a method for sample preparation to visualize biofilm-forming bacteria on fungal hyphae

A Method for Sample Preparation to Visualize Biofilm-Forming Bacteria on Fungal Hyphae

After growing the bacteria overnight, centrifuge the culture at 5,000 times g for three minutes, and then suspend the pellet in 25 milliliters of sterile 0.1 molar potassium phosphate buffer. After repeating the centrifugation, use the same buffer to adjust the final bacterial concentration to 10 to the ninth cells per milliliter.
Fill a 6-well microplate with 5 milliliters of the bacterial suspension. Use a sterile razor blade to cut the cellophane membrane of the fungal culture into squares, with a single colony on each membrane. Then with forceps, carefully remove cellophane squares containing hyphae from the solid medium, and transfer them to individual wells of the bacterial suspension.
Gently shake the microplate until the fungal colonies are detached from the cellophane. Then, remove the cellophane sheets leaving the fungal colonies in the plate. Incubate the microplate with gentle agitation at 20 degrees Celsius for a time depending on the strains used and the stage to be analyzed.
For P. fluorescens BBc6/L. bicolor, incubate the cultures for 30 minutes to get early-stage biofilms and for up to 16 hours for mature biofilms. To remove planktonic bacteria and bacteria electrostatically attached to the hyphae, rinse the fungus by transferring it to a new 6-well microplate filled with strong salt solution. Gently shake the plate for 1 minute.
Transfer the fungus to a new 6-well microplate containing 5 milliliters of sterile 0.1 molar potassium phosphate buffer. Gently shake the plate for two minutes, and then transfer the fungus to fresh buffer.
While keeping the fungal colony in buffer, use a scalpel to cut the colony approximately in half. Transfer half of the colony to be stained to a Petri dish containing 1 milliliter of sterile water supplemented with an appropriate fluorescent dye to visualize the fungal network, such as wheat germ agglutinin conjugated to Alexa Fluor633. Then, incubate the sample in the dark.
After staining, rinse the sample by transferring it to a Petri dish lid containing 10 milliliters of sterile 0.1 molar potassium phosphate buffer, and gently shake for 1 minute. Half submerge a slide in the Petri dish lid and delicately position the cut section to float above the slide. Then, slowly lift the side from the buffer solution allowing the sample to gently settle on the slide.
The most delicate aspect is the positioning of the sample on the slide. To avoid biofilm disruption, the sample and the slide must be immersed while positioning the sample, and the sample must not be moved anymore before imaging.
Finally, add 10 microliters of anti-fading mounting medium to the sample and cover it with a glass coverslip.

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A Method for Sample Preparation to Visualize Biofilm Forming Bacteria on Fungal Hyphae

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1. Preparation of an In Vitro Biofilm of Bacteria on the Fungal Colony
<ol>
	<li>Centrifuge the bacterial culture at 5,000 x g for 3 min and suspend the pellet in 25 ml of sterile 0.1 M potassium phosphate buffer (25 g/L KH2PO4 and 2.78 g/L K2HPO4, pH 5.8). Repeat this step once and adjust the final bacterial concentration to 109 cells/ml with the same buffer.</li>
	<li>Fill a 6-well microplate with 5 ml of the bacterial suspension (or sterile 0.1 M potassium phosphate buffer for the negative control).</li>
	<li>Cut the cellophane membrane of the fungal culture with a sterile razor blade to obtain squares of cellophane with a single fungal colony on each membrane. Carefully remove the cellophane squares containing hyphae from the solid medium using forceps and transfer the square of cellophane containing hyphae to a well of the microplate containing the bacterial suspension.</li>
	<li>Gently shake the microplate while the fungal colonies are still attached to the cellophane; then, remove the cellophane sheets, leaving the fungal colonies in the plate.</li>
	<li>Incubate the microplate with gentle agitation (~60 rpm) at a temperature adapted to the studied microorganisms. 
	Note: The time of incubation depends on the speed of establishment of the biofilm and the stage to be analyzed. For P. fluorescens BBc6/L. bicolor, incubate at 20 &#176;C for 30 min to get early-stage biofilms and up to 16 hr to get mature biofilms.</li>
</ol>
2. Laser Scanning Confocal Microscopy Analysis of the Biofilm Formation
<ol>
	<li>Sample preparation
	<ol>
		<li>To remove planktonic bacteria and bacteria electrostatically attached to the hyphae, rinse the fungus by transferring it to a new 6-well microplate filled with 5 ml of a strong salt solution (NaCl, 17 g/L); gently shake for 1 min.</li>
		<li>Transfer the fungus to a new 6-well microplate containing 5 ml of sterile 0.1 M potassium phosphate buffer, gently shake for 2 min, and transfer the fungus to fresh potassium phosphate buffer.</li>
		<li>Cut the part of the fungal colony to be imaged with a scalpel while keeping it in the potassium phosphate buffer, such that the obtained section contains about half of the fungal colony.</li>
		<li>To stain the sample, transfer it to a Petri dish filled with sterile water containing an appropriate fluorescent dye (depending on the aim of the analysis), and incubate it in the dark.
		<ol>
			<li>To visualize the fungal network, use, for example, Congo Red (0.1% final concentration, 5 min of incubation), wheat germ agglutin (WGA) lectin conjugated to Alexa Fluor 633 (10 &#181;g/ml final concentration, 10 min of incubation) or FUN1 (10 &#181;M final concentration, 10 min of incubation).</li>
			<li>If using a non-fluorescent-tagged bacterium, counterstain the bacterial cells with a DNA-specific fluorescent probe among the numerous cell-permeant DNA dyes commercially available. For example, use DAPI (0.25 &#181;g/ml final concentration, 10 min of incubation).</li>
			<li>To visualize the matrix proteins, use a protein stain.</li>
		</ol>
		</li>
		<li>After staining, rinse the sample by transferring it to a Petri dish lid containing 10 ml of sterile 0.1 M potassium phosphate and gently shake for 1 min.</li>
		<li>Half submerge a slide in the Petri dish lid and delicately bring the cut section to float above the slide. Then, slowly remove the slide from the buffer solution, allowing the sample to gently settle on the slide. 
		Note: For this step, it is very important to proceed gently to avoid biofilm disturbance.</li>
		<li>Finally, add 10 &#956;l of anti-fading mounting medium to the sample and cover it with a glass coverslip. 
		Note: Once the slide is prepared, the microscopic analysis must be performed as soon as possible (within at most 30 min) to avoid modification in the biofilm structure.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>6 well Falcon Tissue Culture Plates&#160;</td>
			<td>Fisher Scientific</td>
			<td>08-772-33</td>
			<td>Used in 2.2 &#38; 3.1</td>
		</tr>
		<tr>
			<td>Congo Red</td>
			<td>Fisher Scientific</td>
			<td>C580-25</td>
			<td>Used in 3.1.4.1</td>
		</tr>
		<tr>
			<td>FUN 1 Cell Stain</td>
			<td>Thermo Fisher Scientific</td>
			<td>F7030</td>
			<td>Used in 3.1.4.1</td>
		</tr>
		<tr>
			<td>Wheat Germ Agglutinin, Alexa Fluor 633 Conjugate</td>
			<td>Thermo Fisher Scientific</td>
			<td>W21404</td>
			<td>Used in 3.1.4.1</td>
		</tr>
		<tr>
			<td>DAPI solution</td>
			<td>Thermo Fisher Scientific</td>
			<td>62248</td>
			<td>Used in 3.1.4.2</td>
		</tr>
		<tr>
			<td>Propidium iodide</td>
			<td>Thermo Fisher Scientific</td>
			<td>P3566</td>
			<td>Used in 3.1.4.3</td>
		</tr>
		<tr>
			<td>FilmTracer SYPRO Ruby Biofilm Matrix protein Stain</td>
			<td>Thermo Fisher Scientific</td>
			<td>F10318</td>
			<td>Used in 3.1.4.4</td>
		</tr>
		<tr>
			<td>Fluoromount-G Slide Mounting Medium</td>
			<td>Fisher Scientific</td>
			<td>OB100-01</td>
			<td>Used in 3.1.7</td>
		</tr>
		<tr>
			<td>LSM780 Axio Observer Z1</td>
			<td>Zeiss</td>
			<td></td>
			<td>Used in 3.2.1</td>
		</tr>
		<tr>
			<td>ZEN 2.1 lite black software</td>
			<td>Zeiss</td>
			<td></td>
			<td>Used in 3.2.1</td>
		</tr>
		<tr>
			<td>High Vacuum Coater Leica EM ACE600</td>
			<td>Leica</td>
			<td></td>
			<td>Used in 4</td>
		</tr>
		<tr>
			<td>GeminiSEM-FEG</td>
			<td>Zeiss</td>
			<td></td>
			<td>Used in 4</td>
		</tr>
	</tbody>
</table>

a method for sample preparation to visualize biofilm forming bacteria on fungal hyphae

Source: Guennoc, Cora Miquel, et al, <a href="https://app.jove.com/v/54771">A New Method for Qualitative Multi-scale Analysis of Bacterial Biofilms on Filamentous Fungal Colonies Using Confocal and Electron Microscopy.</a> J. Vis. Exp. (2017).
This video describes a method to grow and qualitatively analyze bacterial biofilms on fungal hyphae using microscopy. Bacterial biofilms frequently form on fungal surfaces and involve numerous bacterial-fungal interaction processes, such as metabolic cooperation, competition, or predation.

1. Preparation of an In Vitro Biofilm of Bacteria on the Fungal Colony

	Centrifuge the bacterial culture at 5,000 x g for 3 min and suspend the pellet in ...

Introduce a cellophane-bound fungal colony into a fluorescently labeled bacterial suspension. Remove the cellophane and incubate.
The bacterial cells adhere to the fungal surface via surface molecules and receptors. The adhered bacteria secrete extracellular polymeric substances consisting of polysaccharides, proteins, and DNA, trapping the bacterial cells and forming the structural foundation of the biofilm.
Rinse the section in a strong salt solution to remove loosely bound bacteria
In a phosphate buffer, cut a section of the colony.
Transfer the section to sterile water containing wheat germ agglutinin, or WGA bound to a red fluorescent dye.
WGA, a plant protein, binds to N-acetylglucosamine and sialic acid residues, allowing the attached dye to fluoresce on the fungal cell wall.
Rinse the section in a phosphate buffer.
Partially immerse a slide in the buffer. Let the section float on it. Lift gently for the section to settle without biofilm disruption.
Place a mounting medium, followed by a coverslip for microscopic analysis.

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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.

去除生物膜使用二氧化碳气溶胶没有氮气吹扫

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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.

方法学研究<em&gt;乙。枯草</em&gt;生物膜作为模型的表征小分子抑制剂生物膜

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Assessing Biofilm Dispersal in Murine Wounds

Biofilm-related infections are implicated in a wide array of chronic conditions such as non-healing diabetic foot ulcers, chronic sinusitis, reoccurring otitis media, and many more. Microbial cells within these infections are protected by an extracellular polymeric substance (EPS), which can prevent antibiotics and host immune cells from clearing the infection. To overcome this obstacle, investigators have begun developing dispersal agents as potential therapeutics. These agents target various components within the biofilm EPS, weakening the structure, and initiating dispersal of the bacteria, which can theoretically improve antibiotic potency and immune clearance. To determine the efficacy of dispersal agents for wound infections, we have developed protocols that measure biofilm dispersal both ex vivo and in vivo. We use a mouse surgical excision model that has been well-described to create biofilm-associated chronic wound infections. To monitor dispersal in vivo, we infect the wounds with bacterial strains that express luciferase. Once mature infections have established, we irrigate the wounds with a solution containing enzymes that degrade components of the biofilm EPS. We then monitor the location and intensity of the luminescent signal in the wound and filtering organs to provide information about the level of dispersal achieved. For ex vivo analysis of biofilm dispersal, infected wound tissue is submerged in biofilm-degrading enzyme solution, after which the bacterial load remaining in the tissue, versus the bacterial load in solution, is assessed. Both protocols have strengths and weaknesses and can be optimized to help accurately determine the efficacy of dispersal treatments.

Here, we describe ex vivo and in vivo methods for assessing bacterial dispersal from a wound infection in mice. This protocol can be utilized to test the efficacy of topical antimicrobial and anti-biofilm therapies, or to assess the dispersal capacity of different bacterial strains or species.

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Biofilm-related infections are implicated in a wide array of chronic conditions such as non-healing diabetic foot ulcers, chronic sinusitis, reoccurring ...

A Method for Sample Preparation to Visualize Biofilm Forming Bacteria on Fungal Hyphae

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A Method for Sample Preparation to Visualize Biofilm-Forming Bacteria on Fungal Hyphae