&#160;Listeria monocytogenes,&#160;&#160;Bacillus subtilis,&#160;etc.,&#160;from the frozen glycerol stock and grow them in Tryptic Soy Broth (TSB, 3 mL) in a shaking incubator at 37 &#176;C overnight maintained at 250 rpm.</li>
	<li>T...

bacterial cell staining and preparation for immunological studies

Source: Yang, J. et al., <a href="https://app.jove.com/v/62764">Automated, High-Throughput Detection of Bacterial Adherence to Host Cells.</a>&#160;J. Vis. Exp. (2021)
This video demonstrates the staining and preparation of bacterial cells for immunological studies using red fluorescent dye. The stained bacteria are diluted and resuspended in a growth medium to achieve the desired bacterial concentration for host cell infection, representing the multiplicity of infection.

Bacterial Cell Staining and Preparation for Immunological Studies

1. Bacterial growth and staining

	Perform all bacterial work in a Biosafety Cabinet, Biosafety Level 2 laboratory.
	Inoculate all bacterial cultures, ...

Begin with a tube containing a bacterial culture in the exponential phase, ensuring uniform and actively growing bacteria for consistent results.
Centrifuge to collect the cells; aspirate the debris-containing supernatant, and resuspend the cells.
Introduce a cell-permeable red fluorescent dye. Dye molecules penetrate the bacterial cell membrane and reach the nucleoid, binding with DNA and imparting red fluorescence to the bacteria.
Centrifuge to pellet the stained bacteria and aspirate unbound dye-containing supernatant.
Resuspend the bacteria into a growth medium to maintain cell viability.
Using a photometer, measure the absorbance of the diluted bacterial suspension at 600 nanometers.
Compare the absorbance value with a standard curve to determine bacterial concentration.
Transfer the desired volume of the stained bacterial suspension into a fresh tube containing a growth medium. This is to achieve the desired bacterial concentration for the host cell infection representing the multiplicity of infection, or MOI.
The stained bacteria with the desired MOI are ready for immunological studies.