1. Preparation of in vitro biofilm
<ol>
	<li>Obtain isolated colonies of S. aureus from a cryopreserved stock using a streak-plate technique on a nutrient-rich agar plate, such as Tryptic Soy Agar (see Table of Materials).</li>
	<li>Coat individual wells of a 96-well plate with 100 &#181;L of 0.001% (v/v) poly-L-Lysine (PLL) diluted in sterile H2O and incubate at room temperature for 30 min. Aseptically, aspirate the PLL solution using a vacuum-assisted aspiration trap. Allow the wells to dry overnight at room temperature. 
	NOTE: All aspiration steps in the protocol are performed using a vacuum-assisted aspiration trap unless otherwise stated.</li>
	<li>Prepare an overnight culture by inoculating a colony of S. aureus in minimal essential media alpha (MEM&#945;) supplemented with 2% glucose and incubate at 37 &#176;C, shaking at 200 rpm for 16-18 h.</li>
	<li>Dilute the overnight culture by transferring 50 &#181;L to 5 mL of fresh MEM&#945; supplemented with 2% glucose and incubate at 37 &#176;C, shaking at 200 rpm, until mid-logarithmic phase, generally between optical density 600 (OD600nm) of 0.5-0.8. Use MEM&#945; to normalize mid-logarithmic culture to an OD600nm of 0.1.</li>
	<li>Transfer 150 &#181;L of normalized culture to each well of the PLL-treated 96-well plate. Incubate statically for 18-20 h in a humidified chamber at 37 &#176;C. 
	NOTE: The biofilms can also be grown in other formats such as &#181;-channel slides (see Table of Materials).</li>
	<li>Aspirate the supernatant to remove the planktonic cells. Gently wash the remaining biomass with 150 &#181;L of Hanks' Balanced Salt Solution (HBSS) to remove the unattached cells. Add HBSS dropwise to avoid disrupting the biofilm. 
	NOTE: While aspirating the supernatant and HBSS during washes, leave just enough liquid (supernatant or HBSS) in the wells containing biofilm such that the biofilm is still immersed. This prevents the disruption of the biofilm structure when HBSS is added dropwise to wash the biofilm.</li>
	<li>Repeat step 1.6 at least two more times to remove all the planktonic cells. At this point, biofilms are ready for immediate downstream experiments. 
	NOTE: If the biofilms are not used for neutrophil experiments, HBSS can be substituted with phosphate-buffered saline (PBS). HBSS is preferred over PBS as HBSS contains components, including glucose, that provide optimum conditions for neutrophil activation.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BD Bacto Tryptic Soy Broth</td>
			<td>BD</td>
			<td>DF0370-07-5</td>
			<td>Combine with 1.5% agar to make Tryptic Soy Agar</td>
		</tr>
		<tr>
			<td>Clear bottom 96-well flat bottom polystyrene plates</td>
			<td>Costar</td>
			<td>3370</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks&#39; balanced salt solution (HBSS) 1x</td>
			<td>Corning cellgro</td>
			<td>21-022-CV</td>
			<td>Without calcium, magnesium, and phenol red</td>
		</tr>
		<tr>
			<td>Poly-L-lysine solution</td>
			<td>Sigma</td>
			<td>P4707-50ML</td>
			<td></td>
		</tr>
		<tr>
			<td>White opaque 96-well plates</td>
			<td>Falcon</td>
			<td>353296</td>
			<td>Tissue culture treated and flat-bottom plate</td>
		</tr>
		<tr>
			<td>Minimal essential media (MEM) Alpha 1x</td>
			<td>Gibco</td>
			<td>41061-029</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro biofilm synthesis by staphylococcus aureus

In Vitro Biofilm Synthesis by Staphylococcus aureus

Source: Rana, P. S. et al., <a href='https://app.jove.com/v/63773'>Standardized In vitro Assays to Visualize and Quantify Interactions between Human Neutrophils and Staphylococcus aureus Biofilms.</a> J. Vis. Exp. (2022)
In this video, we demonstrate the preparation of in vitro biofilm by S. aureus in a poly-L-Lysine-coated multi-well plate. The biofilm-formed plate can be used for downstream experiments.

Source: Rana, P. S. et al., Standardized In vitro Assays to Visualize and Quantify Interactions between Human Neutrophils and Staphylococcus aureus ...

Bacterial biofilms are three-dimensional structures consisting of bacterial communities embedded in a self-generated extracellular matrix, which evade the host immune response.
For in vitro Staphylococcus aureus biofilm preparation, transfer an actively growing Staphylococcus aureus suspension of the desired cell density into the poly-L-lysine-coated wells of a multi-well plate.
During incubation under static conditions, the positively-charged poly-L-lysine coating facilitates electrostatic interactions with the negatively-charged glycopolymers, such as teichoic acids, on the bacterial surface, promoting bacterial attachment.
With available nutrient sources, Staphylococcus aureus divides and accumulates, forming microcolonies. Additionally, the bacteria secrete an extracellular matrix comprising polysaccharides, proteins, and extracellular DNA, which encases cells and promotes bacterial cohesion and surface adhesion.&#160;
With continued cell division and matrix secretion, the biofilm matures into a three-dimensional structure with efficient signaling molecule distribution for cell-to-cell communication.
Upon reaching a threshold bacterial density, Staphylococcus aureus secretes proteases and nucleases, degrading the biofilm matrix and releasing a few Staphylococcus aureus into the media. Remove the media containing free-floating, planktonic bacteria.
Gently add a buffer to prevent biofilm disruption. Remove the buffer containing any remaining unattached bacteria.
The generated biofilm is ready for downstream experiments.

Begin by obtaining isolated colonies of Staphylococcus aureus from a cryopreserved stock using a streak plate technique on a nutrient-rich agar plate such as tryptic soy agar. Coat individual wells of a 96-well plate with 100 microliters of PLL diluted in sterile water, and incubate at room temperature for 30 minutes. Aspirate the PLL solution aseptically, using a vacuum-assisted aspiration trap. Allow the wells to dry overnight at room temperature.
Prepare an overnight culture by inoculating a colony of S. aureus in MEM-alpha supplemented with 2% glucose, and incubate it at 37 degrees Celsius for 16 to 18 hours at 200 rotations per minute. Dilute the overnight culture by transferring 50 microliters to 5 milliliters of fresh MEM-alpha supplemented with 2% glucose. Then, incubate it at 37 degrees Celsius at 200 rotations per minute until mid-logarithmic phase is achieved. Use MEM-alpha to normalize mid-logarithmic culture to an OD of 0.1.
Transfer 150 microliters of normalized culture to each well of the PLL-treated 96-well plate. Incubate the plate in a humidified chamber at 37 degrees Celsius for 18 to 20 hours. Aspirate the supernatant to remove the planktonic cells. Gently, wash the remaining biomass with 150 microliters of HBSS to remove the unattached cells. Repeat at least twice to remove all the planktonic cells.

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1.7K Views. Bacterial biofilms are three-dimensional structures consisting of bacterial communities embedded in a self-generated extracellular matrix, which evade the host immune response. For in vitro Staphylococcus aureus biofilm preparation, transfer an actively growing Staphylococcus aureus suspension of the desired cell density into the poly-L-lysine-coated wells of a multi-well plate. During incubation under static conditions, the positively-charged poly-L-l...

In Vitro Biofilm Synthesis by Staphylococcus aureus

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Abstract

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Materials

Tags

Name	Company	Catalog Number	Comments
BD Bacto Tryptic Soy Broth	BD	DF0370-07-5	Combine with 1.5% agar to make Tryptic Soy Agar
Clear bottom 96-well flat bottom polystyrene plates	Costar	3370
Hanks' balanced salt solution (HBSS) 1x	Corning cellgro	21-022-CV	Without calcium, magnesium, and phenol red
Poly-L-lysine solution	Sigma	P4707-50ML
White opaque 96-well plates	Falcon	353296	Tissue culture treated and flat-bottom plate
Minimal essential media (MEM) Alpha 1x	Gibco	41061-029