Name: In vitro Biofilm Formation in an 8-well Chamber Slide (Video) | JoVE Journal
Uploaded: 2011-01-20T18:30:00.000+00:00
Duration: 6 min 14 s
Description: Learn to grow and visualize bacterial biofilms using an 8-well chamber slide with confocal microscopy. This method enables study of biofilm kinetics and morphology while minimizing experimental compound volumes.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

The work was funded by NIDCD/ NIH R01DC003915 to L.O. Bakaletz.

1. In Vitro Biofilm Formation
<ol>
<li>Bacterial colonies (which have been grown on appropriate agar overnight), were suspended in media and the OD490 was adjusted to 0.65</li>
<li>The resulting bacterial suspension was then diluted 1:6 (1 mL bacterial suspension + 5 mL pre-warmed medium) and incubated at 37°C with 5% CO2 for approximately 3 hours in order to reach mid-log phase.</li>
<li>Dilute the mid-log growth suspension 1:2500 with pre-warmed media and place 200 μL of dilution into each well of an 8-well chamber slide.</li>
<li>Incubate at 37°C with 5% CO2.</li>
<li>After approximately 16 hours, aspirate medium from corner of each chamber and add 200 μL fresh, pre-warmed medium - dispense along wall of chamber so as not to create shear forces within the chamber which could disrupt the biofilm.</li>
<li>If incubating longer than 24 hrs, change medium every 12 hrs or as needed to maintain bacterial viability.</li>
</ol>
2. 	Visualization of Biofilm
<ol>
<li>Aspirate medium from each chamber, as described above, and wash resident biofilm twice gently with sterile saline.</li>
<li>Add 200 μL of BacLight Live/Dead stain (3 μL component A + 3 μL component B per mL of saline) to each well and incubate at room temperature for 15 minutes. Protect culture from light from this point onward.</li>
<li>Aspirate the stain and wash twice gently with sterile saline as before.</li>
<li>Add 200 μL of neutral buffered formalin to each well and incubate for 30 minutes at room temperature to fix specimen.</li>
<li>Remove fixative and properly dispose of in accordance with institutional guidelines.</li>
<li>Wash twice with saline and dispose of the wash fluids that contain formalin as above.</li>
<li>Remove the plastic wells from the slide, add enough saline to each well so that when a coverslip is placed on the gasket, no air bubbles are present.</li>
<li>Coverslip and seal the edges of the coverslip with mounting medium. Nail polish can be used to seal the slides however the slides will not be as permanent.</li>
<li>Allow the sealant to dry for approximately 1 hour prior to examining via microscopy.</li>
</ol>
3. Results

 <img src="/files/ftp_upload/2481/2481fig1.jpg" alt="Fluorescence microscopy, biofilm structure, 3D diagram, green emission, microbial analysis." data-altupdatedat="1788235205000" data-alt="Figure 1"> 
Figure 1. Representative 3D composite images of a biofilm formed in an 8-well chamber slide. Bacteria were labeled with Live / Dead stain wherein live bacteria fluoresce green and dead bacteria fluoresce red. (A) Composite image of entire biofilm showing distinct architecture with towers and water channels. (B) Same biofilm as in (A), now seen in cross section.

<ol><li>Bakaletz, L. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bacterial+biofilms+in+otitis+media%3A+evidence+and+relevance%5BTitle%5D)+AND+%22Pediatr+Infect+Dis+J%22%5BJournal%5D)">Bacterial biofilms in otitis media: evidence and relevance.</a> Pediatr Infect Dis J. 26, Suppl 10. S17-S19 (2007).</li>
<li>Heydorn, A., Nielsen, A. T., Hentzer, M., Sternberg, C., Givskov, M., Ersboll, B. K., Molin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantification+of+biofilm+structures+by+the+novel+computer+program+COMSTAT%5BTitle%5D)+AND+%22Microbiology%22%5BJournal%5D)">Quantification of biofilm structures by the novel computer program COMSTAT.</a> Microbiology. 146, 2395-2407 (2000).</li>
</ol>

No conflicts of interest declared.

Understanding the mechanism by which biofilms form, as well as characterizing their matrix components, is an area of intense interest. This protocol can be used to help identify proteins or other constituents that contribute to the structural integrity of the biofilm, as well as help identify and target proteins which may lend themselves to a therapeutic application.
The incubation times and dilutions in the above described protocol have been established for optimal biofilm formation by nontypeable Haemophilus influenzae (NTHI). Some preliminary work may be needed to optimize these steps for other bacterial species (i.e. initial dilution and incubation time needed to achieve mid-log phase growth). The 1:2500 dilution for the initial seeding of the chambers was established to ensure that a robust biofilm would develop in the chamber slides as induced by this organism and within the incubation times stated, without the biofilm overgrowing the chamber and/or exhausting available nutrients. Accordingly, the initial dilution and or incubation times may need to be adjusted for other bacterial species.

<table><tbody><tr><td>Chocolate II agar</td><td>Fisher Scientific</td><td>B21267X</td><td></td></tr><tr><td>8-well chamber slides</td><td>Fisher Scientific</td><td>12-656-18</td><td></td></tr><tr><td>BacLight Live/Dead bacterial viability kit</td><td>Fisher Scientific</td><td>NC9439023</td><td></td></tr><tr><td>BHI</td><td>Fisher Scientific</td><td>211059</td><td></td></tr><tr><td>Hemin</td><td>Sigma-Aldrich</td><td>H5533</td><td></td></tr><tr><td>&amp;#946;-NAD</td><td>Sigma-Aldrich</td><td>N7004</td><td></td></tr><tr><td>Permaslip mounting media</td><td>Fisher Scientific</td><td>NC9693613</td><td></td></tr></tbody></table>

in vitro biofilm formation in an 8-well chamber slide

In vitro Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.

This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

The overall goal of this procedure is to grow bacterial biofilms in the wells of a chamber Slide. Each well is first inoculated with bacteria in growth medium, then cultured at 37 degrees Celsius. Once the biofilms have formed, a vital dye is used to stain the cells.The biofilms are then washed and fixed, and the wells are sealed using a cover slip. Finally, the cells can be observed by confocal microscopy and the morphology of different biofilms characterized. One advantage of this technique over existing methods such as flow cells, is that the volumes are much smaller and therefore reduce the amount of any experimental compound that will be used.Also, more variables can be compared at one time in an eight well chamber slide compared to three chamber flow cell. This method could help answer several key questions in the field of microbiology, such as kinetics of biofilm formation. This method can also provide insight into multiple diseases that include a biofilm component such as chronic otitis media, cystic fibrosis, and chronic bladder infections to name a few.Generally individuals new new to this method will struggle because there are numerous steps where the existing biofilm can be artificially disrupted. So therefore, visual demonstration of this method is critical as a removal and addition of fluid steps are difficult to learn, and the chance of disrupting the biofilm due to sheer forces is greatest at these points in the protocol demonstrating the procedure today will be Liz, a grad student from the laboratory, Inoculate bacteria in liquid culture medium, and incubate the culture until it reaches mid log phase. Dilute the mid log phase suspension at one to 2, 500 in prewarm medium for nontypeable hemophilus influenza, which is being used in this example.This will provide for 40, 000 colony forming units per milliliter as a starting concentration. Aseptically transfer 200 microliters of diluted culture into each well of a sterile eight well chamber slide. Incubate the slide at 37 degrees Celsius in a 5%carbon dioxide incubator to allow the bacteria to begin forming biofilms 16 to 18 hours later.Remove the slide from the incubator and aspirate the medium from the corner of each chamber. Then replace it with 200 microliters of fresh prewarm medium. Be sure to dispense the new medium along the wall of the chambers in order to avoid disrupting the biofilms.Return the slide to the incubator and allow the cultures to grow to the desired density. Replacing the medium every eight to 16 hours to maintain bacterial viability. To visualize the biofilms, first, remove the culture supernatants as described earlier and gently wash the biofilms twice using 200 microliters of sterile saline.Once the biofilms have been washed, add 200 microliters of backlight live dead stain to each well for one eight well chamber slide. Make up a total of two milliliters of the stain solution. Incubate the slide in the dark at room temperature for 15 minutes.From this step forward, be sure to protect the slide from light. Incubate the slide in the dark at room temperature for 15 minutes. Once the cells have been stained, remove the live dead stain and wash each well twice with saline.As demonstrated earlier, add 200 microliters of neutral buffered formin to each well and incubate the slide at room temperature for at least 30 minutes to fix the specimen. Following this incubation, remove and discard the fixative and wash the biofilms twice Using saline, remove the plastic wells from the slide and add approximately 150 microliters of saline to each. Well cover the wells by placing a cover slip on the gasket, making sure no bubbles are present and seal the edges of the cover slip with mounting medium.Alternatively, nail polish can be used to seal the cover slip. In this example, we are using nail polish For better visualization of the procedure, allow the sealant to dry at room temperature for at least one hour before examining the biofilms under a microscope. This image is a three dimensional reconstruction of Zack images of a live dead stained H influenza biofilm collected using a confocal microscope.The biofilm shown here is an example of a biofilm formed by NTHI after 24 hours of growth in the chamber slide. Using this technique, the bacteria were viable at the time of fixation as indicated by the green fluorescence and the absence of red fluorescence. The architecture of the biofilm can be seen quite clearly with numerous towers within the biofilm structure and many water channels present.The side view in panel B demonstrates the relative height of the biofilm formed at this time point. While attempting this procedure, it's important to remember to add and remove the fluids from the well slowly to minimize the generation of sheer forces that may disrupt the biofilm present in the well. By following this procedure, other methods such as immuno labeling for specific proteins of interest or lectin labeling can be performed in order to answer additional questions regarding biochemical components present within the matrix formed by the bacteria in the biofilm.After watching this video, you should have a good understanding of how to use an eight well chamber slide to study the formation of bacterial biofilms.

in-vitro-biofilm-formation-in-an-8-well-chamber-slide

Research

JoVE Journal

Immunology and Infection

The assay describes a rapid means to measure early biofilm formation in bacteria and fungi.  This method uses a microtiter plate as the substratum for microbial biofilm formation, and the biofilm is visualized using crystal violet strain.  The assay provides either a qualitative or quantitative assay for early biofilm formation.

Microtiter Dish Biofilm Formation Assay

Source: Beaudoin, T., et al. Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model. J. Vis. Exp. (2016)This video demonstrates the use of a chambered coverglass to study the effect of cystic fibrosis patient-derived sputum host factors on bacterial biofilm development.

A Chambered Coverglass Method for In Vitro Bacterial Biofilm Formation

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.

In Vitro Biofilm Synthesis by Staphylococcus aureus

The video demonstrates a solid-phase adherence assay for assessing biofilm formation. In a multi-well plate, bacteria in bile salt-supplemented media adhere to solid surfaces, forming biofilms, which is confirmed by colorimetry through crystal violet staining and ethanol treatment.

A Solid Phase Adherence Assay to Assess Biofilm Formation

The goal of this methods paper is to describe the use of a microfluidic system for the development of multi-species biofilms that contain species typically identified in human supragingival dental plaque. Methods to describe biofilm architecture, biofilm viability, and an approach to harvest biofilm for culture-dependent or culture-independent analyses are highlighted.

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

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

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

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

This protocol describes the use of a customizable automated microfluidic device to visualize biofilm formation in Candida albicans under host physiological conditions.

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

This protocol presents methodology to perform biofilm growth and biomass measurements using self-assembled deep well PCR-plate devices for high-throughput 96-well pegged lid static biofilm screening.

Generation of Greater Bacterial Biofilm Biomass using PCR-Plate Deep Well Microplate Devices

This protocol delivers a modular, BSL-2 workflow combining crystal-violet biomass assays, time-lapse phase-contrast kinetics, confocal 3-D/matrix mapping, SEM ultrastructure, and an in vivo hamster infection module to cultivate, quantify, characterize and investigate Leptospira biofilm functional role, enabling standardized evaluation of mutants and anti-biofilm interventions across laboratories.

A Modular Workflow for Quantitative, Structural and Functional Analysis of Leptospira Biofilms

Learn to grow and visualize bacterial biofilms using an 8-well chamber slide with confocal microscopy. This method enables study of biofilm kinetics and morphology while minimizing experimental compound volumes.

In vitro Biofilm Formation in an 8-well Chamber Slide

Explore More Videos