Name: A Modular Workflow for Quantitative, Structural and Functional Analysis of Leptospira Biofilms (Video) | JoVE Journal
Uploaded: 2025-12-19T13:45:00.000+00:00
Duration: 8 min 51 s
Description: Learn a modular workflow for quantitative, structural and functional analysis of Leptospira biofilms using crystal-violet assays, microscopy, and in vivo infection models. This standardized protocol enhances reproducibility in biofilm research.

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We gratefully acknowledge the financial support provided by the AXA Research Fund through a postdoctoral fellowship (reference: 15-AXA-PDOC-037), by the Institut Pasteur of New Caledonia (IPNC) and the University of New Caledonia (UNC) through a PhD fellowship, and by the French National Research Agency (ANR) under grant number SPIraL-19-CE35-0006-01. The mention of trade names or commercial products in this publication is exclusively intended to document the materials used in the experimental procedures. Such references do not constitute endorsement, recommendation, or indication of commercial interest by the Institut Pasteur of New Caledonia.

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<li>Chiani, Y. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Presence+of+Leptospira+spp.+in+a+mosaic+of+wetlands+used+for+livestock+raising+under+differing+hydroclimatic+conditions%5BTitle%5D)+AND+%22Appl+Environ+Microbiol%22%5BJournal%5D)">Presence of Leptospira spp. in a mosaic of wetlands used for livestock raising under differing hydroclimatic conditions.</a> Appl Environ Microbiol. 89 (6), e0197122(2023).</li>
<li>Yamaguchi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterizing+interactions+of+Leptospira+interrogans+with+proximal+renal+tubule+epithelial+cells%5BTitle%5D)+AND+%22BMC+Microbiol%22%5BJournal%5D)">Characterizing interactions of Leptospira interrogans with proximal renal tubule epithelial cells.</a> BMC Microbiol. 18 (1), 64(2018).</li>
<li>Santos, A. A. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Leptospira+interrogans+biofilm+formation+in+Rattus+norvegicus+%28Norway+rats%29+natural+reservoirs%5BTitle%5D)+AND+%22PLoS+Negl+Trop+Dis%22%5BJournal%5D)">Leptospira interrogans biofilm formation in Rattus norvegicus (Norway rats) natural reservoirs.</a> PLoS Negl Trop Dis. 15 (9), e0009736(2021).</li>
<li>Ackermann, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+biofilm+formation+of+pathogenic+Leptospira+spp.+in+the+vitreous+humor+of+horses+with+recurrent+uveitis%5BTitle%5D)+AND+%22Microorganisms%22%5BJournal%5D)">In vivo biofilm formation of pathogenic Leptospira spp. in the vitreous humor of horses with recurrent uveitis.</a> Microorganisms. 9 (9), (2021).</li>
<li>Dos Santos Ribeiro, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Environmental+biofilms+from+an+urban+community+in+Salvador%2C+Brazil%2C+shelter+previously+uncharacterized+saprophytic%C2%A0Leptospira%5BTitle%5D)+AND+%22Microb+Ecol%22%5BJournal%5D)">Environmental biofilms from an urban community in Salvador, Brazil, shelter previously uncharacterized saprophytic Leptospira.</a> Microb Ecol. 86 (4), 2488-2501 (2023).</li>
<li>Vinod Kumar, K., Lall, C., Raj, R. V., Vedhagiri, K., Vijayachari, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+detection+of+pathogenic+leptospiral+protein+encoding+gene+%28lipL32%29+in+environmental+aquatic+biofilms%5BTitle%5D)+AND+%22Lett+Appl+Microbiol%22%5BJournal%5D)">Molecular detection of pathogenic leptospiral protein encoding gene (lipL32) in environmental aquatic biofilms.</a> Lett Appl Microbiol. 62 (4), 311-315 (2016).</li>
<li>Davignon, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Leptospirosis%3A+Toward+a+better+understanding+of+the+environmental+lifestyle+of+Leptospira%5BTitle%5D)+AND+%22Front+Water%22%5BJournal%5D)">Leptospirosis: Toward a better understanding of the environmental lifestyle of Leptospira.</a> Front Water. 5, 1195094(2023).</li>
<li>Dias, C. S., Pinna, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Leptospira+biofilms%3A+Implications+for+survival%2C+transmission%2C+and+disease+management%5BTitle%5D)+AND+%22Appl+Environ+Microbiol%22%5BJournal%5D)">Leptospira biofilms: Implications for survival, transmission, and disease management.</a> Appl Environ Microbiol. 91 (2), e0191424(2025).</li>
<li>Gomes, D. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biofilm+production+of+Leptospira+spp.+strains%5BTitle%5D)+AND+%22Acta+Sci+Vet%22%5BJournal%5D)">Biofilm production of Leptospira spp. strains.</a> Acta Sci Vet. 46 (1), 5(2018).</li>
<li>Thibeaux, R., Kainiu, M., Goarant, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biofilm+formation+and+quantification+using+the+96-microtiter+plate%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Biofilm formation and quantification using the 96-microtiter plate.</a> Methods Mol Biol. 2134, 207-214 (2020).</li>
<li>Vinod Kumar, K., Lall, C., Vimal Raj, R., Vedhagiri, K., Vijayachari, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Co-existence+and+survival+of+pathogenic+Leptospires+by+formation+of+biofilm+with+Azospirillum%5BTitle%5D)+AND+%22FEMS+Microbiol+Ecol%22%5BJournal%5D)">Co-existence and survival of pathogenic Leptospires by formation of biofilm with Azospirillum.</a> FEMS Microbiol Ecol. 91 (6), fiv051(2015).</li>
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<li>Rollet, C., Gal, L., Guzzo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biofilm-detached+cells%2C+a+transition+from+a+sessile+to+a+planktonic+phenotype%3A+A+comparative+study+of+adhesion+and+physiological+characteristics+in+Pseudomonas+aeruginosa%5BTitle%5D)+AND+%22FEMS+Microbiol+Lett%22%5BJournal%5D)">Biofilm-detached cells, a transition from a sessile to a planktonic phenotype: A comparative study of adhesion and physiological characteristics in Pseudomonas aeruginosa.</a> FEMS Microbiol Lett. 290 (2), 135-142 (2009).</li>
<li>Haake, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hamster+model+of+leptospirosis%5BTitle%5D)+AND+%22Curr+Protoc+Microbiol%22%5BJournal%5D)">Hamster model of leptospirosis.</a> Curr Protoc Microbiol. Chapter 12 (Unit 12E.12), (2006).</li>
<li>Cagliero, J., Huet, K., Matsui, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Use+of+golden+Syrian+hamster+as+an+animal+model+to+study+leptospirosis-associated+immune+responses%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Use of golden Syrian hamster as an animal model to study leptospirosis-associated immune responses.</a> Methods Mol Biol. 2134, 243-255 (2020).</li>
<li>Gomes-Solecki, M., Santecchia, I., Werts, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Animal+models+of+leptospirosis%3A+Of+mice+and+hamsters%5BTitle%5D)+AND+%22Front+Immunol%22%5BJournal%5D)">Animal models of leptospirosis: Of mice and hamsters.</a> Front Immunol. 8, 58(2017).</li>
<li>Guedes, I. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Improvement+of+the+enrichment+used+in+the+EMJH+medium+%28Ellinghausen-McCullough-Johnson-Harris%29+for+the+cultivation+of+Leptospira+spp%5BTitle%5D)+AND+%22Rev+Argent+Microbiol%22%5BJournal%5D)">Improvement of the enrichment used in the EMJH medium (Ellinghausen-McCullough-Johnson-Harris) for the cultivation of Leptospira spp.</a> Rev Argent Microbiol. 54, 95-99 (2022).</li>
<li>Iraola, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transcriptome+sequencing+reveals+wide+expression+reprogramming+of+basal+and+unknown+genes+in+Leptospira+biflexa+biofilms%5BTitle%5D)+AND+%22mSphere%22%5BJournal%5D)">Transcriptome sequencing reveals wide expression reprogramming of basal and unknown genes in Leptospira biflexa biofilms.</a> mSphere. 1 (2), e00042-e00116 (2016).</li>
<li>Davignon, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Leptospira+interrogans+biofilm+transcriptome+highlights+adaption+to+starvation+and+general+stress+while+maintaining+virulence%5BTitle%5D)+AND+%22NPJ+Biofilms+Microbiomes%22%5BJournal%5D)">Leptospira interrogans biofilm transcriptome highlights adaption to starvation and general stress while maintaining virulence.</a> NPJ Biofilms Microbiomes. 10 (1), 95(2024).</li>
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<li>Werner, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stratified+growth+in+Pseudomonas+aeruginosa+biofilms%5BTitle%5D)+AND+%22Appl+Environ+Microbiol%22%5BJournal%5D)">Stratified growth in Pseudomonas aeruginosa biofilms.</a> Appl Environ Microbiol. 70 (10), 6188-6196 (2004).</li>
<li>Neu, T. R., Lawrence, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Advanced+techniques+for+in+situ+analysis+of+the+biofilm+matrix+%28structure%2C+composition%2C+dynamics%29+by+means+of+laser+scanning+microscopy%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Advanced techniques for in situ analysis of the biofilm matrix (structure, composition, dynamics) by means of laser scanning microscopy.</a> Methods Mol Biol. 1147, 43-64 (2014).</li>
<li>Neu, T. R., Lawrence, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Innovative+techniques%2C+sensors%2C+and+approaches+for+imaging+biofilms+at+different+scales%5BTitle%5D)+AND+%22Trends+Microbiol%22%5BJournal%5D)">Innovative techniques, sensors, and approaches for imaging biofilms at different scales.</a> Trends Microbiol. 23 (4), 233-242 (2015).</li>
<li>Relucenti, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microscopy+methods+for+biofilm+imaging%3A+Focus+on+SEM+and+VP-SEM+pros+and+cons%5BTitle%5D)+AND+%22Biology+%28Basel%29%22%5BJournal%5D)">Microscopy methods for biofilm imaging: Focus on SEM and VP-SEM pros and cons.</a> Biology (Basel). 10 (1), (2021).</li>
<li>Braet, F., De Zanger, R., Wisse, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Drying+cells+for+SEM%2C+AFM+and+TEM+by+hexamethyldisilazane%3A+A+study+on+hepatic+endothelial+cells%5BTitle%5D)+AND+%22J+Microsc%22%5BJournal%5D)">Drying cells for SEM, AFM and TEM by hexamethyldisilazane: A study on hepatic endothelial cells.</a> J Microsc. 186 (Pt 1), 84-87 (1997).</li>
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The authors declare no competing financial or non-financial interests. All authors have reviewed and approved this disclosure.

This protocol presents a modular and integrative workflow for studying Leptospira biofilms, integrating quantitative biomass (crystal violet)9,27, dynamics (time-lapse phase contrast), three-dimensional composition (confocal laser-scanning microscopy with targeted probes)38, ultrastructure (scanning electron microscopy), and functional assessment (hamster infection). By using the same culture series across modules, batch effects are minimized and within-experiment cross-validation is enabled, which is especially important for slow-growing, fragile spirochetes. Each module complements the others: CV quantifies &#34;how much,&#34; time-lapse shows &#34;how fast,&#34; CLSM reveals &#34;what&#39;s inside,&#34; SEM resolves &#34;how it&#39;s built,&#34; and in vivo testing provides &#34;proof-of-function.&#34; Inclusion of L. biflexa demonstrates adaptability across species, highlighting faster and denser biofilm formation and emphasizing methodological flexibility35.
The success of each module depends on inoculum quality and physiological state. Mid-log planktonic cultures (3-5 days), highly motile and free of aggregates, yield reproducible adhesion and biofilm development. Only low-passage isolates should be used to ensure robust biofilm formation and reproducibility.
The CV assay anchors the workflow through its speed, scalability, and throughput9,27. It enables rapid screening of mutants, media, or anti-biofilm compounds. However, because Leptospira biofilms are fragile and heterogeneous, gentle manipulation and replicate validation are essential. CV cannot distinguish compact vs porous architectures or detect matrix composition -- hence its role as a screening gate for deeper structural analyses.
Time-lapse imaging bridges that gap by revealing biofilm kinetics in real time. It captures the emergence, coalescence, and immobilization of motile aggregates, exposing transient phenomena that endpoint assays miss. Environmental stability (temperature, humidity, autofocus) is critical. These recordings have shown that even when CV biomass appears stable, dynamics can diverge -- indicating structural rearrangements or viability loss in deeper strata.
CLSM provides volumetric and chemical insight without dehydration artefacts39. Live/dead staining reveals cell viability gradients within the biofilm, consistent with reports of limited oxygen diffusion and metabolic stratification6,40. Lectins and nucleic-acid dyes expose abundant extracellular DNA and specific glycan motifs, allowing quantification and characterization of matrix components41,42. CLSM data frequently reveal internal voids, echoing the collective rearrangements seen in time-lapse38. Still, limitations include probe specificity, photobleaching, and diffraction-limited axial resolution; therefore, microscope settings and dye performance should be standardized and tested beforehand to ensure reliable, comparable results across experiments.
SEM offers complementary ultrastructural resolution43. At early stages, it resolves nascent aggregates; at maturity (&#8776; 15-21 days), it reveals a polarized architecture: a rough, channeled basal layer for anchorage and exchange, and a smoother apical matrix-rich surface37. Careful fixation, HMDS drying, and correlation with confocal data mitigate artefacts due to dehydration or collapse44.
Together, these four modules enable internal cross-validation: when CV, CLSM, and SEM converge, conclusions are robust; when they diverge, the discrepancies themselves are informative -- revealing, for example, increased biomass with reduced viability or unchanged biomass with altered matrix composition.
Several intrinsic limitations remain. Leptospira biofilms are heterogeneous and delicate, making them sensitive to handling and dehydration. CV integrates total biomass but not viability16; CLSM cannot exceed optical limits38; SEM risks preparation artefacts. Mortality in deeper strata is expected due to oxygen gradients6, but this bias is systematic and comparable across conditions. Biofilm maturation reaches a plateau around 15-21 days, associated with transcriptional signatures of nutrient limitation36. Later stages remain poorly characterized.
In vivo assays provide functional context. Injecting aggregates intraperitoneally tests whether biofilm-derived cells differ in virulence or persistence from planktonic counterparts. Although intraperitoneal inoculation is not a natural route, it offers a controlled comparative model36. Aggregate heterogeneity introduces some variance, but consistent handling and matched inoculum size mitigate this. Importantly, biofilm persistence traits (e.g., ECM-mediated stress tolerance) do not necessarily translate into enhanced acute virulence, highlighting the ecological rather than pathogenic role of biofilm formation24.
The modular design enables scalable use. For rapid screening, CV and time-lapse suffice. For mechanistic insight, CLSM and SEM add compositional and architectural resolution. Modules can integrate enzymatic or chemical perturbations (DNase, glycosidase), alternative probes for glycans or nucleic acids, or microfluidic systems to test flow responses. The same inoculum can feed transcriptomic or proteomic analyses, directly linking biofilm phenotype to regulation (e.g., c-di-GMP signaling, starvation response). This framework also accommodates mutant screening, environmental perturbation tests, and anti-biofilm or anti-virulence compound evaluation.
This integrated workflow transforms isolated observations into a coherent multidimensional understanding of Leptospira biofilms. By combining throughput (CV), dynamics (time-lapse), chemistry and depth (CLSM), and ultrastructure (SEM), the approach captures the mobile, porous, and polarized nature of Leptospira communities with fidelity. Extending earlier studies17,35,36, it demonstrates that coordinated, modular experimentation reveals phenomena invisible to single-method studies -- such as rising biomass despite falling viability, or divergent kinetics among species. Ultimately, this approach supports comparative, reproducible, and functionally relevant analyses across species, mutants, and conditions, providing a foundation for mechanistic microbiology, ecological resilience, and anti-biofilm strategy design.

Biofilms are structured microbial communities in which cells are embedded in a self-secreted extracellular polymeric substance (EPS) matrix composed of polysaccharides, proteins, nucleic acids and lipids1. This matrix provides mechanical stability, mediates adhesion to surfaces and enhances microbial survival by conferring tolerance to environmental stresses such as desiccation, oxidative stress, and antimicrobials2,3. In pathogenic bacteria biofilms facilitate persistence, immune evasion, and chronic infection4,5.
Compared to planktonic cells, which are free-floating and metabolically more homogeneous, biofilm-associated cells exhibit altered gene expression, growth rates, and metabolic activity6. These differences confer enhanced tolerance to environmental challenges, antibiotics, and host defenses, while allowing coordinated behaviors such as quorum sensing, nutrient retention, and spatial organization7,8.
Biofilm formation has been extensively characterized in model organisms such as Pseudomonas aeruginosa, Staphylococcus aureus, and Vibrio cholerae, which have collectively shaped our understanding of the genetic, structural, and physiological bases of the biofilm lifestyle. Studies on Pseudomonas have revealed that exopolysaccharides and quorum sensing work together to shape complex three-dimensional biofilm architectures9,10. Research on Staphylococcus has emphasized the roles of surface proteins and extracellular DNA in enhancing biofilm cohesion and antibiotic tolerance11,12. Investigations into Vibrio species further highlighted the remarkable diversity of biofilm morphologies and their ecological significance in aquatic environments&#185;&#179;. Collectively, these systems have provided a robust conceptual and methodological framework for biofilm research, reinforced by standardized protocols14 and critical evaluations of existing techniques15,16, which together highlight the need for harmonized approaches when investigating biofilm formation in less-studied bacteria such as Leptospira.
Members of the spirochete genus Leptospira, the causative agents of leptospirosis, were long presumed to be predominantly planktonic. Recent studies have experimentally demonstrated robust biofilm formation across multiple species17. While some studies suggest biofilm formation in environmental18 and host-associated19 contexts, others have experimentally demonstrated the ability of leptospires to form biofilm in the renal tubules of Rattus norvegicus20 and the vitreous humor of horses21, as well as detecting leptospiral species within environmental biofilms22,23. Deciphering the conditions and mechanisms that govern Leptospira biofilms is therefore critical to understanding environmental transmission, reservoir persistence, and disease pathogenesis24,25.
Existing Leptospira biofilm assays are fragmented, ranging from qualitative microscopic observations17,26 to endpoint colorimetric or crystal violet stains27,28. While these studies have provided valuable insights into biofilm formation, they often lack both the kinetic resolution required to monitor biofilm maturation and dispersal in real time and the ultrastructural context provided by confocal or electron microscopy. Continuous monitoring and 3D structural analysis are considered essential to capture the dynamic nature of biofilm formation and dispersal14,15. These limitations hamper comparability across studies and limit the systematic evaluation of factors or interventions affecting biofilm formation and dispersal, highlighting the need for a standardized, multi-readout workflow that captures both structural and functional dynamics of Leptospira biofilms in a reproducible and comprehensive manner.
To address these gaps, we developed an integrated, modular workflow that unifies six complementary readouts drawn from the same culture series. First, a high-throughput CV microtiter assay quantifies attached biomass at multiple time points. Second, a time-resolved fractionation assay in 96-well plates partitions total, liquid-phase (non-attached or partially detached), and attached (biofilm) populations by OD&#8324;&#8320;&#8325; to track their evolution during formation and dispersal. The term liquid-phase is used here to encompass both planktonic-like and detached cells, acknowledging the dynamic continuum between free-living and surface-associated phenotypes29,30. Third, time-lapse phase-contrast imaging provides non-destructive kinetics of adhesion, aggregate motility, and coalescence. Fourth, confocal laser-scanning microscopy (CLSM) yields full 3D reconstructions with live/dead and matrix-probe readouts, enabling depth-dependent viability and matrix mapping. Fifth, membrane- or coverslip-supported scanning electron microscopy (SEM) resolves extracellular architecture and basal-apical polarity at high resolution. In addition, an in vivo module was incorporated to evaluate virulence using intraperitoneal injection of biofilm aggregates into the susceptible golden Syrian hamster model, a sensitive and well-established model for leptospirosis31,32,33. This approach links biofilm phenotypes to pathogenic potential, providing functional context that complements in vitro analyses.
Compared with single-modality approaches, this platform offers several advantages: (i) synchronized quantitative (CV and fractionated OD) and structural (CLSM/SEM) readouts; (ii) non-destructive kinetic monitoring of early adhesion, growth, maturation, and dispersal; (iii) 3-D viability and matrix characterization using targeted probes; (iv) ultrastructural visualization of extracellular matrix architecture ; and (v) direct functional assessment of biofilm-associated versus planktonic virulence in a susceptible hamster model, each achievable with standard BSL-2 infrastructure. By leveraging multi-well formats and removable glass or polycarbonate substrates, the workflow supports replicate screens of environmental variables, antimicrobial compounds, or mutant libraries while maintaining sterility, throughput, and cross-validation across modules.
Laboratories focused on ecology, persistence, host-pathogen interactions, or anti-biofilm discovery can adopt individual modules or the full pipeline. Required resources are limited to a plate reader, an inverted microscope with environmental control for time-lapse, access to a confocal microscope and SEM facility, and standard animal facilities for the hamster model, enabling broad adoption and reproducible comparisons across studies.

<table><tbody><tr><td>0.1 &amp;micro;m sterile hydrophilic polycarbonate membrane</td><td>it4ip</td><td>1000M25/861N101/13</td><td></td></tr><tr><td>12 mm sterile glass coverslip</td><td>SPL</td><td>330164</td><td></td></tr><tr><td>16% paraformaldehyde (PFA)&amp;nbsp;</td><td>Thermo Scientific</td><td>28908</td><td></td></tr><tr><td>21G needle</td><td>BD Medical</td><td>304432</td><td></td></tr><tr><td>24-well microplate</td><td>NUNC</td><td>143982</td><td></td></tr><tr><td>35 mm glass-bottom dish</td><td>Ibidi</td><td>81218-200</td><td></td></tr><tr><td>35-mm glass bottom Hi-Q4 dish</td><td>Ibidi</td><td>81156</td><td></td></tr><tr><td>96-well microplate</td><td>NEST</td><td>701001</td><td></td></tr><tr><td>Antifade mounting medium&amp;nbsp;</td><td>Biorad</td><td>29410</td><td></td></tr><tr><td>Carbon coater</td><td>Leica Microsystem</td><td>Em ACE600</td><td></td></tr><tr><td>CFDA/SE tracer</td><td>Biolegend</td><td>423801</td><td></td></tr><tr><td>Conductive carbone adhesive</td><td>Electron Microscopy Science</td><td>77816</td><td></td></tr><tr><td>Crystal violet (CV)</td><td>Sigma</td><td>C3886</td><td></td></tr><tr><td>Dark field microscope</td><td>Leica Microsystem</td><td>11888846</td><td>Leica DM4000 B equiped with a dark fiel condenseur (cat n&amp;deg; 11505142)</td></tr><tr><td>Ethanol</td><td>VWR Chemicals</td><td>83813.36</td><td></td></tr><tr><td>FM4-64</td><td>Invitrogen</td><td>T3166</td><td></td></tr><tr><td>Glacial acetic acid</td><td>Supelco</td><td>1.00063</td><td></td></tr><tr><td>Glutaraldehyde solution</td><td>Sigma-Aldrich</td><td>G5882</td><td></td></tr><tr><td>Hexamethyldisilazane</td><td>Acros Organics</td><td>120581000</td><td></td></tr><tr><td>Incubator</td><td>Memmert</td><td>IN30</td><td></td></tr><tr><td>Inverted confocal microscope</td><td>Leica Microsystem</td><td>Leica DMI6000 TCS SP8 X</td><td>Confocal microscope SP8</td></tr><tr><td>Inverted microscope fitted with phase-contrast optics</td><td>Nikon</td><td>CELL-S2</td><td>BioStation IM-Q</td></tr><tr><td>Leptospira Medium Base EMJH&amp;nbsp;</td><td>Becton Dickinson</td><td>BD&amp;nbsp;279410</td><td>EMJH medium for Leptospira</td></tr><tr><td>Osmium tetroxide (OsO&lt;sub&gt;4&lt;/sub&gt;)&amp;nbsp;</td><td>Sigma</td><td>BCCG9181</td><td></td></tr><tr><td>Propidium iodide&amp;nbsp;</td><td>Biotium</td><td>40017</td><td></td></tr><tr><td>Scanning electron microscope</td><td>JEOL</td><td>JSM-IT300</td><td></td></tr><tr><td>Sodium cacodylate buffer&amp;nbsp;</td><td>Thermo Scientific Chemicals</td><td>15453149</td><td></td></tr><tr><td>Spectrophotometer Varioskan Lux</td><td>Thermo Scientific</td><td>VLBL00GD0</td><td></td></tr><tr><td>Sterile Phosphate-buffered Saline</td><td>Biosolve</td><td>0016232301BS</td><td></td></tr><tr><td>Syringe (1 mL)</td><td>Chirana</td><td>CH03002L</td><td></td></tr><tr><td>SYTO9 green fluorescent nucleic acid stain&amp;nbsp;</td><td>Invitrogen</td><td>S34854</td><td></td></tr></tbody></table>

a modular workflow for quantitative, structural and functional analysis of leptospira biofilms

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

Here, we present an integrated suite of protocols for the cultivation, quantitative monitoring, structural and functional characterization of Leptospira spp. biofilms over time. This workflow combines crystal violet microtiter assays to measure biofilm biomass at multiple time points, with a time-resolved fractionation approach that distinguishes attached (biofilm) and non-attached (liquid-phase) bacterial populations, time-lapse phase-contrast imaging for non-destructive kinetic observation, confocal laser-scanning microscopy to generate full 3D reconstructions with matrix-probe readouts, and membrane-supported scanning electron microscopy for ultrastructural analysis. In parallel, we detail a standardized procedure for harvesting intact biofilm aggregates and preparing them for intraperitoneal injection into the susceptible golden Syrian hamster model, enabling direct assessment of biofilm-associated virulence in vivo alongside matched planktonic controls.
Optimized for the pathogenic strain Leptospira interrogans Manilae L495, each module is readily transferable to other Leptospira species and mutant libraries to compare biofilm-forming capacity. Together, these coordinated modules provide a robust foundation for screening anti-biofilm strategies, probing genetic determinants, and clarifying the contribution of biofilms to Leptospira persistence and pathogenesis.

When the inoculum is prepared correctly, cultures enter mid-log phase within 3-5 days and exhibit OD405 values of ~ 0.2-0.4 with bright, highly motile fields under dark-field microscopy (≥ 90% of cells motile) and no visible clumps. Suboptimal preparations present as sluggish bacteria, and heterogeneous motility across the field; such cultures frequently yield weak biofilms and should be discarded. In practice, confirming motility and OD side-by-side immediately before seeding minimizes failed runs. Establishing these quality control gates at the inoculum stage is the single best predictor of success across downstream applications. Although the methodology was optimized for L. interrogans serovar Manilae L495, the same procedures were also applied to Leptospira biflexa Patoc strain to demonstrate cross-species applicability. L. biflexa generally forms less cohesive and thinner biofilms compared to L. interrogans, yet the characteristic developmental sequence and structural features remain detectable with appropriate parameter adjustments. Including both species thus underscores the adaptability of the workflow to pathogenic and saprophytic Leptospira alike.
After 21 days of static incubation at 30 °C in a humidified chamber (or the appropriate duration for the Leptospira species used), biofilms become visible to the naked eye on both glass coverslips and hydrophilic polycarbonate membranes. Successful growth produces characteristic CV patterns after 2-3 weeks such as dot-like, branching, or reticulated footprints attached to the surface (Figure 2A).
In a typical run, wild-type L. interrogans Manilae L495 reaches ~ 50% surface coverage by week 3, while low-biofilm mutants' plateau near ~ 20% and high-biofilm phenotypes approach ~ 70-80%, establishing a practical dynamic range for screening. The extent of biofilm formation may also vary depending on the Leptospira species and strain; for instance, L. biflexa Patoc strain develops visible biofilms more rapidly, with previous studies considering structures as early as 120 h post-inoculation to be mature biofilms35.
Crystal violet staining provides a reliable and reproducible method for quantifying biofilm biomass. In well-developed biofilms, staining results in deep purple coloration localized to the coverslip or membrane area, indicating high levels of attached biomass (Figure 2A). Visual cues during the staining and solubilization steps also provide useful indicators of protocol success. For instance, uneven CV distribution or pale staining may be due to under-seeding, evaporation, or aggressive washing that detaches early biofilms.
Absorbance readings at 570 nm reflect the amount of retained stain, and therefore, the relative biofilm density (Figure 2B). In representative experiments, L. interrogans cultures grown under optimal conditions show consistent and reproducible OD₅₇₀ readings across replicates, reflecting stable biofilm formation. In contrast, large variations between replicates are often observed when samples undergo excessive pipetting during medium changes, indicating poor adhesion or partial biofilm detachment. Such variability should be considered a sign of technical issues, and the affected samples should be excluded or the protocol carefully reviewed. Notably, L. biflexa Patoc forms more extensive biofilms on both glass coverslips and polycarbonate membranes under similar conditions, which is reflected in higher CV absorbance values, demonstrating that the protocol is adaptable and effective across Leptospira species.
Successful SEM preparation is able to reveal extracellular matrix deposits as early as day 3, followed by a strikingly polarized architecture in mature biofilms: a rough, channeled basal face (often with &gt; 5 µm channels) that anchors a porous inner structure, and a smoother apical face where spirochetes lie enmeshed in dense matrix (Figure 2C, D, E, F). High-magnification fields frequently capture branching extracellular filaments and occasional mushroom-like protrusions; features that correspond to the coalescence dynamics seen by time-lapse (Supplemental video 1). In suboptimal preparations, collapsed matrices, charging, and indistinct cell outlines may be observed, usually reflecting inadequate post-fixation, insufficient conductive coating, or poor drying. When basal-apical polarity and pervasive channels are evident, SEM readouts align closely with confocal estimates of thickness and porosity, confirming successful execution of both preparation and imaging.
In effective time-lapse series, isolated puncta appear within 24-72 h and progressively coalesce into larger aggregates that sweep across the surface before space limitations slow their motion (Figure 3A, B, C). Quantitative segmentation yields a monotonic increase in total covered area, while aggregate counts rise, peak, and then decline as collisions and mergers dominate between ~12 and 216 h. These kinetics (area up, aggregates number down), indicate active accretion rather than simple sedimentation. Failed or borderline runs lack early puncta, show flat area-over-time curves, or suffer focus drift linked to unstable temperature/humidity. Maintaining a stabilized 30 °C environment with 95% humidity and using autofocus at each time point typically restores clear trajectories suitable for comparing mutants or treatments.
Representative Z-stacks from mature biofilms show foam-like, multilayered architecture typically exceeding 50 µm in thickness, with most cells staining live (SYTO 9-positive) and occasional central voids indicative of collective rearrangements during growth (Figure 3D). Matrix probes can be used to investigate matrix composition: WGA highlights polysaccharide epitopes, BOBO-3 labels abundant extracellular DNA, and protein-selective stains contribute little outside cell-associated signal (Figure 3E). Suboptimal results include thin or discontinuous stacks dominated by propidium iodide, strong photobleaching, or inconsistent gain settings, each of which undermines quantitative comparability. Interpreting thickness, biovolume, and live/dead ratios together (while holding laser power, detector gain, and pinhole constant across conditions) confirms maturation status and supports direct comparisons with SEM ultrastructure and CV biomass.
The biofilm formation process of Leptospira typically follows distinct phases, though exact timings and values may vary depending on the species or strain. Using L. interrogans strain Manilae L495 as a reference, the expected progression over 21 days includes an initial phase (days 0-3) where bacteria remain mostly planktonic with minimal biofilm (Figure 4A). This is followed by an exponential growth phase (days 3-7) during which both planktonic and biofilm-associated bacteria increase, reaching around 9 x 10⁸ cells/mL, accompanied by the formation and expansion of biofilm aggregates. Between days 7 and 12, planktonic bacteria decline significantly, while biofilm-associated cells peak, representing roughly 80% of the population. Finally, during the maturation phase (days 12-21), biofilm bacterial numbers decrease without a rise in planktonic cells, yet biofilm size and complexity continue to grow. Observing this sequence of changes indicates that the protocol effectively captures the dynamic development and maturation of Leptospira biofilms.
When properly performed, the infection protocol uses inocula containing ~2 x 10⁸ leptospires in 200 µL EMJH, prepared from well-defined planktonic (5-day) or biofilm (21-day) cultures. Although these two culture types represent distinct physiological states, this difference is intentional, as the experiment aims to determine whether Leptospira biofilms -- characterized by reduced metabolic activity and structural differentiation -- retain the ability to initiate infection. This contrast is supported by transcriptomic studies showing major shifts in gene expression between planktonic and biofilm Leptospira35,36. Biofilm aggregates are carefully harvested to preserve their structure and remain intact after passage through a 21 G needle, as confirmed before injection (Figure 4C, D). Following intraperitoneal injection, golden Syrian hamsters typically display clinical signs of leptospirosis within 3 to 5 days (e.g., lethargy, ruffled fur, prostration). Negative controls injected with EMJH medium alone show no signs of disease. The progression and severity of clinical signs, as well as time-to-euthanasia, vary in accordance with the inoculum: planktonic bacteria often induce earlier and more acute symptoms, whereas biofilm-derived aggregates may cause a delayed but persistent infection (Figure 4B). Monitoring twice daily for up to 21 days allows capturing the full disease course.
<img alt="Bacterial growth analysis; absorbance graph, agar plate colony images, SEM results of biofilm structures." class="xfigimg" src="/files/ftp_upload/69511/69511fig2.jpg" data-altupdatedat="1784001604000" data-alt="Figure 2"> 
Figure 2. Crystal violet staining, quantification, and ultrastructural imaging of Leptospira biofilms. (A) Crystal violet (CV) staining of biofilms grown on different substrates. CV staining allows visualization of biofilm architecture and initial attachment patterns for different species and substrates. i. L. interrogans on polycarbonate filter; ii. L. biflexa on polycarbonate filter; iii. L. interrogans on glass coverslip; iv. L. biflexa on glass coverslip. (B) Example of quantitative biofilm formation assessed by CV absorbance (OD570 nm) over time for L. interrogans and L. biflexa. This kinetic readout captures both early adhesion and biomass accumulation dynamics, highlighting differences in biofilm growth between pathogenic and saprophytic species. This figure has been modified from27. (C-E) Scanning electron microscopy (SEM) of L. interrogans biofilms: (c) 3-day-old biofilm showing early microcolony formation and initial extracellular matrix deposition;(d) 14-day-old biofilm displaying mature architecture with extensive matrix and three-dimensional organization;(e-f) 3-week-old biofilm illustrating structural consolidation and matrix maturation over prolonged culture. This combination of CV staining, quantitative OD measurements, and SEM imaging provides a comprehensive overview of biofilm development, from early attachment to mature ultrastructural organization, enabling direct comparison across species and culture durations. <a href="https://www.jove.com/files/ftp_upload/69511/69511fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Cell growth and morphology microscopy images; panel sequence shows tissue structure over time." class="xfigimg" src="/files/ftp_upload/69511/69511fig3.jpg" data-altupdatedat="1784001604000" data-alt="Figure 3"> 
Figure 3. Visualization of Leptospira biofilm formation. Phase-contrast images acquired with a BioStation show biofilm development at (A) 48 h, (B) 96 h, and (C) 144 h. (D) CLSM reconstruction of a Leptospira biofilm displaying total biovolume with orthogonal slices for 3D visualization. (E) Confocal staining of a mature biofilm with DAPI (green) and WGA (red), highlighting bacterial cells and extracellular matrix components. This figure has been modified from37. <a href="https://www.jove.com/files/ftp_upload/69511/69511fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Biofilm growth chart and survival graph, 405 nm absorbance vs. time, experiment setup tube, sample." class="xfigimg" src="/files/ftp_upload/69511/69511fig4.jpg" data-altupdatedat="1784001604000" data-alt="Figure 4"> 
Figure 4. Time-Resolved Quantification of Planktonic and Biofilm Fractions and functional assessment of Leptospira biofilms. (A) Kinetics of biofilm formation measured by absorbance at 405 nm. For each time point, readings were taken from the total well, the biofilm fraction, and the supernatant containing planktonic leptospires, allowing distinction between attached and free-swimming bacteria. (B) Example survival curves of hamsters injected with biofilm aggregates, planktonic leptospires, or EMJH control. Biofilm-injected animals show reduced virulence, with some surviving 21 days, whereas planktonic leptospires cause rapid mortality. (C-D) Preliminary validation of biofilm integrity: aggregates are visible in the syringe prior to injection (C) and remain intact after passage through a 21 G needle (D, white arrows), confirming that biofilm structure is preserved during handling. This figure has been modified from36. <a href="https://www.jove.com/files/ftp_upload/69511/69511fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Here, we present an integrated suite of protocols for the cultivation, quantitative monitoring, structural and functional characterization of Leptospira ...

We study biofilms as protective structures enabling environmental and host persistence, investigating the formation dynamics, molecular composition, adaptative features and contribution to infection. Combining crystal bio assay, time text imaging, confocal microscopy, scanning electron microscopy and transcriptomics in our labs, advances biofilm research through integrated multi dimension analysis. To begin, grow Leptospira cells in EMJH medium in flat bottomed screw cap glass tubes.Incubate the cultures under aerobic conditions at 30 degrees Celsius without shaking until they reach mid logarithmic phase. Ensure the cultures reach an optical density between 0.2 and 0.4 at 405 nanometers, which corresponds to two to five times 10 to the power of eight cells per milliliter. Using a dark field microscope at 20 x magnification, verify that the cells are modal and not clumped.Dilute the verified mid log arrhythmic phase culture at a one to 100 ratio in fresh EMJH medium to obtain approximately one times 10 to the power of six cells per milliliter and mix gently by inversion without vortexing. Now use sterile forceps to place one 0.1 micrometer sterile hydrophilic polycarbonate membrane flat at the bottom of each well in a sterile 24 well plate with lid. Add one milliliter of sterile EMJH medium to each well and pre-soak the membrane for two hours at 30 degrees Celsius.Next, remove the soaking solution from each well without displacing the membrane and add 1.5 milliliters of diluted bacterial suspension, ensuring the membrane remains firmly in place at the bottom. Then, place a water-filled tray inside the incubator to maintain humidity. Incubate the plate at 30 degrees Celsius under static conditions to allow biofilm formation.Leaving the plate for up to three weeks for slow growing strains. At the desired time point, carefully aspirate as much culture medium as possible without disturbing the biofilm. Rinse each well gently with one milliliter of sterile PBS while keeping the membrane flat against the bottom.Add one milliliter of 4%paraform aldehyde in PBS to each well and incubate at 37 degrees Celsius for 30 minutes to fix the biofilm samples. After incubation, remove the fixative and gently rinse twice with one milliliter PBS. Add one milliliter of 0.1%weight by volume crystal violet solution to each well and incubate at room temperature for 15 minutes, making sure the cover slip is fully submerged.Then discard the crystal violet dye from each well and rinse twice with one milliliter of PBS. Tilt the plate and drain all remaining liquid. Leave the plate at room temperature to air dry until the substrate appears completely dry, preferably overnight.Next, add 500 microliters of elucian buffer composed of 50%ethanol and 50%glacial acetic acid by volume to each well. Pipette up and down to fully dissolve the stain bound to the biofilm. Now, transfer 200 microliters of each sample to an optically clear 96 well microplate.Measure absorbance at 570 nanometers and subtract background values from uninoculated controls processed through all steps. Record the mean and standard deviation for at least three technical replicates. Obtain the biofilms in well plates as demonstrated earlier.Add a solution of 4%paraform aldehyde, and 1%glutaraldehyde in 0.2 molar sodium buffer at pH 7.4 into the wells. Incubate the fixed samples for 30 minutes at 37 degrees Celsius. Remove the fixative and rinse the sample twice with PBS to preserve the surface attached biofilm while minimizing detachment.Now immerse the cover slip in 1%osmium tetroxide diluted in PBS and incubate for one hour to enhance scanning electron microscopy contrast. Then rinse the substrate twice with PBS. Dehydrate the samples by immersing them sequentially for 10 minutes each in graded ethanol series.Next, add 500 microliters of hexamethyldisilazane and incubate for five minutes. Then replace with fresh hexamethyldisilazane and incubate for an additional five minutes. Afterward, remove the excess solution and let the sample air dry completely under a fume hood.Now mount the dried samples onto scanning electron microscopy stubs using double-sided conductive carbon tape. Sputter coat the samples with a thin layer, approximately 10 nanometers of gold or platinum to enhance electron contrast for imaging. Finally, load the prepared stubs into the scanning electron microscope using the appropriate sample holder.Evacuate the chamber overnight if possible to improve imaging quality. Then acquire secondary electron images at five to 15 kilovolts using suitable magnifications to visualize the ultra structure of the biofilm. After 21 days of incubation, crystal violet staining revealed visible biofilm patterns on both polycarbonate filters and glass cover slips for leptospira interrogans and leptospira biflexa.With each species displaying distinct architectural footprints such as dotlike, branching or reticulated forms. Absorbance measurements at 570 nanometers confirmed greater crystal violet retention in leptospira byflexa biofilms compared to leptospira interrogans across all time points indicating higher biomass accumulation in the strain. Scanning electron microscopy of leptospira interrogans biofilms captured early extracellular matrix deposits in three day old biofilms, a mature and channeled basal face with three dimensional structure at 14 days and fully developed matrix consolidation with dense architecture by 21 days.Our optimized protocol synchronizes complementary readouts from identical cultures, increasing robustness, reducing variability and revealing dynamic and structural information and available with single method approaches. By integrating complementary analysis, our findings standardized biofilm assessment, clarify leptospira dynamics and architecture. They link structure to virulence and strengthen reproducible comparative research.Future mutants to link regulation with biofilm morphology and dynamics, clarifying environmental persistence and evaluate strategies disrupting biofilm information or promoting dispersal.

a-modular-workflow-for-quantitative-structural-functional-analysis

Research

JoVE Journal

Biology

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An Analytical Tool-box for Comprehensive Biochemical, Structural and Transcriptome Evaluation of Oral Biofilms Mediated by Mutans Streptococci

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Engineering Adherent Bacteria by Creating a Single Synthetic Curli Operon

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Concurrent Quantification of Cellular and Extracellular Components of Biofilms

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

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

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

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A New Method for Qualitative Multi-scale Analysis of Bacterial Biofilms on Filamentous Fungal Colonies Using Confocal and Electron Microscopy

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Paraffin Embedding and Thin Sectioning of Microbial Colony Biofilms for Microscopic Analysis

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Applying Live Cell Imaging and Cryo-Electron Tomography to Resolve Spatiotemporal Features of the Legionella pneumophila Dot/Icm Secretion System

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.

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

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Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

Learn a modular workflow for quantitative, structural and functional analysis of Leptospira biofilms using crystal-violet assays, microscopy, and in vivo infection models. This standardized protocol enhances reproducibility in biofilm research.

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

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