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. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtiter+dish+biofilm+formation+assay.">Microtiter dish biofilm formation assay.</a> J Vis exp. 47, e2437 (2011).</li><li>Chakraborty, P., Bajeli, S., Kaushal, D., Radotra, B. D., Kumar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm+formation+in+the+lung+contributes+to+virulence+and+drug+tolerance+of+Mycobacterium+tuberculosis.">Biofilm formation in the lung contributes to virulence and drug tolerance of Mycobacterium tuberculosis.</a> Nat Comm. 12 (1), 1606 (2021).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.2 &#181;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 &#181;L tips</td>
			<td>Genetix</td>
			<td>GXM-6110 C</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#181;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>M. smegmatis 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.

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

Growing Mycobacterial Biofilm as a Model to Study Antimicrobial Resistance

Our research focuses on bacterial metabolism, especially energy metabolism, which is crucial for growth and survival. Despite extensive biochemical and structural data, systems level understanding and adaptive mechanisms remain unclear. We aim to deepen this knowledge and to address antimicrobial resistance.Our protocol enhances the physiological relevance of lab experiments by using biofilms, which mimics the natural bacterial growth better than the planktonic methods. Adapting microbacterial biofilms will offer a great system to understand the gene's lifestyle, and it'll also offer a screening system for antimicrobials. We are not the first ones to produce mycobacterial biofilms.There are several groups using this as a model. Our motivation behind producing this protocol was to simplify the procedures as much as possible so many labs can adapt this system as a model.

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...

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.

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

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Research

JoVE Journal

Biology

Culture of Mycobacterium smegmatis for Biofilm Production

To begin, with a serological pipette dispense two milliliters of autoclave lysogeny broth media into two sterile 14-milliliter test tubes. inside a laminar hood. Add 20 microliters of filter sterilized 5%Tween 80 to the test tubes with a micro pipette.Next, add the cryostock of Mycobacterium smegmatis into a tube with an inoculation loop. Incubate the tubes for 48 hours in a shaker incubator at 300 rotations per minute and 37 degrees Celsius. To prepare the secondary cultures, dispense two milliliters of autoclave lysogeny broth media into two sterile 14-milliliter test tubes.Then use a micro pipette to add 20 microliters of filter sterilized 5%Tween 80 to the test tubes. Next, with a micro pipette, transfer 20 microliters of the primary culture to one of the tubes. Incubate the cultures in a shaking incubator at 300 rotations per minute and 37 degrees Celsius until they reach an OD600 of 0.6 to 0.8.

Setting Up and Estimating the Pellicle Biofilm Development in Mycobacterium smegmatis Cultures

To begin, in a laminar hood, dispense six milliliters of Soutan's media supplemented with 2%glucose into the wells of a six-well plate. Inoculate the wells with 120 microliters of the secondary cultures of mycobacterium smegmatis. Keep one un-inoculated well as a media control.Next, pipette the culture up and down with a 1-milliliter micropipette for even mixing. Cover the lid and carefully seal the plate with paraffin film. Incubate the plate in a static incubator at 37 degrees Celsius for seven days.For a visual estimation of biofilm growth, place the plates in a gel documentation system under epi white light illumination. To measure dry weight, weigh a sheet of blotting paper. Extract the biofilm from the six-well plate and transfer it onto the paper with a spatula.Carefully collect most of the biofilm from the culture. Place the biofilm on the blotting paper. Place the blotting paper in an incubator until the biofilm dries completely.Then measure the combined weight of the paper and biofilm. Biofilm pellicles were visible from Day 3 onwards. Reticulation of the cultures improved with the addition of 2%glucose to Soutan's media.Biofilm development was visible between days 3 to 6. A film with slight reticulation was observed on Day 3. This film matured by Day 5 and disintegrated from Day 6.

368 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.

Developing and Assessing Pellicle Biofilms in Mycobacteria

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