eoe sub articles

EoE Sub Articles

1. Biofilm Initiation
<ol>
	<li>Grow a culture of biofilm-producing organisms in a nutrient-rich medium. Inoculate&#160;Staphylococcus aureus&#160;strain Newman in 10 ml of Mueller-Hinton medium from a glycerol stock. Perform all work involving handling of&#160;S. aureus&#160;with gloves and within a biosafety cabinet.</li>
	<li>Incubate for 16 hr at 37 &#176;C on a rotary shaker (100-200 rpm).</li>
	<li>Determine OD600&#160;of the culture using a spectrophotometer and a standard cuvette (path length: 1cm).</li>
	<li>Calculate the volume (V) of culture needed to prepare 20 ml of inoculum with OD600(inoculum)&#160;of 0.1 in chelexed Roswell Park Memorial Institute media (CRPMI) using the formula: [V = 20 ml*0.1/OD600(culture)].
	<ol>
		<li>Add the calculated culture volume (from above) to a centrifuge tube and pellet cells by spinning for 1 min at 10,000 x g. To prepare biofilm inoculum, aspirate the spent growth medium and resuspend the cell pellet in 20 ml CRPMI.</li>
	</ol>
	</li>
	<li>Pour inoculum into a 25 ml reservoir.</li>
	<li>Carefully remove the lid from a sterile 96-well peg plate by pulling it straight up. 
	Note: Care must be taken not to contaminate the pegs hanging down from the lid. The lid can be placed upside down on a clean surface within a biosafety cabinet.</li>
	<li>Using a 200 &#181;l multi-channel pipette, add 125 &#181;l inoculum to each well of rows A to G of the bottom plate.</li>
	<li>Fill 125 &#181;l sterile CRPMI medium in each well of row H as a sterility control.</li>
	<li>Take the peg lid and hold it above the plate bottom with the pegs facing downwards. Carefully align the individual pegs with their respective wells. Avoid physical contact between the plate and the pegs. Once aligned, lower the lid carefully down. Avoid misalignments as readjustments performed after the pegs have touched the bottom plate may contaminate sterility controls in row H.</li>
	<li>Seal the lid to the plate with paraffin film to prevent evaporation and place it in a sealable plastic bag, sealing tightly. Keep the air volume in the bag as low as possible to minimize evaporation.</li>
	<li>Incubate without shaking at 37 &#176;C for 48 hr in a 5% CO2&#160;incubator. 
	Note: 48 hr of growth has been optimal for&#160;S. aureus, but will vary based upon the organism used. 
	Note: Although static incubation is a good starting point to optimize the assay, gently shaking at 150 rpm on a microplate shaker is a possible variation that may enhance the biofilm formation of some&#160;S. aureus&#160;isolates.</li>
</ol>
2. Preparation of the Biofilm Challenge Plate
<ol>
	<li>On the day of the biofilm challenge take a fresh 96-well plate and add 100 &#181;l of CRPMI, equilibrated to RT, to each well of rows B to H. To avoid inconsistent results, utilize a 96-well plate that can hold 200 &#181;l of media when the pegs are inside, and features wells with dimensions identical to those of the biofilm initiation plate.</li>
	<li>Dilute up to 4 test compounds separately in 1.0 ml of CRPMI to 2x the desired final concentration, in order to account for a final dilution in step 2.7. 
	Note: A good starting point is 8 fold above the inhibitory concentration of planktonic cells.</li>
	<li>Add 200 &#181;l of compound 1 in wells A1 to A3, compound 2 in wells A4 to A6, compound 3 in wells A7 to A9, and compound 4 in wells A10 to A12.</li>
	<li>Serially dilute the test compounds by using a multichannel pipette and transfer 100 &#181;l from row A to row B and mix.</li>
	<li>In that manner, continue to transfer 100 &#181;l well to well. Mix after each transfer and stop in row F.</li>
	<li>After mixing, expel 100 &#181;l from row F into a waste container. Do not transfer any liquid to rows G and H.</li>
	<li>Add 100 &#181;l of CRPMI to each well of the plate using a multichannel pipette to bring the volume to 200 &#181;l. See&#160;Figure 1&#160;for the final plate layout.</li>
</ol>
3. Biofilm Challenge
<ol>
	<li>Add 200 &#181;l of phosphate-buffered saline (PBS) to a fresh sterile 96-well plate that features wells with dimensions identical to those of the biofilm initiation plate.</li>
	<li>Remove the plastic bag and paraffin film from the incubated biofilm initiation plate.</li>
	<li>Lift the lid straight up. Avoid contact between the pegs and the plate bottom as this may damage the biofilm. Keep the bottom part for subsequent OD600&#160;analysis (see 3.8).</li>
	<li>Rinse off planktonic cells by placing the peg-lid onto the plate containing PBS (from step 3.1). Submerge pegs for 5 sec, lift out of PBS, and submerge once more, being careful not to hit the pegs against the well sides.</li>
	<li>Place the rinsed peg lid into the challenge plate. Avoid unnecessary contact between pegs and bottom plate as this will damage the biofilm leading to inconsistent results.</li>
	<li>Seal the plate with paraffin film and place it into a sealable plastic bag as described in step 1.10.</li>
	<li>Incubate the plate without shaking for 24 hr at 37 &#176;C in a 5% CO2&#160;incubator.</li>
	<li>Take the bottom of the biofilm initiation plate from 3.3. Resuspend the bacteria in each well using a multichannel pipette and measure the OD600&#160;with a suitable microplate reader to confirm growth occurring in all wells but the sterility controls in row H.</li>
</ol>
4. Biofilm Determination
<ol>
	<li>Use a plate reader equipped with an incubation chamber and capable of taking kinetic fluorescence readings for detecting resazurin conversion. Set up a 24-hour kinetic fluorescence measurement using a 530 nm excitation and 590 nm emission.</li>
	<li>Set the chamber temperature to 37 &#176;C and have the plate read every 20 min. Set the plate reader to measure from the bottom of the plate according to the manufacturer&#39;s instructions.</li>
	<li>Prepare the wash plate by adding 200 &#181;l of PBS with a multichannel pipette to a sterile 96-well plate.</li>
	<li>Prepare biofilm recovery medium by adding 400 &#181;l of 0.8 mg/ml resazurin stock solution to 20 ml of CRPMI medium to a final concentration of 16 &#181;g/ml resazurin. 
	Note: Resazurin is a known skin irritant. Use a lab coat, splash goggles, and gloves while handling. CRPMI as the biofilm recovery media was used because it provides the lowest background signal, but it can be replaced by Mueller Hinton media if appropriate.</li>
	<li>Add 150 &#181;l of biofilm recovery medium with a multichannel pipette to each well of a fresh 96-well plate.</li>
	<li>Transfer the challenge plate from the incubator into a biosafety cabinet and carefully remove the plastic bag and sealing film.</li>
	<li>Remove the peg lid from the challenge plate and rinse off planktonic cells in PBS as described in 3.4. Keep the bottom of the challenge plate for subsequent OD600&#160;analysis (see 4.10).</li>
	<li>After rinsing, place the peg lid into the plate bottom containing the biofilm recovery medium.</li>
	<li>Wrap the side of the plate with paraffin film, being careful not to obstruct the bottom of the perimeter wells. Immediately after wrapping the plate, place it on the plate reader and start a kinetic read over a 24-hour period by initiating the previously made resazurin kinetic protocol from step 4.1.</li>
	<li>To quantify the growth of planktonic cells that may or may not occur during the challenge, read the OD600&#160;of the entire challenge plate bottom using a microplate reader. Follow the instructions given in step 3.8. 
	Note: Carefully resuspend the content of each well as cells shedding off the biofilm tend to grow in clumps at the bottom of the well. Any bubbles within the well will strongly interfere with OD600&#160;readings and must be avoided or removed prior to the read.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Mueller-Hinton Medium</td>
			<td>Oxoid</td>
			<td>CM0405</td>
			<td>Follow recommendations of manufacturer</td>
		</tr>
		<tr>
			<td>RPMI-medium</td>
			<td>Corning</td>
			<td>17-105-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>CRPMI</td>
			<td>Ref 9</td>
			<td></td>
			<td>RPMI-1640 medium chelexed for 1 hr with Chelex 100 resin and then supplemented with 10% unchelexed RPMI-1640</td>
		</tr>
		<tr>
			<td>Chelex 100 Resin</td>
			<td>Bio Rad</td>
			<td>142-2822</td>
			<td></td>
		</tr>
		<tr>
			<td>MBEC-plates</td>
			<td>Innovotech</td>
			<td>19111</td>
			<td></td>
		</tr>
		<tr>
			<td>Resazurin Sodium Salt</td>
			<td>Sigma</td>
			<td>R7017</td>
			<td>800 &#181;g/ml in DI water Filter sterile</td>
		</tr>
		<tr>
			<td>Micro Plate Shaking Platform</td>
			<td>Heidolph Titramax 1000</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cytation 3 Plate Reader</td>
			<td>Biotek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gen5 software</td>
			<td>Biotek</td>
			<td></td>
			<td>Recording and analysis of resazurin conversion</td>
		</tr>
		<tr>
			<td>Neocuproine</td>
			<td>Sigma</td>
			<td>N1501</td>
			<td>Prepare 10 mM stock in 100% Ethanol, store at -80 &#186;C</td>
		</tr>
		<tr>
			<td>Copper sulfate</td>
			<td>Acros Organics</td>
			<td>7758-99-8</td>
			<td>Prepare a 100 mM stock solution in water, store at 4 &#186;C</td>
		</tr>
		<tr>
			<td>Cu-Neocuproine</td>
			<td>Self-Made</td>
			<td></td>
			<td>Generated by mixing equal molarities of neocuproine and copper sulfate. Mix was diluted in CRPMI medium to desired concentration.</td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Sigma</td>
			<td>G3632-1G</td>
			<td></td>
		</tr>
	</tbody>
</table>

a peg plate biofilm assay for screening antibacterial agents

Source: Dalecki, A. G., et al. <a href="https://app.jove.com/v/53925">Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay.</a> J. Vis. Exp. (2016)
This video demonstrates an assay for screening antimicrobial compounds that efficiently eliminate biofilms using a peg-plate. The methodology monitors the biofilm&#39;s metabolic state after treatment with antimicrobial compounds, facilitating the determination of the minimum biofilm eradication concentration.

<img alt="Figure 1" src="/files/ftp_upload/21980/21980_Figure1.jpg" /> 
Figure 1. Challenge plate layout. Example layout of challenge plate accommodating parallel testing of four compounds in triplicate at six distinct concentrations. Untreated and sterility controls are also included. The layout design supports convenient in-plate preparation of serial dilutions using a multichannel pipet. Concentrations are in &#181;M.

A Peg Plate Biofilm Assay for Screening Antibacterial Agents

1. Biofilm Initiation

	Grow a culture of biofilm-producing organisms in a nutrient-rich medium. Inoculate&#160;Staphylococcus aureus&#160;strain Newman ...

Take a microtiter plate with a lid featuring cylindrical supports, or pegs, coated in bacterial biofilms.
Place the biofilm-coated peg-lid onto microtiter plate wells with buffer to eliminate loosely attached or planktonic bacteria, focusing on biofilm-attached measurements.
Transfer the rinsed biofilm-coated peg-lid to a challenge plate with increasing test antibacterial agent concentrations. Incubate without shaking.
Depending on the agent&#39;s concentration and antibacterial potential, it disrupts the bacterial metabolic activity and biofilm integrity.
Post-incubation, place the peg lid onto buffer-containing plate wells to remove planktonic bacteria.
Transfer it to microtiter plate wells with a biofilm recovery medium containing resazurin, a cell-permeable, weakly fluorescent dye. Incubate.
In metabolically active bacteria, dehydrogenase enzymes reduce blue-colored resazurin into pink-colored, fluorescent resorufin.
Lack of resazurin conversion indicates biofilm eradication, while conversion to resorufin suggests the presence of viable cells.
The lowest test antibacterial agent concentration yielding no resazurin conversion denotes the minimal biofilm-eradicating concentration.

a-peg-plate-biofilm-assay-for-screening-antibacterial-agents

Research

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Specialized Assays

197 Görüntüleme. Kaynak: Dalecki, AG, et al. Resazurin Bazlı İlaç Duyarlılık Testi Kullanılarak Biyofilm İlişkili Staphylococcus aureus'un Hedeflenmesi. J. Vis. Uzm. (2016) Bu videoda, bir peg plakası kullanarak biyofilmleri etkili bir şekilde ortadan kaldıran antimikrobiyal bileşiklerin taranması için bir test gösterilmektedir. Metodoloji, antimikrobiyal bileşiklerle muamele edildikten sonra biyofilmin metabolik durumunu izleyerek minimum biyofilm eradikasyon konsantrasyonunun belirlenmesini ko...

Video: Antibakteriyel Ajanların Talınması için Bir Peg Plaka Biyofilm Testi - Kavram

Biofilm Removal Using Carbon Dioxide Aerosols without Nitrogen Purge

Biofilms can cause serious concerns in many applications. Not only can they cause economic losses, but they can also present a public health hazard. Therefore, it is highly desirable to remove biofilms from surfaces. Many studies on CO2 aerosol cleaning have employed nitrogen purges to increase biofilm removal efficiency by reducing the moisture condensation generated during the cleaning. However, in this study, periodic jets of CO2 aerosols without nitrogen purges were used to remove Pseudomonas putida biofilms from polished stainless steel surfaces. CO2 aerosols are mixtures of solid and gaseous CO2 and are generated when high-pressure CO2 gas is adiabatically expanded through a nozzle. These high-speed aerosols were applied to a biofilm that had been grown for 24 hr. The removal efficiency ranged from 90.36% to 98.29% and was evaluated by measuring the fluorescence intensity of the biofilm as the treatment time was varied from 16 sec to 88 sec. We also performed experiments to compare the removal efficiencies with and without nitrogen purges; the measured biofilm removal efficiencies were not significantly different from each other (t-test, p &gt; 0.55). Therefore, this technique can be used to clean various bio-contaminated surfaces within one minute.

Biofilms on surfaces can be effectively and rapidly removed by using a periodic jet of carbon dioxide aerosols without a nitrogen purge.

Biyofilm Kaldırma azotla temizleme karbon dioksiti aerosollerle

Biofilms can cause serious concerns in many applications. Not only can they cause economic losses, but they can also present a public health hazard. ...

JoVE Journal

Çevre

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

Araştırma yöntemleri<em&gt; B. subtilis</emKüçük Molekül Biyofilm İnhibitörleri Karakterizasyonu için Bir Model Olarak&gt; Biyofilmler

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

İmmünoloji ve Enfeksiyon

High-resolution Patterned Biofilm Deposition Using pDawn-Ag43

Spatial structure and patterning play an important role in bacterial biofilms. Here we demonstrate an accessible method for culturing E. coli biofilms into arbitrary spatial patterns at high spatial resolution. The technique uses a genetically encoded optogenetic construct&#8212;pDawn-Ag43&#8212;that couples biofilm formation in E. coli to optical stimulation by blue light. We detail the process for transforming E. coli with&#160;pDawn-Ag43, preparing the required optical set-up, and the protocol for culturing patterned biofilms using pDawn-Ag43 bacteria. Using this protocol, biofilms with a spatial resolution below 25 &#956;m can be patterned on various surfaces and environments, including enclosed chambers, without requiring microfabrication, clean-room facilities, or surface pretreatment. The technique is convenient and appropriate for use in applications that investigate the effect of biofilm structure, providing tunable control over biofilm patterning. More broadly, it also has potential applications in biomaterials, education, and bio-art.

We demonstrate a method for depositing Escherichia coli bacterial biofilms in arbitrary spatial patterns with a high resolution using optical stimulation of a genetically encoded surface-adhesion construct.

Yüksek çözünürlüklü desenli biyofilm ifade kullanarak pDawn-Ag43

Spatial structure and patterning play an important role in bacterial biofilms. Here we demonstrate an accessible method for culturing E. coli biofilms ...

A Peg Plate Biofilm Assay for Screening Antibacterial Agents

TRANSKRIPT