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.

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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JoVE Journal

Biology

620 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

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

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 ...

Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

Plants can resist herbivore damage through three broad mechanisms: antixenosis, antibiosis and tolerance1. Antixenosis is the degree to which the plant is avoided when the herbivore is able to select other plants2. Antibiosis is the degree to which the plant affects the fitness of the herbivore feeding on it1.Tolerance is the degree to which the plant can withstand or repair damage caused by the herbivore, without compromising the herbivore's growth and reproduction1. The durability of herbivore resistance in an agricultural setting depends to a great extent on the resistance mechanism favored during crop breeding efforts3.
We demonstrate a no-choice experiment designed to estimate the relative contributions of antibiosis and tolerance to spittlebug resistance in Brachiaria spp. Several species of African grasses of the genus Brachiaria are valuable forage and pasture plants in the Neotropics, but they can be severely challenged by several native species of spittlebugs (Hemiptera: Cercopidae)4.To assess their resistance to spittlebugs, plants are vegetatively-propagated by stem cuttings and allowed to grow for approximately one month, allowing the growth of superficial roots on which spittlebugs can feed. At that point, each test plant is individually challenged with six spittlebug eggs near hatching. Infestations are allowed to progress for one month before evaluating plant damage and insect survival. Scoring plant damage provides an estimate of tolerance while scoring insect survival provides an estimate of antibiosis. This protocol has facilitated our plant breeding objective to enhance spittlebug resistance in commercial brachiariagrases5.

Characterizing Herbivore Resistance Mechanisms: Spittlebugs on Brachiaria spp. as an Example

This video explains mechanisms of host plant resistance to herbivory and demonstrates a no-choice test that estimates the relative contributions of antibiosis and tolerance to spittlebug resistance in Brachiaria spp.

Plants can resist herbivore damage through three broad mechanisms: antixenosis, antibiosis and tolerance1. Antixenosis is the degree to which the plant is ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The initiation step, which involves the assembly of the ribosomal subunits at the mRNA initiation codon, involved initiation factor including eIF4G1. Defects in this rate limiting step of translation are linked to diverse disorders. To study the potential consequences of such deregulations, Xenopus laevis oocytes constitute an attractive model with high degrees of conservation of essential cellular and molecular mechanisms with human. In addition, during meiotic maturation, oocytes are transcriptionally repressed and all necessary proteins are translated from preexisting, maternally derived mRNAs. This inexpensive model enables exogenous mRNA to become perfectly integrated with an effective translation. Here is described a protocol for assessing translation with a factor of interest (here eIF4G1) using stored maternal mRNA that are the first to be polyadenylated and translated during oocyte maturation as a physiological readout. At first, mRNA synthetized by in vitro transcription of plasmids of interest (here eIF4G1) are injected in oocytes and kinetics of oocyte maturation by Germinal Vesicle Breakdown detection is determined. The studied maternal mRNA target is the serine/threonine-protein-kinase mos. Its polyadenylation and its subsequent translation are investigated together with the expression and phosphorylation of proteins of the mos signaling cascade involved in oocyte maturation. Variations of the current protocol to put forward translational defects are also proposed to emphasize its general applicability. In light of emerging evidence that aberrant protein synthesis may be involved in the pathogenesis of neurological disorders, such a model provides the opportunity to easily assess this impairment and identify new targets.

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1.

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The ...

An In Vitro Model to Study the Effect of 5-Aminolevulinic Acid-mediated Photodynamic Therapy on Staphylococcus aureus Biofilm

Staphylococcus aureus (S. aureus) is a common human pathogen, which causes pyogenic and systemic infections. S. aureus infections are difficult to eradicate not only due to the emergence of antibiotic-resistant strains but also its ability to form biofilms. Recently, photodynamic therapy (PDT) has been indicated as one of the potential treatments for controlling biofilm infections. However, further studies are required to improve our knowledge of its effect on bacterial biofilms, as well as the underlying mechanisms. This manuscript describes an in vitro model of PDT with 5-aminolevulinic acid (5-ALA), a precursor of the actual photosensitizer, protoporphyrin IX (PpIX). Briefly, mature S. aureus biofilms were incubated with ALA and then exposed to light. Subsequently, the antibacterial effect of ALA-PDT on S. aureus biofilm was quantified by calculating the colony forming units (CFUs) and visualized by viability fluorescent staining via confocal laser scanning microscopy (CLSM). Representative results demonstrated a strong antibacterial effect of ALA-PDT on S. aureus biofilms. This protocol is simple and can be used to develop an in vitro model to study the treatment of S. aureus biofilms with ALA-PDT. In the future, it could also be referenced in PDT studies utilizing other photosensitizers for different bacterial strains with minimal adjustments.

An In Vitro Model to Study the Effect of 5-Aminolevulinic Acid-mediated Photodynamic Therapy on Staphylococcus aureus Biofilm

This manuscript describes a protocol to study the antimicrobial effect of 5-aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) on a Staphylococcus aureus biofilm. This protocol can be used to develop an in vitro model to study the treatment of bacterial biofilms with PDT in the future.

Staphylococcus aureus (S. aureus) is a common human pathogen, which causes pyogenic and systemic infections. S. aureus infections are difficult to ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

Harvesting and Disaggregation: An Overlooked Step in Biofilm Methods Research

Biofilm methods consist of four distinct steps: growing the biofilm in a relevant model, treating the mature biofilm, harvesting the biofilm from the surface and disaggregating the clumps, and analyzing the sample. Of the four steps, harvesting and disaggregation are the least studied but nonetheless critical when considering the potential for test bias. This article demonstrates commonly used harvesting and disaggregation techniques for biofilm grown on three different surfaces. The three biofilm harvesting and disaggregation techniques, gleaned from an extensive literature review, include vortexing and sonication, scraping and homogenization, and scraping, vortexing and sonication. Two surface types are considered: hard non-porous (polycarbonate and borosilicate glass) and porous (silicone). Additionally, we provide recommendations for the minimum information that should be included when reporting the harvesting technique followed and an accompanying method to check for bias.

This paper details methods that demonstrate three common biofilm harvesting and disaggregation techniques on two surface types, ruggedness testing of a harvesting method and minimum information to consider when choosing and optimizing harvesting and disaggregation techniques to increase reproducibility.

Biofilm methods consist of four distinct steps: growing the biofilm in a relevant model, treating the mature biofilm, harvesting the biofilm from the ...

Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds

Staphylococcus aureus expresses virulence factors to trigger its internalization into eukaryote cells and to survive inside different subcellular compartments. This paper describes an enzyme protection assay to study the extent of S. aureus internalization and its intracellular survival in adherent non-professional phagocytic cells (NPPCs) as well as the intracellular efficacy of antimicrobial compounds. NPPCs are grown in a multi-well plate until they reach 100% confluence. S. aureus cultures are grown overnight in cell culture medium. The bacterial suspension is diluted according to the number of cells per well to inoculate the cells at a controlled multiplicity of infection. Inoculated cells are incubated for 2 h to allow the bacteria to be internalized by the NPPCs, following which lysostaphin is added to the culture medium to selectively kill extracellular bacteria. Lysostaphin is present in the culture medium for the rest of the experiment. 
At this point, the infected cells could be incubated with antimicrobial compounds to assess their intracellular activities against S. aureus. Next, the cells are washed three times to remove the drugs, and intracellular S. aureus load is then quantified by culturing on agar plates. Alternatively, for studying staphylococcal virulence factors involved in intracellular survival and cell toxicity, lysostaphin could be inactivated with proteinase K to eliminate the need for washing steps. This tip improves the reliability of the intracellular bacterial load quantification, especially if cells tend to detach from the culture plate when they become heavily infected because of the multiplication of intracellular S. aureus. These protocols can be used with virtually all types of adherent NPPCs and with 3D cell culture models such as organoids.

Improved Enzyme Protection Assay to Study Staphylococcus aureus Internalization and Intracellular Efficacy of Antimicrobial Compounds

This protocol aims to describe how to study the extent of Staphylococcus aureus internalization and its ability to survive inside the human host cell, as well as the intracellular efficacy of antimicrobial compounds.

Staphylococcus aureus expresses virulence factors to trigger its internalization into eukaryote cells and to survive inside different subcellular ...

A Simple and Inexpensive Running Wheel Model for Progressive Resistance Training in Mice

Previously developed rodent resistance-based exercise models, including synergistic ablation, electrical stimulation, weighted-ladder climbing, and most recently, weighted-sled pulling, are highly effective at providing a hypertrophic stimulus to induce skeletal muscle adaptations. While these models have proven invaluable for skeletal muscle research, they are either invasive or involuntary and labor-intensive. Fortunately, many rodent strains voluntarily run long distances when given access to a running wheel. Loaded wheel running (LWR) models in rodents are capable of inducing adaptations commonly observed with resistance training in humans, such as increased muscle mass and fiber hypertrophy, as well as stimulation of muscle protein synthesis. However, the addition of moderate wheel load either fails to deter mice from running great distances, which is more reflective of an endurance/resistance training model, or the mice discontinue running nearly entirely due to the method of load application. Therefore, a novel high-load wheel running model (HLWR) has been developed for mice where external resistance is applied and progressively increased, enabling mice to continue running with much higher loads than previously utilized. Preliminary results from this novel HLWR model suggest it provides sufficient stimulus to induce hypertrophic adaptations over the 9 week training protocol. Herein, the specific procedures to execute this simple yet inexpensive progressive resistance-based exercise training model in mice are described.

This procedure describes a translatable progressive loaded running wheel resistance training model in mice. The primary advantage of this resistance training model is that it is entirely voluntary, thus reducing stress for the animals and the burden on the researcher.

Previously developed rodent resistance-based exercise models, including synergistic ablation, electrical stimulation, weighted-ladder climbing, and most ...