This work was supported by the DBT-Ramalingaswami Fellowship awarded to Amitesh Anand.

The details of all the reagents and equipment used for the study are listed in the Table of Materials.
1. Sauton&#39;s media preparation
<ol>
	<li>Prepare 50 mL of 2.5% potassium dihydrogen phosphate solution by carefully weighing 1.25 g of potassium dihydrogen phosphate and dissolving it in 50 mL of deionized water.</li>
	<li>Prepare 50 mL of 2.5% magnesium sulfate solution by weighing 2.56 g of magnesium sulfate and dissolving it in 50 mL of deionized water.</li>
	<li>Prepare 10 mL of 5% ferric ammonium citrate solution by weighing 0.5 g of ferric ammonium citrate and dissolving it in 10 mL of deionized water. Store it in an amber tube.</li>
	<li>Prepare 100 mL of 20% glucose solution by weighing 20 g of glucose and dissolving it in 100 mL of deionized water. Filter-sterilize using a 50 mL sterile syringe and a 0.2 &#181;M PVDF syringe filter in a tube.</li>
	<li>Prepare 10 mL of sterile 1% zinc sulfate solution by carefully weighing 0.1 g of zinc sulfate and dissolving it in 10 mL of deionized water. Filter-sterilize using a 10 mL sterile syringe and a 0.2 &#181;M PVDF syringe filter in the laminar hood.</li>
	<li>Mix the prepared components in 750 mL of deionized water as described in Table 1. Measure the pH of the solution and adjust it to 7. Make up the volume to 900 mL with deionized water.</li>
	<li>Autoclave the media at 15 PSI and 121 &#176;C for 15-20 min. Let it cool down. Then, add 100 &#181;L of filter-sterilized 1% zinc sulfate. Add 100 mL of filter-sterilized 20% glucose and dissolve it well.</li>
</ol>
2. Preparation of filter-sterilized 5% Tween-80
<ol>
	<li>Dissolve 0.5 mL of Tween-80 in 9.5 mL of deionized water.</li>
	<li>Filter-sterilize the solution using a 10 mL sterile syringe and a 0.2 &#181;M PVDF syringe filter in the laminar hood.</li>
</ol>
3. Preparing the primary culture
<ol>
	<li>In the laminar hood, use a serological pipette to dispense 2 mL of autoclaved LB media into two sterile 14&#160;mL test tubes.</li>
	<li>Add 20 &#181;L of filter-sterilized 5% Tween-80 to the test tubes using a micropipette.</li>
	<li>Inoculate M. smegmatis into one of the tubes by adding its cryo-stock using an inoculation loop. The other test tube serves as a media control.</li>
	<li>Incubate the tubes for 48 h in a shaker incubator at 300 rpm and 37 &#176;C. 
	NOTE: The media control tube should not show any growth.</li>
</ol>
4. Preparing the secondary culture
<ol>
	<li>In the laminar hood, use a serological pipette to dispense 2 mL of autoclaved LB media into two sterile 14 mL test tubes.</li>
	<li>Add 20 &#181;L of filter-sterilized 5% Tween-80 to the test tubes using a micropipette.</li>
	<li>Add 20 &#181;L of the primary culture to one of the tubes using a micropipette. Keep the other tube as a media control.</li>
	<li>Incubate the cultures in a shaker&#160;incubator at 300 rpm and 37 &#176;C until they reach an OD600 of 0.6 to 0.8. 
	NOTE: It generally takes 12 to 14 hours to reach an OD600 of 0.6 to 0.8. Other cell densities may also be used, but a defined OD600 value helps with comparative studies.</li>
</ol>
5. Setting up the biofilm
<ol>
	<li>In the laminar hood, dispense 6 mL of Sauton&#39;s media supplemented with 2% glucose into each well of a 6-well plate using a serological pipette.</li>
	<li>Inoculate the respective wells with 120 &#181;L (i.e., 2%) of the secondary inoculum using a micropipette. Keep one uninoculated well as a media control.</li>
	<li>Mix the media and culture by pipetting up and down with a 1 mL micropipette. Cover the lid and carefully seal the plate with paraffin film.</li>
	<li>Incubate the plate undisturbed in a static incubator at 37 &#176;C for 7 days.</li>
</ol>
6. Setting up biofilms for stage-specific observations
NOTE: The overall biofilm setup is the same as described in step 5. To harvest or image biofilm at different points in time, it is suggested that the same biofilm be set up in multiple sets on separate plates.
<ol>
	<li>To observe biofilms between day 3 and day 6, prepare four plates with identical conditions and use one plate per day for imaging. 
	NOTE: The biofilm layer starts appearing on the air-medium interface beginning on the third day of plate setup.</li>
</ol>
7. Estimating the biofilm development
<ol>
	<li>Visual estimate
	<ol>
		<li>Image the plates using a gel documentation system with epi-white light illumination (Figure 1). 
		NOTE: Imaging gives a gross qualitative and quantitative estimate of the biofilm formed. Any good quality camera can be used as an alternative.</li>
	</ol>
	</li>
	<li>Dry weight estimate
	<ol>
		<li>Perform dry-weight measurement for more precise quantification.</li>
		<li>To measure dry weight, begin by weighing a blotting paper. 
		NOTE: Filter paper can also be used instead of blotting paper.</li>
		<li>Extract the biofilm from the 6-well plate and transfer it onto the paper using a spatula.</li>
		<li>Carefully collect most of the biofilm from the culture, being cautious not to transfer excessive amounts of the media onto the paper.</li>
		<li>Place the biofilm on the blotting paper.</li>
		<li>Place it in an incubator at 50 &#176;C for 0.5-1 h until the biofilm dries completely.</li>
		<li>Measure the combined weight of the paper and biofilm. 
		NOTE: Dry Weight of biofilm=(Weight of biofilm +paper)-(Weight of paper)</li>
	</ol>
	</li>
</ol>
8. Antibiotic tolerance assay in biofilms
<ol>
	<li>Estimate the MIC90 of rifampicin in M. smegmatis planktonic culture following the protocol described by Irith Wiegand et al. using Sauton&#39;s media11. Add the antibiotic when the cultures reach an OD600 of 0.2.</li>
	<li>Set up the plates for biofilm growth as described in step 5 of the protocol.</li>
	<li>On the fourth day of incubation, add rifampicin to cultures at the MIC90 concentration (64 &#181;g/mL) from the sides of the well using a micropipette in the laminar hood. 
	NOTE: Do this carefully without disturbing the biofilm too much. Leave one of the wells with biofilm untreated.</li>
	<li>Slowly swirl the plates to mix the antibiotics.</li>
	<li>Cover the sides of the plates with paraffin film and keep them in the incubator for further growth.</li>
	<li>After 24 h, remove the plates from the laminar hood and add Tween-80 to a final concentration of 0.2% in all the wells containing biofilm.</li>
	<li>Once again, cover the plates with paraffin film and keep them at 4 &#176;C and 100 rpm for 12 h.</li>
	<li>Take the plates out in a laminar hood and homogenize the constituents with a 1 mL micropipette.</li>
	<li>Wash the constituents with Sauton&#39;s media using the protocol described in Anand et al.12.</li>
	<li>Finally, resuspend the cells in 6 mL Sauton&#39;s media and dilute to final concentrations of 10-4, 10-5, 10-6, and 10-7.</li>
	<li>Spread 100 &#181;L of each dilution on LB-agar plates using autoclaved glass beads.</li>
	<li>Seal all the plates using paraffin film and keep them in an incubator at 37 &#176;C.</li>
	<li>After 3 days of incubation, count colonies on each plate. Only consider the plates with a colony count between 30 and 300.</li>
	<li>Calculate CFU/mL and the relative decrease in CFU/mL using the provided formulas13. 
	NOTE: CFU/mL = (No. of colonies &#215; Dilution factor) / (Volume plated (in mL)) 
	Relative decrease in CFU/mL = ((CFU/mL in Untreated - CFU/mL in Treated)) / ((CFU/mL in Untreated)) &#215; 100</li>
</ol>

Developing and Assessing Pellicle Biofilms in Mycobacteria

<ol>
	<li>Davey, M. E., O'toole, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+biofilms:+From+ecology+to+molecular+genetics.">Microbial biofilms: From ecology to molecular genetics.</a> Microbiol Mol Biol Rev. 64 (4), 847-867 (2000).</li><li>Rumbaugh, K. P., Sauer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm+dispersion.">Biofilm dispersion.</a> Nat Rev Microbiol. 18 (10), 571-586 (2020).</li><li>Davies, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+biofilm+resistance+to+antibacterial+agents.">Understanding biofilm resistance to antibacterial agents.</a> Nat Rev Drug Discov. 2 (2), 114-122 (2003).</li><li>Hern&#225;ndez-Jim&#233;nez, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm+vs.+planktonic+bacterial+mode+of+growth:+Which+do+human+macrophages+prefer.">Biofilm vs. planktonic bacterial mode of growth: Which do human macrophages prefer.</a> Biochem Biophys Res Commun. 441 (4), 947-952 (2013).</li><li>Nadell, C. D., Drescher, K., Foster, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+structure,+cooperation+and+competition+in+biofilms.">Spatial structure, cooperation and competition in biofilms.</a> Nat Rev Microbiol. 14 (9), 589-600 (2016).</li><li>Jo, J., Price-Whelan, A., Dietrich, L. E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gradients+and+consequences+of+heterogeneity+in+biofilms.">Gradients and consequences of heterogeneity in biofilms.</a> Nat Rev Microbiol. 20 (10), 593-607 (2022).</li><li>Keating, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycobacterium+tuberculosis+modifies+cell+wall+carbohydrates+during+biofilm+growth+with+a+concomitant+reduction+in+complement+activation.">Mycobacterium tuberculosis modifies cell wall carbohydrates during biofilm growth with a concomitant reduction in complement activation.</a> Cell Surf. 7, 100065 (2021).</li><li>Ojha, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+Mycobacterium+tuberculosis+biofilms+containing+free+mycolic+acids+and+harbouring+drug-tolerant+bacteria.">Growth of Mycobacterium tuberculosis biofilms containing free mycolic acids and harbouring drug-tolerant bacteria.</a> Mol Microbiol. 69 (1), 164-174 (2008).</li><li>Dietrich, L. E. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+community+morphogenesis+is+intimately+linked+to+the+intracellular+redox+state.">Bacterial community morphogenesis is intimately linked to the intracellular redox state.</a> J Bacteriol. 195 (7), 1371-1380 (2013).</li><li>Kulka, K., Hatfull, G., Ojha, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+Mycobacterium+tuberculosis+biofilms.">Growth of Mycobacterium tuberculosis biofilms.</a> J Vis Exp. (60), e3820 (2012).</li><li>Wiegand, I., Hilpert, K., Hancock, R. E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agar+and+broth+dilution+methods+to+determine+the+minimal+inhibitory+concentration+(MIC)+of+antimicrobial+substances.">Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances.</a> Nat Protoc. 3 (2), 163-175 (2008).</li><li>Anand, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyketide+quinones+are+alternate+intermediate+electron+carriers+during+mycobacterial+respiration+in+oxygen-deficient+niches.">Polyketide quinones are alternate intermediate electron carriers during mycobacterial respiration in oxygen-deficient niches.</a> Mol Cell. 60 (4), 637-650 (2015).</li><li>. Growth Curves: Generating Growth Curves Using Colony Forming Units and Optical Density Measurements Available from: <a target="_blank" href="https://www.jove.com/v/10511/growth-curves-cfu-and-optical-density-measurements">https://www.jove.com/v/10511/growth-curves-cfu-and-optical-density-measurements</a> (2019)</li><li>H&#248;iby, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+personal+history+of+research+on+microbial+biofilms+and+biofilm+infections.">A personal history of research on microbial biofilms and biofilm infections.</a> Pathog Dis. 70 (3), 205-211 (2014).</li><li>Rajewska, M., Maci&#261;g, T., Jafra, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbon+source+and+surface+type+influence+the+early-stage+biofilm+formation+by+rhizosphere+bacterium+Pseudomonas+donghuensis+P482.">Carbon source and surface type influence the early-stage biofilm formation by rhizosphere bacterium Pseudomonas donghuensis P482.</a> bioRxiv. , (2023).</li><li>O'Toole, G. 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The authors declare no competing interests.

The multicellular lifestyle of microbes was described almost a century ago; however, clinical studies remain sparse, mostly due to the lack of robust methods14. Methods described in works on biofilm biology are often difficult to adapt. Here, the detailed methodology, aided by demonstrations of critical steps, is expected to improve the reproducibility of the protocols.
The method of biofilm production described in this article is scalable, requiring a proportional incr...

Bacteria are able to survive as single-celled entities; however, in most physiologically relevant conditions, they organize into community mimetics. Biofilm is a widely recognized community organization of bacteria formed by aggregated cells encased in a self-produced matrix1. Such assembly possesses signatures of early multicellularity and provides higher stress resilience to bacterial systems. Biofilms are often tolerant to antimicrobials and are estimated to be responsible for almost 80% of microbial infections2,3.
Shake flask and plate-based cultures have traditionally been the usual practices for bacterial culturing. Their enormous acceptability and success can be attributed to their ease of handling, reproducibility, and scalability. However, the lack of physiological context limits the translational potential of the knowledge generated using such systems4. Therefore, biofilms are becoming an attractive model system to study bacterial pathophysiology. Biofilms provide a dynamic model system, closely mirroring natural conditions, allowing researchers to replicate physiological aspects such as nutrient gradients and spatial heterogeneity5,6.
The biofilm lifestyle is particularly pertinent in mycobacterial studies, as mycobacteria, including the notorious Mycobacterium tuberculosis, are adept biofilm formers7. Their ability to thrive within biofilms contributes to their persistence in host tissues during infections. It poses a formidable challenge in treating mycobacterial diseases, given the inherent antibiotic resistance associated with biofilm lifestyles8. Biofilms also provide an ideal model system to study mycobacterial metabolism, as they allow for the investigation of the unique metabolic adaptations and nutrient utilization strategies employed by mycobacteria within complex microbial communities9.
While biofilm is increasingly being accepted as a better model system for mycobacterial studies10, there is a need for consistent and reproducible standard operating procedures, especially for drawing parallels among studies conducted in different laboratories. The method outlined here describes biofilm formation procedures for a mycobacterial species, M. smegmatis. M. smegmatis is a more accessible model for studying mycobacterial biofilms, given its non-pathogenicity and faster biofilm formation kinetics. The method can be modified to suit applications like antimycobacterial screening, metabolite extraction, and omics studies.

<table><tbody><tr><td>0.2 &amp;micro;M PVDF syringe filter</td><td>Axiva</td><td>SFNY04 R</td><td></td></tr><tr><td>1 mL tips</td><td>Genetix</td><td>GXM-611000 C</td><td></td></tr><tr><td>10 &amp;micro;L tips</td><td>Genetix</td><td>GXM-6110 C</td><td></td></tr><tr><td>200 &amp;micro;L tips</td><td>Genetix</td><td>GXM-61200C</td><td></td></tr><tr><td>6-well polypropylene plates</td><td>Tarsons</td><td>980010</td><td></td></tr><tr><td>Amber tubes</td><td>Tarsons</td><td>546051</td><td></td></tr><tr><td>Autoclave</td><td>Hospharma</td><td></td><td></td></tr><tr><td>Biosafety Cabinet A II</td><td>MSET</td><td></td><td></td></tr><tr><td>Blotting paper</td><td>Any suitable vendor</td><td></td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Citric acid</td><td>Sigma</td><td>251275</td><td></td></tr><tr><td>Cuvettes</td><td>Bio-Rad</td><td>2239955</td><td></td></tr><tr><td>Ferric ammonium citrate</td><td>Sigma</td><td>F5879</td><td></td></tr><tr><td>Gel documentation system</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>Glass Beads</td><td>Sigma</td><td>G8772</td><td></td></tr><tr><td>Glucose</td><td>Sigma</td><td>49139</td><td></td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G5516</td><td></td></tr><tr><td>Inoculation loops</td><td>Genaxy</td><td>HS81121C</td><td></td></tr><tr><td>L-Aspargine</td><td>Sigma</td><td>A0884</td><td></td></tr><tr><td>LB-agar</td><td>Himedia</td><td>M1151</td><td></td></tr><tr><td>LB-media</td><td>Himedia</td><td>M575</td><td></td></tr><tr><td>&lt;em&gt;M. smegmatis&lt;/em&gt; mc2155 cryo-stock</td><td>ATCC</td><td>700084</td><td></td></tr><tr><td>Magnesium sulfate</td><td>Sigma</td><td>M2643</td><td></td></tr><tr><td>Micropipettes</td><td>Gilson</td><td></td><td></td></tr><tr><td>Parafilm</td><td>Tarsons</td><td></td><td></td></tr><tr><td>Petri Dish</td><td>Tarsons</td><td>460020</td><td></td></tr><tr><td>pH meter</td><td>Labman Scientific Instruments</td><td></td><td></td></tr><tr><td>Plate Reader</td><td>Tecan</td><td></td><td></td></tr><tr><td>Polypropylene test tubes</td><td>Genaxy</td><td>GEN-14100-PS</td><td></td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma</td><td>P5379</td><td></td></tr><tr><td>Rifampicin</td><td>MedchemExpress</td><td>HY-B0272</td><td></td></tr><tr><td>Serological pipette</td><td>SPL Life Sciences</td><td>95210</td><td></td></tr><tr><td>Shaker Incubator</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Spatula</td><td></td><td></td><td></td></tr><tr><td>Spectrophotometer</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Static Incubator</td><td>CARON</td><td></td><td></td></tr><tr><td>Sterile 10 mL syringe</td><td>Becton Dickinson</td><td>309642</td><td></td></tr><tr><td>Sterile 50 mL syringe</td><td>Becton Dickinson</td><td>309653</td><td></td></tr><tr><td>Tween-80</td><td>Sigma</td><td>P1754</td><td></td></tr><tr><td>Weighing balance</td><td>Sartorius</td><td></td><td></td></tr><tr><td>Zinc sulfate</td><td>Sigma</td><td>Z0251</td><td></td></tr></tbody></table>

growing mycobacterial biofilm as a model to study antimicrobial resistance

Many bacteria thrive in intricate natural communities, exhibiting key attributes of multicellularity such as communication, cooperation, and competition. The most prevalent manifestation of bacterial multicellular behavior is the formation of biofilms, often linked to pathogenicity. Biofilms offer a haven against antimicrobial agents, fostering the emergence of antimicrobial resistance. The conventional practice of cultivating bacteria in shake flask liquid cultures fails to represent their proper physiological growth in nature, consequently limiting our comprehension of their intricate dynamics. Notably, the metabolic and transcriptional profiles of bacteria residing in biofilms closely resemble those of naturally growing cells. This parallelism underscores the significance of biofilms as an ideal model for foundational and translational research. This article focuses on utilizing Mycobacterium smegmatis as a model organism to illustrate a technique for cultivating pellicle biofilms. The approach is adaptable to various culture volumes, facilitating its implementation for diverse experimental objectives such as antimicrobial studies. Moreover, the method&#39;s design enables the qualitative or quantitative evaluation of the biofilm-forming capabilities of different mycobacterial species with minor adjustments.

Biofilm pellicles become visible to the naked eye from the third day onwards. Although biofilm grows on Sauton&#39;s media without 2% glucose, an improvement was observed in the reticulation when it was added. We obtained 10.48 mg &#177; 3.13 mg (n = 4) of biofilm dry weight from each well of a 24-well plate with 1.5 mL of Sauton&#39;s media (supplemented with 2% glucose) grown for four days. In Figure 2, biofilm development was visible from day 3 to day 6. It starts forming a film with slig...

Author Spotlight: Advancing Mycobacterial Biofilm Protocols for Enhanced Bacterial Metabolism Research

This protocol describes a robust method for developing pellicle biofilm. The method is scalable to different culture volumes, allowing easy adoption for various experimental objectives. The method's design enables qualitative or quantitative assessment of the biofilm-forming potential of several mycobacterial species.

Growing Mycobacterial Biofilm as a Model to Study Antimicrobial Resistance

Many bacteria thrive in intricate natural communities, exhibiting key attributes of multicellularity such as communication, cooperation, and competition. ...

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Biology

647 Views.  Tata Institute of Fundamental Research. This protocol describes a robust method for developing pellicle biofilm. The method is scalable to different culture volumes, allowing easy adoption for various experimental objectives. The method's design enables qualitative or quantitative assessment of the biofilm-forming potential of several mycobacterial species.

Video: Author Spotlight: Advancing Mycobacterial Biofilm Protocols for Enhanced Bacterial Metabolism Research

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living surfaces. In this paper, we describe protocols for forming Pseudomonas aeruginosa biofilms on human airway epithelial cells (CFBE cells) grown in culture. In the first method (termed the Static Co-culture Biofilm Model), P. aeruginosa is incubated with CFBE cells grown as confluent monolayers on standard tissue culture plates. Although the bacterium is quite toxic to epithelial cells, the addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFBE cells. The second method (termed the Flow Cell Co-culture Biofilm Model), involves adaptation of a biofilm flow cell apparatus, which is often used in biofilm research, to accommodate a glass coverslip supporting a confluent monolayer of CFBE cells. This monolayer is inoculated with P. aeruginosa and a peristaltic pump then flows fresh medium across the cells. In both systems, bacterial biofilms form within 6-8 hours after inoculation. Visualization of the biofilm is enhanced by the use of P. aeruginosa strains constitutively expressing green fluorescent protein (GFP). The Static and Flow Cell Co-culture Biofilm assays are model systems for early P. aeruginosa infection of the Cystic Fibrosis (CF) lung, and these techniques allow different aspects of P. aeruginosa biofilm formation and virulence to be studied, including biofilm cytotoxicity, measurement of biofilm CFU, and staining and visualizing the biofilm.

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity.

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

Immunology and Infection

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis is one of the likely contributors to the 6-month long chemotherapy of tuberculosis 1, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms 2-4. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) 5,6. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms 7.
In a series of recent papers we established that M. tuberculosis and Mycobacterium smegmatis have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin 8-10. M. tuberculosis, however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere 9. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis, mc27000, with deletion in the two loci, panCD and RD1, that are critical for in vivo growth of the pathogen 9. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species.
Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis.

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

Establishing the Minimal Bactericidal Concentration of an Antimicrobial Agent for Planktonic Cells (MBC-P) and Biofilm Cells (MBC-B)

This protocol allows for a direct comparison between planktonic and biofilm resistance for a bacterial strain that can form a biofilm in vitro. Bacteria are inoculated into the wells of a 96-well microtiter plate. In the case of the planktonic assay, serial dilutions of the antimicrobial agent of choice are added to the bacterial suspensions. In the biofilm assay, once inoculated, the bacteria are left to form a biofilm over a set period of time. Unattached cells are removed from the wells, the media is replenished and serial dilutions of the antimicrobial agent of choice are added. After exposure to the antimicrobial agent, the planktonic cells are assayed for growth. For the biofilm assay, the media is refreshed with fresh media lacking the antimicrobial agent and the biofilm cells are left to recover. Biofilm cell viability is assayed after the recovery period. The MBC-P for the antimicrobial agent is defined as the lowest concentration of drug that kills the cells in the planktonic culture.&#160; In contrast, the MBC-B for a strain is determined by exposing preformed biofilms to increasing concentrations of antimicrobial agent for 24 hr. The MBC-B is defined as the lowest concentration of antimicrobial agent that kills the cells in the biofilm.

Establishing the Minimal Bactericidal Concentration of an Antimicrobial Agent for Planktonic Cells MBC-P and Biofilm Cells MBC-B

This protocol allows for a direct comparison between planktonic and biofilm resistance for a bacterial strain that can form a biofilm in vitro using a 96-well microtiter plate. Planktonic or biofilm bacteria are exposed to serial dilutions of the antimicrobial agent of choice. Viability is assayed by growth on agar plates.

This protocol allows for a direct comparison between planktonic and biofilm resistance for a bacterial strain that can form a biofilm in vitro. Bacteria ...

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

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

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.

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

Quantifying the Effects of Antimicrobials on In vitro Biofilm Architecture using COMSTAT Software

Biofilms are aggregates of microorganisms that rely on a self-produced matrix of extracellular polymeric substance for protection and structural integrity. The nosocomial pathogen, Pseudomonas aeruginosa, is known to adopt a biofilm mode of growth, causing chronic pulmonary infection in patients with cystic fibrosis (CF). The computer program, COMSTAT, is a useful tool for quantifying antimicrobial-induced changes in P. aeruginosa biofilm architecture by extracting data from three-dimensional confocal images. However, standardized operation of the software is less commonly addressed, which is important for optimal reporting of biofilm behavior and cross-center comparison. Thus, the aim of this protocol is to provide a simple and reproducible framework for quantifying in vitro biofilm structures under varying antimicrobial conditions via COMSTAT. The technique is modeled using a CF P. aeruginosa isolate, grown in the form of biofilm replicates, and exposed to tobramycin and the anti-Psl monoclonal antibody, Psl0096. The step-by-step approach aims to reduce user ambiguity and minimize the chance of overlooking crucial image-processing steps. Specifically, the protocol emphasizes the elimination of subjective variations associated with the manual operation of COMSTAT, including image segmentation and the selection of appropriate quantitative analysis functions. Although this method requires users to spend additional time processing confocal images prior to running COMSTAT, it helps minimize misrepresented biofilm heterogenicity in automated outputs.

Quantifying the Effects of Antimicrobials on In vitro Biofilm Architecture using COMSTAT Software

Antimicrobial-induced changes to Pseudomonas aeruginosa biofilm architecture differ among clinical isolates cultured from patients with cystic fibrosis and chronic pulmonary infection. Following confocal microscopy, COMSTAT software can be utilized to quantify variations in biofilm architecture (e.g., surface area, thickness, biomass) for individual isolates to assess the efficacy of anti-infective agents.

Biofilms are aggregates of microorganisms that rely on a self-produced matrix of extracellular polymeric substance for protection and structural ...

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a poor correlation between antibiotic susceptibility testing (AST) results using standard diagnostic methods (e.g., broth microdilution, disk diffusion, or Etest) and clinical outcomes after antibiotic treatment. Attempts to improve AST by the use of off-the-shelf biofilm growth platforms show little improvement in results. The limited ability of in vitro biofilm systems to mimic the physicochemical environment of the CF lung and, therefore bacterial physiology and biofilm architecture, also acts as a brake on the discovery of novel therapies for CF infection. Here, we present a protocol to perform AST of CF pathogens grown as mature, in vivo-like biofilms in an ex vivo CF lung model comprised of pig bronchiolar tissue and synthetic CF sputum (ex vivo&#160;pig lung, EVPL).
Several in vitro&#160;assays exist for biofilm susceptibility testing, using either standard laboratory medium or various formulations of synthetic CF sputum in microtiter plates. Both growth medium and biofilm substrate (polystyrene plate vs. bronchiolar tissue) are likely to affect biofilm antibiotic tolerance. We show enhanced tolerance of clinical Pseudomonas aeruginosa and Staphylococcus aureus isolates in the ex vivo model; the effects of antibiotic treatment of biofilms is not correlated with the minimum inhibitory concentration (MIC) in standard microdilution assays or a sensitive/resistant classification in disk diffusion assays.
The ex vivo platform could be used for bespoke biofilm AST of patient samples and as an enhanced testing platform for potential antibiofilm agents during pharmaceutical research and development. Improving the prescription or acceleration of antibiofilm drug discovery through the use of more in vivo-like testing platforms could drastically improve health outcomes for individuals with CF, as well as reduce the costs of clinical treatment and discovery research.

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

This workflow can be used to perform antibiotic susceptibility testing using an established&#160;ex vivo model of bacterial biofilm in the lungs of individuals with cystic fibrosis. Use of this model could enhance the clinical validity of MBEC (minimal biofilm eradication concentration) assays.

The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a ...

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

Bacterial biofilms are difficult to eradicate from surfaces using conventional antimicrobial interventions. High-throughput 96-well microplate methods are frequently used to cultivate bacterial biofilms for rapid antimicrobial susceptibility testing to calculate minimal biofilm eradication concentration (MBEC) values. Standard biofilm devices consist of polystyrene pegged-lids fitted to 96-well microplates and are ideal for measuring biofilm biomass and MBEC values, but these devices are limited by available peg surface area for biomass accumulation and cost. Here, we outline a protocol to use self-assembled polypropylene 96-well deep well PCR-plate pegged-lid device to grow Escherichia coli BW25113 and Pseudomonas aeruginosa PAO1 biofilms. A comparison of 24-hour biofilms formed on standard and deep well devices by each species using crystal violet biomass staining and MBEC determination assays are described. The larger surface area of deep well devices expectedly increased overall biofilm formation by both species 2-4-fold. P. aeruginosa formed significantly greater biomass/mm2 on deep well pegs as compared to the standard device. E. coli had greater biomass/mm2 on standard polystyrene devices as compared the deep well device. Biofilm eradication assays with disinfectants such as sodium hypochlorite (bleach) or benzalkonium chloride (BZK) showed that both compounds could eliminate E. coli and P. aeruginosa biofilms from both devices but at different MBEC values. BZK biofilm eradication resulted in variable E. coli MBEC values between devices, however, bleach demonstrated reproducible MBEC values for both species and devices. This study provides a high throughput deep well method for growing larger quantities of biofilms on polypropylene devices for downstream studies requiring higher amounts of static biofilm.

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.

Bacterial biofilms are difficult to eradicate from surfaces using conventional antimicrobial interventions. High-throughput 96-well microplate methods are ...

Adaptable CF Model for Antimicrobial Testing and Microbial Dynamics Analysis

Development of a Polymicrobial Colony Biofilm Model to Test Antimicrobials in Cystic Fibrosis

A range of bacteria biofilm models exist for the testing of antibiotics. However, many of these are limited to a single experimental output, such as colony-forming units or metabolic activity. Furthermore, many biofilm models do not reflect the biological and physiochemical properties of the human host environment. This is an important issue in many conditions, but most noticeably in cystic fibrosis (CF). A large proportion of people with CF suffer from both chronic and intermittent infections, and in vitro, antibiotic susceptibility testing poorly correlates with patient treatment outcomes. Some biofilm models incorporate CF lung-relevant media, including synthetic sputum mimics, but do not consider the polymicrobial nature of the environment, which alters biofilm architecture, physiology, and the way microbes respond to treatment. The solid-air interface colony biofilm model described here is highly adaptable and incorporates both CF-relevant media and a polymicrobial context. This model can also be used for mid-throughput screening of antimicrobials and to study their effect on polymicrobial dynamics. Output measurements from the model can be colony-forming units, metabolic activity, and confocal microscopy analysis. The model can easily be adapted to different microorganisms, media, temperatures, and variable oxygen conditions and can be used to test a wide range of chemical, biological, and physical treatments.

This methodology allows for the establishment of a polymicrobial biofilm model in cystic fibrosis for antimicrobial sensitivity testing in research and clinical laboratories. This model provides accurate and reliable results over a range of outputs.

A range of bacteria biofilm models exist for the testing of antibiotics. However, many of these are limited to a single experimental output, such as ...

In Vitro Polymicrobial Interactions in Cystic Fibrosis Lung Infections

Growing a Cystic Fibrosis-Relevant Polymicrobial Biofilm to Probe Community Phenotypes

Most in vitro models lack the capacity to fully probe bacterial phenotypes emerging from the complex interactions observed in real-life environments. This is particularly true in the context of hard-to-treat, chronic, and polymicrobial biofilm-based infections detected in the airways of individuals living with cystic fibrosis (CF), a multiorgan genetic disease. While multiple microbiome studies have defined the microbial compositions detected in the airway of people with CF (pwCF), no in vitro models thus far have fully integrated critical CF-relevant lung features. Therefore, a significant knowledge gap exists in the capacity to investigate the mechanisms driving the pathogenesis of mixed species CF lung infections. Here, we describe a recently developed four-species microbial community model, including Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus sanguinis, and Prevotella melaninogenica grown in CF-like conditions. Through the utilization of this system, clinically relevant phenotypes such as antimicrobial&#160;recalcitrance of several pathogens were observed and explored at the molecular level. The usefulness of this in vitro model resides in its standardized workflow that can facilitate the study of interspecies interactions in the context of chronic CF lung infections.

This protocol describes a cystic fibrosis (CF) lung-relevant four-species polymicrobial biofilm model that can be used to explore the impact of bacterial interspecies interactions.

Most in vitro models lack the capacity to fully probe bacterial phenotypes emerging from the complex interactions observed in real-life environments. This ...

Growing Mycobacterial Biofilm as a Model to Study Antimicrobial Resistance

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Chapters in this video

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Computer-Generated Animal Model Stimuli

Growth of Mycobacterium tuberculosis Biofilms

Establishing the Minimal Bactericidal Concentration of an Antimicrobial Agent for Planktonic Cells (MBC-P) and Biofilm Cells (MBC-B)

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

Quantifying the Effects of Antimicrobials on In vitro Biofilm Architecture using COMSTAT Software

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

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

Development of a Polymicrobial Colony Biofilm Model to Test Antimicrobials in Cystic Fibrosis

Growing a Cystic Fibrosis-Relevant Polymicrobial Biofilm to Probe Community Phenotypes