Scientific illustration as a multiscale framework for representing biofilms

Abstract

Biofilms are widely recognised as a dominant mode of microbial life, yet their visual representation in the scientific literature still often relies on simplified diagrams derived from laboratory systems. While these schemes have strong pedagogical value, they do not adequately reflect the heterogeneity, structural complexity, and environmental dependence of biofilms encountered in real settings. Because visual representations shape both scientific communication and conceptualisation, the way biofilms are illustrated matters for how they are understood.

Here, we present the outcome of a collaboration between a biofilm microbiologist and a scientific illustrator to develop a conceptual visual framework for representing the classical biofilm life cycle across scales and environments. Rather than replacing the classical sequence, the framework embeds it within complex microbial scenes and uses graphical guidance, including colour coding, to make its most explicit trajectory readable within heterogeneous biofilm organisation. In this way, adhesion, growth, matrix production, remodelling, and dispersal are represented not as rigidly separated stages, but as processes that may coexist locally within structured communities.

To connect microscopic mechanisms with real-world situations, these processes were integrated into domestic environments such as bathrooms and kitchens. The illustrations include both undesirable and beneficial biofilms, and selected domestic biofilm structures were informed by real microscopy observations to preserve biological plausibility while maintaining graphical coherence. Particular attention was given to microbial and structural diversity across domestic habitats.

The visual framework relies on isometric projection and multiscale transitions that connect experimental systems, colonised surfaces, biofilm architectures, individual cells, and extracellular matrix components within a coherent visual continuum. This approach is introduced through a progressive view extending from a laboratory flow-cell device to the molecular network surrounding cells within the biofilm. The framework was implemented across complementary media, including static figures, animated slides, a simplified movie, and a large-format poster. Together, these materials form a multiformat visual framework intended to support reasoning, teaching, and interdisciplinary communication about biofilm organisation.

Highlights

• •The biofilm life cycle is reinterpreted within heterogeneous microbial contexts.
• •A multiscale visual sequence connects the experimental device, biofilm architecture, individual cells, and extracellular matrix components.
• •Domestic environments are used as model ecosystems for contextual biofilm representation.
• •Scientific illustration functions as a conceptual tool for biofilm reasoning, teaching, and communication.

1. Introduction

Although biofilms are now widely understood as heterogeneous, dynamic, and spatially organised microbial systems, they are still commonly represented through simplified diagrams that portray a linear succession of developmental stages. This contrast between current biological knowledge and conventional visual models is not trivial. Scientific images do not merely accompany knowledge after the fact: they help organise, stabilise, and communicate it. In the case of biofilms, the way microbial communities are illustrated may therefore influence not only how they are taught, but also how they are conceptually framed and discussed across disciplines.

Biofilms are recognised as a predominant mode of microbial life in natural, domestic, industrial, and clinical environments [[1], [2], [3]]. Rather than existing mainly as isolated planktonic cells, microorganisms often form structured communities attached to interfaces and embedded in extracellular material. Extensive experimental work has shown that such communities are physiologically heterogeneous, continuously remodelled, and strongly shaped by local gradients of nutrients, oxygen, hydration, signalling molecules, and mechanical constraints [[4], [5], [6]]. Their organisation emerges from interactions among cells, matrix, surfaces, and surrounding environmental conditions rather than from the behaviour of individual cells alone. This heterogeneity is therefore not a secondary property of mature biofilms, but a central feature of their biology.

Despite this complexity, the dominant visual language used to describe biofilms often remains derived from early laboratory models. Classical representations typically organise the biofilm life cycle into successive stages, including initial adhesion, microcolony formation, maturation, and dispersal [7,8]. These schemes have played an important pedagogical role and remain highly effective for introducing the field. However, they also suggest a regular and predictable developmental pathway that does not fully reflect many real systems. In environmental, food-associated, and domestic settings, biofilms are rarely uniform structures and do not necessarily follow discrete, universally ordered trajectories [3,9]. Attachment, proliferation, matrix production, local cell death, remodelling, detachment, and recolonisation may occur simultaneously within different regions of the same community [4,10,11]. Rather than abandoning the classical cycle, there is therefore value in asking how it can be visually retained as a readable explanatory framework while being reinserted into a more realistic and heterogeneous microbial context. This question is central to the present work. Our objective was not to replace the classical biofilm life cycle with a new biological model, but to reinterpret its graphical use. In the visual framework developed here, the classical cycle remains a deliberate guide for reading the image, but it is embedded within a broader polymicrobial and environmentally situated scene. Colour coding is used to make the most explicit classical trajectory visually traceable, while the surrounding composition shows that this trajectory is only one of many readable pathways within a more complex organisation. In this sense, the image does not present biofilm development as a rigid temporal script. Rather, it offers a spatially contextualised reading of the classical cycle by showing how processes classically described in sequence may coexist locally within a structured biofilm system.

Scientific illustration offers a useful intermediate approach between schematic abstraction and direct imaging. Unlike microscopy, it allows selective emphasis on relevant structures, integration of observations obtained at different scales, and the representation of processes that are distributed, overlapping, or difficult to observe simultaneously in a single field of view. In the present work, however, illustration was not conceived as a free artistic reconstruction detached from experimental observation. Some microbial structures depicted in the domestic scenes were redrawn from real microscopy images and then harmonised graphically to ensure coherence across the visual system. Other elements were developed as biologically informed visual syntheses based on published knowledge, domestic microhabitat conditions, and biofilm imaging expertise. The resulting images are therefore not literal reproductions, but interpretative reconstructions grounded in observed biofilm morphologies and reworked so that biologically relevant organisation remains visible within a coherent illustrative language.

The need for such an approach becomes particularly clear when biofilms are considered in real-world environments. Domestic spaces contain numerous surfaces repeatedly exposed to moisture, nutrients, aerosols, human contact, and fluctuating cleaning pressures, thereby creating favourable conditions for microbial attachment and persistence [[12], [13], [14], [15]]. Bathrooms and kitchens can therefore be viewed as distributed microbial landscapes composed of interconnected habitats differing in hydration, material properties, disturbance regimes, and nutrient availability. These settings are immediately recognisable to broad audiences, yet they also capture many of the ecological variables that govern biofilm establishment and persistence. Domestic environments were therefore used here not as anecdotal illustrations, but as model ecosystems in which multiscale microbial organisation could be represented in relation to familiar objects and surfaces.

Within these domestic scenes, particular attention was given to the diversity of microbial compositions and extracellular matrices. In the bathroom, the framework includes moisture-associated biofilms with distinct matrix appearances and architectures, as well as polymicrobial communities involving both bacterial and fungal forms. The kitchen offers a complementary setting in which beneficial and undesirable biofilms can coexist, thereby avoiding an exclusively negative view of the biofilm mode of life. Fermented foods provide especially relevant examples of structured microbial communities that contribute to preservation, flavour development, texture, and ecosystem stability [16,17], while food-contact surfaces and humid domestic reservoirs illustrate persistence, recolonisation, and cross-contamination under everyday conditions [13,14,18].

In this context, the graphical technique is consequential in itself. The framework developed here relies on isometric projection, a representational method widely used in technical and architectural drawing in which parallel lines remain parallel and spatial relationships are preserved without perspective distortion. This geometry allows cells, matrix, surfaces, objects, and environmental conditions to coexist within a stable composition and facilitates continuity across figures and formats. For biofilms, whose properties emerge across multiple levels of organisation, such multiscale continuity is particularly useful. Isometry does not make the image more realistic in an optical sense; rather, it makes it more navigable and conceptually coherent. The graphical principles and construction steps underlying this isometric framework are illustrated in Supplementary Material S4.

The present study originated from a sustained collaboration between a biofilm microbiologist and a scientific illustrator. Through iterative exchanges between experimental knowledge, published literature, microscopy-derived forms, and graphical experimentation, illustration became part of the reasoning process itself rather than a final aesthetic step. Constructing coherent images required explicit choices about scale, hierarchy, heterogeneity, uncertainty, and readability. These constraints, in turn, helped clarify the assumptions embedded in conventional representations of the biofilm life cycle and reinforced the need for a framework that preserves pedagogical clarity without reducing biofilms to rigidly ordered, visually simplified systems.

We therefore propose a conceptual and visual framework for representing biofilms across spatial scales and environmental contexts. The multiscale logic of the framework is introduced in Fig. 1 through a progressive transition from a laboratory flow-cell device to the colonised flow channel, to structured biofilm architectures, to individual microbial cells, and to extracellular matrix components that contribute to their spatial cohesion. This sequence is not intended to represent a quantitative magnification series or the direct observation of a single experimental specimen. Rather, it provides a conceptual reading path that connects the experimental, architectural, cellular, and molecular levels of biofilm organisation. The subsequent figures apply this multiscale visual logic to the classical biofilm life cycle and to biofilms situated within domestic environments. Rather than depicting a single realistic system or introducing a new universal developmental model, the framework synthesises current knowledge through a set of coherent visual outputs, including article figures, extracted thematic scenes, animated slide sequences, a simplified video, and a large-format poster. By linking microbial processes to material surfaces, experimental devices, and familiar domestic settings, this work argues that scientific illustration can function not only as a communication tool but also as a conceptual instrument for representing and discussing structured microbial life.

Fig. 1.

Multiscale representation of biofilm organisation from the experimental system to the extracellular matrix. Conceptual sequence connecting complementary levels of biofilm observation and representation. (A) A laboratory operator handles a flow-cell device used for controlled biofilm cultivation under defined hydrodynamic conditions. (B) A closer view of a single flow channel reveals surface-associated microbial material, but does not resolve its detailed organisation. (C) At the biofilm scale, heterogeneous, spatially structured microbial communities become visible, including clusters at different stages of development and surrounding matrix-rich regions. (D) Higher magnification of a three-dimensional cluster reveals individual microbial cells embedded within the biofilm structure. (E) At the cellular scale, neighbouring cells are shown in close spatial association within extracellular material. (F) At the molecular scale, extracellular polymeric substances, including polysaccharides, proteins, extracellular DNA, and other matrix-associated components, form an interconnected network that contributes to cell-cell cohesion and biofilm architecture. The panels constitute a conceptual progression across levels of organisation rather than a quantitative magnification series or a sequence of measurements obtained from a single specimen. The figure introduces the multiscale visual logic used throughout the article to connect experimental systems, microbial structures, individual cells, and molecular components.

Status and reading sequence of the visual materials: The article is built around a set of complementary visual outputs. The annotated animated PowerPoint presentation (Supplementary Material S1) and the high-resolution poster (Supplementary Material S3) constitute the most complete versions of the framework, whereas the static article figures are reduced extracts designed to anchor specific concepts in the text. Readers are therefore encouraged to explore the animated presentation and poster alongside the manuscript figures, as each format offers a distinct level of spatial, temporal, and pedagogical interpretation.

Methodological basis of the visual framework: The visual framework was developed through iterative exchanges between biofilm microbiology expertise and scientific illustration practice. Microbial forms, matrix architectures, and spatial arrangements were informed by published biofilm literature, previous microscopy observations, and the authors' experience with biofilm imaging. The illustrations combine different levels of empirical anchoring. Some elements in the domestic environment scenes were directly redrawn or adapted from real electron microscopy images, whereas others, such as the coffee machine and sponge examples, were developed as biologically informed visual syntheses based on published knowledge, domestic microhabitat conditions, and the authors’ experience with biofilm imaging. The biofilm life-cycle illustration was not derived from a single microscopy image, but was constructed as a composite conceptual synthesis of classical biofilm processes, microscopy-informed morphology, and current knowledge of biofilm heterogeneity. The resulting illustrations should therefore be understood as interpretative reconstructions rather than literal reproductions of single experimental fields. The graphical construction process, including isometric projection principles, early drawing attempts, composition trials, project sketches, and successive refinements, is documented in Supplementary Material S4. Together, these steps define the illustrations as biologically informed conceptual syntheses rather than decorative representations.

2. Visualising the biofilm life cycle: a spatialised reading of the classical sequence

Biofilms are commonly depicted in schematic diagrams that describe a succession of developmental stages, typically including initial adhesion, microcolony formation, maturation, and dispersal [7,8]. This classical framework has been highly influential in teaching and structuring the field because it provides a simple, readable narrative of biofilm development. Its enduring success reflects an important pedagogical strength: it offers an accessible entry point into a complex mode of microbial life. However, this sequential representation only partially reflects current understanding of biofilm biology. Experimental observations show that biofilms are not homogeneous structures progressing through a universal series of clearly separated steps. Rather, they are dynamic systems in which attachment, cell proliferation, matrix production, physiological differentiation, local death, remodelling, detachment, and recolonisation may occur simultaneously within different regions of the same community [4,10,11]. Spatial gradients of nutrients, oxygen, hydration, and signalling molecules further generate heterogeneous physiological states over short distances [4,10]. Recent work has reinforced this view by showing both the spatial structuring of transcriptional activity within biofilm communities [19] and the capacity of motile cells to infiltrate and remodel established biofilms from within [20]. These observations make clear that the classical life cycle should not be read as a rigid or universal chronology. The objective of the present work was therefore not to reject the classical biofilm cycle, but to reconsider how it is graphically used. Fig. 1 first establishes the multiscale organisation of the framework by connecting the experimental device, the colonised interface, biofilm architecture, individual cells, and extracellular matrix components. Building on this multiscale reading, Fig. 2 embeds the classical biofilm life-cycle sequence within a heterogeneous and potentially polymicrobial context. The image does not isolate a simplified monospecies trajectory from its surroundings. Instead, it places the familiar sequence within a broader organisation in which multiple structures, taxa, matrix organisations, and local physiological processes may coexist.

Fig. 2.

Spatialised representation of the biofilm life cycle within a heterogeneous microbial context. Building on the multiscale visual framework introduced in Fig. 1, this conceptual illustration represents the main processes commonly associated with biofilm development, including planktonic cell transport, initial surface adhesion, local proliferation, microcolony formation, extracellular matrix production, structural maturation, physiological differentiation, local cell death and remodelling, and dispersal. The classical life-cycle sequence is retained as a pedagogical reading guide, but it is embedded within a heterogeneous and potentially polymicrobial community in which biofilm structures, microbial populations, and matrix organisations coexist. The arrows and the coloured trajectory guide the reader through one canonical developmental pathway, while the surrounding microbial landscape indicates that this pathway represents only one possible trajectory within a broader spatially organised system. Processes conventionally presented as successive stages may overlap in space and time and may occur simultaneously in different regions of the same biofilm. The figure should therefore be interpreted as a spatialised reading of the classical biofilm life cycle rather than as a rigid, universal, or deterministic developmental sequence. This static illustration is a reduced extract from the broader visual framework presented in Supplementary Materials S1 and S3, in which the progressive interpretation and complete spatial organisation of the scene are developed in greater detail.

A key graphical choice was to make this classical trajectory visually traceable through colour coding. Within the larger composition, a distinct colour is used to guide the reader along the most explicit pathway corresponding to the classical cycle. This choice does not imply that a single developmental route exhausts the biology of the system. Rather, it provides a readable thread through a complex image. The function of colour is therefore interpretative and pedagogical: it helps the viewer follow the canonical stages of adhesion, growth, matrix production, remodelling, and dispersal, while simultaneously showing that these processes unfold within a spatially heterogeneous, environmentally situated, and potentially polymicrobial context. The classical cycle is thus not erased by complexity, but made legible within it. The arrows and colour coding are therefore used as graphical aids for reading. They preserve the visual movement and pedagogical clarity of the classical biofilm life-cycle representation, while the surrounding heterogeneous scene indicates that this trajectory should not be interpreted as a single deterministic pathway. Biofilm development remains a dynamic temporal process, but the framework emphasises that processes commonly described as successive stages may also be locally distributed and partially coexisting within structured communities. For this reason, it is more accurate to describe the present approach as a spatialised reading of the classical biofilm cycle than as a strict transformation of a temporal model into a spatial one. The figures do not claim that time can be replaced by space in any absolute sense. Biofilm development remains dynamic, and temporal succession remains biologically relevant. What the visual framework does is to translate a classically sequential description into a composition in which coexistence, overlap, and local variation become visible. Processes that are often introduced one after another in teaching diagrams can here be read as partially simultaneous and locally distributed within the same organised system. The emphasis is therefore less on replacing chronology than on showing that chronology is embedded within spatial heterogeneity. This distinction is important both conceptually and visually. If the classical life cycle is presented solely as a temporal succession of idealised stages, the reader may infer that biofilms develop through a regular and predictable programme. By contrast, if the cycle is made readable within a complex spatial scene, it becomes possible to preserve its explanatory clarity while avoiding the impression of rigid universality. The figure can then be understood as showing a canonical pathway within a biologically richer setting, rather than as depicting a single deterministic script. This approach is particularly useful for representing real biofilms, where local matrix accumulation, dispersal events, differentiated cell states, and recolonisation may occur simultaneously in different regions of the same system [4,11]. The successive processes guiding this spatialised reading of biofilm development are detailed in Fig. 3.

Fig. 3.

Spatialised representation of successive processes involved in biofilm development. Conceptual illustration of the principal events commonly associated with the classical biofilm life cycle, represented here as a readable sequence while acknowledging that these processes may overlap in space and time within real biofilms. (A) Planktonic cells are transported towards a surface by liquid flow, sedimentation, Brownian motion, or active motility, depending on the local physical and biological context. (B) Initial adhesion occurs when cells interact with the surface through reversible contacts that then become more stable, influenced by surface properties, conditioning films, cell appendages, and local hydrodynamic conditions. (C) Attached cells proliferate and form early microcolonies, generating local clusters that may already differ in density, composition, and physiological state. (D) Extracellular matrix production contributes to the consolidation of the developing community by embedding cells in polymeric material, promoting cell-cell cohesion, surface attachment, water retention, and local protection. (E) As the biofilm matures, structural and physiological heterogeneity increases through the formation of gradients, matrix-rich regions, voids, channels, differentiated cell states, and locally distinct microenvironments. (F) Local remodelling, erosion, detachment, or active dispersal releases cells or aggregates that may colonise new surfaces and initiate additional cycles of biofilm development. The figure therefore retains the pedagogical clarity of the classical life cycle while emphasising that, in natural or domestic settings, adhesion, growth, matrix production, maturation, remodelling, and dispersal are not necessarily discrete or strictly ordered stages. This figure should be read as a reduced static extract from the broader visual framework presented in Supplementary Materials S1 and S3, where the progressive interpretation and complete spatial organisation of the scene are more fully developed.

Building on the isometric principles introduced above, the life-cycle scene uses this geometry to maintain continuity between microorganisms, extracellular matrix, surfaces, fluids, and surrounding objects. In this context, isometry supports not only aesthetic coherence but also conceptual continuity across levels of organisation, allowing the classical biofilm cycle to be read within its material and environmental setting. This multiscale continuity is particularly important for the biofilm life cycle. At the microscopic level, the illustration must retain the legibility of cells, clusters, matrix-rich regions, voids, and local dispersal events. At the mesoscopic level, it must suggest how these structures relate to surface topography, humid zones, residues, or cavities. At the environmental level, it must connect these microbial processes to real interfaces and objects. Representing all these dimensions within a single coherent geometry allows the cycle to be read not as an abstract developmental script detached from its habitat, but as a process embedded within material and ecological conditions. In this sense, the framework does not simply depict stages of development; it situates them. The framework was implemented through a set of complementary visual outputs, each designed to emphasise a different aspect of the biofilm life cycle. Static figures included in the article provide concise conceptual syntheses and serve as reference images for readers. Extracted thematic panels allow closer examination of selected structures or habitats. The annotated animated slide sequence progressively reconstructs the organisation of the system and makes explicit relationships that may be less immediately visible in a single static image (Supplementary Material S1). A simplified video restores part of the temporal continuity that static images necessarily compress (Supplementary Material S2), while the large-format poster enables immersive exploration of local details without losing the overall organisation of the scene (Supplementary Material S3). Taken together, these formats suggest that no single representation can fully capture biofilm complexity. Static figures favour synthesis, animation restores process, and large-scale display encourages exploration.

Viewed in this way, the classical biofilm life cycle remains useful, but it is no longer treated as a self-sufficient explanatory diagram. Instead, it becomes a guided pathway within a heterogeneous visual model that makes room for coexistence, polymicrobial context, matrix diversity, and environmental embedding. The purpose of the framework is therefore not to replace one simplified representation with another, but to preserve the clarity of the classical cycle while showing more explicitly the complexity in which it is biologically situated.

3. Biofilms in domestic environments: a distributed microbial landscape

Biofilms are frequently discussed in medical, industrial, or environmental contexts, yet they are equally pervasive in ordinary human environments. Domestic spaces contain numerous surfaces repeatedly exposed to moisture, nutrients, aerosols, human contact, and fluctuating cleaning pressures, thereby creating favourable conditions for microbial attachment and persistence [[12], [13], [14], [15]]. In this sense, the home can be viewed as a distributed microbial landscape composed of multiple interconnected habitats that differ in hydration regime, nutrient availability, material properties, temperature variation, and disturbance frequency. Such environments provide a useful setting for representing biofilms because they combine ecological realism with immediate recognisability. To make domestic biofilms analytically comparable, each illustrated habitat can be interpreted through three recurrent descriptors: microbiota/examples, matrix/cohesion, and biofilm impact. These descriptors are not intended to transform the figures into taxonomic inventories, but to make the ecological logic of each scene more readable. The first descriptor indicates the dominant type of community or representative microbial groups; the second identifies the main extracellular or environmental components contributing to attachment and cohesion; the third links the local biofilm architecture to an observable consequence such as persistence, staining, odour production, aerosol release, fermentation, protection, spoilage, or cross-contamination. This structure provides a common visual language for comparing domestic biofilms across habitats that differ in moisture regime, surface topology, nutrient input, disturbance frequency, and human use, as illustrated in the domestic sequences presented in Fig. 4, Fig. 5.

Fig. 4.

Beneficial food-associated biofilms in the kitchen environment. Conceptual illustrations of fermented foods in which microorganisms develop as spatially organised communities associated with food matrices or surfaces. These examples emphasise that biofilm-like organisation is not restricted to undesirable contamination, but can also contribute to food preservation, maturation, texture, flavour development, and microbial stability. Each panel is interpreted using the same descriptors: microbiota/examples, matrix/cohesion, and biofilm impact. (A) Kefir grains are self-replicating symbiotic biofilm-like consortia composed mainly of yeasts, lactic acid bacteria, and acetic acid bacteria, embedded in an extracellular matrix. (B) Surface-ripened Camembert cheese, where the fungal-dominated surface microbiota can be interpreted, in a broad ecological sense, as a spatially organised protective community contributing to product maturation and helping limit invasion by undesirable microorganisms. (C) Natto, a traditional Japanese fermented soybean product, is represented as a Bacillus subtilis-dominated biofilm-like community that develops on cooked soybeans and is associated with cohesive texture and long-standing culinary use. (D) Kimchi, a traditional Korean fermented vegetable product, illustrates a complex microbial community rich in lactic acid bacteria structured within a plant-based food matrix. Together, these examples show how beneficial microbial communities in food can be represented within the broader conceptual framework of biofilm organisation. This figure is a selected extract from the broader kitchen visual framework presented in Supplementary Materials S1 and S3, where the spatial relationships between domestic habitats and microbial communities are more fully developed.

Fig. 5.

Undesired kitchen biofilms and foodborne contamination routes. Conceptual illustrations of kitchen microhabitats where microbial persistence, surface-associated growth, and cross-contamination may contribute to foodborne risk. Each panel is interpreted using the same descriptors as in Fig. 4: microbiota/examples, matrix/cohesion, and biofilm impact. (A) Cutting board cross-contamination: raw chicken contaminated with Salmonella enterica can transfer cells to scratches, food residues, or hydrated regions of the board, creating a surface-associated reservoir from which ready-to-eat vegetables may subsequently be contaminated. (B) Refrigerated Listeria monocytogenes persistence: This bacterium can grow at refrigeration temperatures and may persist on organic residues or humid refrigerator surfaces, favouring repeated contamination events. These examples show how domestic food-contact surfaces can support undesired biofilms and connect local microbial organisation with practical contamination routes. This figure is a selected extract from the broader domestic visual framework presented in Supplementary Materials S1.

This perspective is valuable both scientifically and pedagogically. Unlike highly specialised industrial or clinical systems, domestic environments are familiar to broad audiences while still encompassing many of the variables known to influence microbial colonisation and biofilm persistence. Surfaces may be intermittently wet or dry, smooth or porous, nutrient-rich or nutrient-poor, frequently cleaned or only occasionally disturbed. They are also repeatedly inoculated by water, food, air, pets, and human handling, generating constant microbial turnover and opportunities for recolonisation. Domestic settings, therefore, provide accessible model ecosystems for illustrating how biofilms emerge, persist, diversify, and interact with their immediate material context. Representing these environments visually raises a specific challenge: microscopic organisation must remain connected to macroscopic objects and habitats. If these scales are separated into independent panels, the relationship between microbial structure and environmental context becomes less immediately visible. For this reason, the domestic scenes developed here were conceived as integrated visual spaces in which microbial communities, surfaces, moisture conditions, residues, and everyday objects can be read within the same composition. This approach allows biofilm processes to be situated rather than presented as isolated microscopic abstractions. A key methodological aspect of these scenes is that some of the represented microbial structures were redrawn from real microscopy observations and then graphically harmonised to maintain coherence across the broader illustrative framework. This was important not only for visual unity but also for preserving morphological plausibility and avoiding arbitrary or generic depictions of environmental biofilms. The domestic scenes should therefore be understood as interpretative reconstructions grounded in observed microbial structures rather than as literal depictions of single systems.

Within this distributed landscape, the bathroom represents a moisture-driven environment in which hydration, stagnation, organic residues and inaccessible recesses favour microbial attachment and persistence (Fig. 3). Several microhabitats can be distinguished rather than treating the bathroom as a single generic wet setting. Toothbrush bristles illustrate an oral-derived bacterial biofilm, in which saliva, EPS, pili and adhesins can contribute to microbial retention after brushing. Faucet surfaces represent organo-mineral biofilms, where bacterial EPS may interact with soap residues and limescale deposits, favouring persistent colonisation and staining; pigmented bacteria such as Serratia marcescens provide a relevant example for pink-to-orange wet-surface deposits. Showerheads illustrate water-system biofilms, in which EPS, hydrophobic cell envelopes and mineral particles may contribute to persistence under flow and to the release of cells or aggregates into aerosols; non-tuberculous mycobacteria, including members of the Mycobacterium avium complex, are used as illustrative examples of showerhead-associated microorganisms. Tile grout and bath toys represent fungal-bacterial reservoirs in humid or confined niches, where EPS, hyphae, pigments or melanised fungal structures contribute to cohesion, dark staining and malodours. Together, these examples show that bathroom biofilms differ not only in location, but also in community composition, matrix structure, cohesion mechanisms and visible impact.

Particular attention is given to variation in matrix appearance, structural texture, and community composition across the domestic scenes shown in Fig. 4, Fig. 5 and in Supplementary Materials S1 and S3. Some represented biofilms are rich in diffuse hydrated material, whereas others appear denser, more filamentous, patchier, mineral-associated, or more compactly attached to surfaces. Several scenes also include polymicrobial communities involving both bacterial and fungal forms. This is important because many domestic biofilms are multispecies assemblages whose architecture depends not only on bacterial proliferation, but also on fungal growth forms, extracellular polymer production, surface chemistry, mineral deposition, organic residues and hydration history. Including this variation helps move away from overly generic depictions of biofilms as uniform amorphous layers and instead emphasises that matrix-rich surface communities may differ markedly in organisation, texture, and biological impact.

The kitchen offers a complementary ecological spectrum, containing both intentionally exploited and undesired biofilm-associated communities. Beneficial food-associated examples include surface-ripened cheese, kefir grains, and natto, in which microbial growth contributes to protection, maturation, flavour development, texture, fermentation stability, or nutritional value [16,17]. In these cases, the matrix is part of the desirable product architecture: fungal hyphae and food-derived proteins and lipids contribute to the organisation of the cheese rind, kefiran-rich extracellular polysaccharides stabilise kefir grains, and extracellular polymers produced during natto fermentation give this product its characteristic cohesive texture [16,17]. These examples counter the frequent reduction of biofilms to contamination or risk, and show that matrix-embedded microbial communities can also be deliberately valued in food systems. These beneficial food-associated biofilm-like communities are presented in Fig. 4.

By contrast, other kitchen habitats illustrate unintended domestic biofilms and contamination-prone reservoirs. Refrigerator surfaces, cutting boards, coffee makers, and sponges combine moisture, food residues, repeated inoculation, surface irregularities, and uneven cleaning, creating conditions conducive to attachment, survival, and persistence [[13], [14], [15]]. In these settings, EPS may interact with proteins, fats, coffee oils, humid deposits, scratches or porous materials to create persistent reservoirs. Their biofilm impact is therefore undesirable: refrigerated persistence and potential infection risk, transfer from raw food to ready-to-eat products, off-flavours and odours, deposits, or cross-contamination. This distinction is illustrated by foodborne pathogen-associated examples such as the refrigerated persistence of Listeria monocytogenes and the transfer of Salmonella enterica via contaminated cutting boards [13,18]. Kitchen sponges are particularly informative because their porous, three-dimensional structure creates protected, hydrated microhabitats, demonstrating that object design and material architecture can contribute to microbial persistence and cross-contamination [[13], [14], [15],18]. Complementary examples of foodborne pathogen-associated contamination routes, including Salmonella enterica cross-contamination via cutting boards and the refrigerated persistence of Listeria monocytogenes, are presented in Fig. 5. The kitchen sequence, therefore, makes visible a central ambivalence of biofilm biology: similar principles of attachment, matrix production and community stabilisation may support either beneficial fermentation or unwanted contamination, depending on the ecological and human context.

The domestic framework is therefore not intended to catalogue household contamination, but to make visible how microbial life is distributed across familiar environments. By placing cells, matrix, surfaces, and objects within a coherent visual system, the illustrations show that domestic biofilms are shaped not only by microbial traits, but also by hydration patterns, surface topology, material properties, human practices, and spatial connectivity between niches. This environmental embedding is essential for understanding why the same broad processes of attachment, growth, matrix formation, remodelling, and dispersal may produce very different local outcomes across habitats.

Selected extracted figures from these domestic scenes focus attention on representative habitats and processes. Static panels provide close-up views of specific niches, including humid reservoirs, food-contact surfaces, and fermentation-associated communities. In the supplementary animated slide sequences, these habitats are progressively revealed and commented upon, allowing viewers to move stepwise through the household landscape and connect local examples into a broader ecological narrative. The simplified video restores part of the temporal continuity of colonisation and dispersal, whereas the poster format encourages prolonged inspection of local details within the global organisation of the domestic environment. Together, these complementary formats underline that no single image can exhaust the complexity of biofilm life across scales. The complete domestic visual framework is therefore best explored through the annotated animated presentation and the high-resolution poster, where spatial continuity between habitats and local microbial details are more fully preserved (Supplementary Materials S1 and S3).

By situating biofilms in bathrooms and kitchens, the proposed framework emphasises that structured microbial communities are not exceptional formations restricted to specialised infrastructures, but a widespread and context-dependent mode of microbial organisation embedded in ordinary life. Domestic environments, therefore, serve here not as anecdotal examples, but as model ecosystems that make visible the diversity, spatial heterogeneity, polymicrobial character, and functional ambivalence of the biofilm mode of life.

4. Scientific illustration as a conceptual tool for representing biofilms

The visual framework presented in this work was developed through a sustained collaboration between a biofilm microbiologist and a scientific illustrator. Rather than considering illustration as a final step occurring after the production of scientific knowledge, the process was conceived as part of the reasoning itself. The images were constructed through iterative exchanges combining experimental knowledge, published literature, microscopy-informed observation, graphical testing, and successive drawing attempts. Examples of these intermediate stages, including project sketches, composition trials, and progressive refinements, are provided in Supplementary Material S4. In this context, illustration did not simply translate established ideas into visual form: it also helped clarify which aspects of biofilm organisation could be made explicit, compared, or connected across scales.

This perspective is consistent with a broader understanding of scientific images as active instruments of knowledge rather than neutral reflections of reality. In many scientific fields, visual representations have long served to select, stabilise, compare, and synthesise phenomena that are otherwise difficult to apprehend directly. Microbiology provides a particularly relevant case, because microbial life is largely invisible to the naked eye and often accessible only through highly mediated forms of observation. Under these conditions, drawing and illustration do not merely embellish data: they can help organise observations into interpretable forms. This is especially important for biofilms, whose properties emerge from interactions among cells, extracellular matrix, surfaces, hydration, flow, and surrounding environmental conditions.

For such systems, direct imaging and scientific illustration are not opposed but complementary. Microscopy provides access to real structures, local arrangements, and experimentally grounded morphologies. Illustration, by contrast, allows combining observations acquired at different scales, suppressing visually distracting detail, and rendering simultaneous relationships more legible within a single composition. In the present work, this complementarity was central. The goal was neither exact reproduction nor free invention, but an interpretative reconstruction anchored in real biofilm observations and organised according to a coherent visual logic.

This methodological position is important because it defines the epistemic status of the illustrations. The figures are not intended to function as literal documents of a single observed biofilm, nor as generic icons detached from empirical evidence. Instead, they operate as conceptual syntheses that integrate experimentally informed forms, known biological processes, and selected spatial relationships into coherent visual models. In this sense, illustration functions here in a way analogous to other scientific models: it does not aim at exhaustive realism, but at intelligible organisation. Its value lies in the explicit selection and arrangement of relevant features rather than in any claim to photographic completeness.

Constructing such images required making explicit a number of choices that often remain implicit in textual descriptions of biofilms. These included the choice of spatial scale, the degree of structural detail to retain, the way to represent matrix-rich regions, the level of polymicrobial complexity to include, and the balance between realism and readability. These constraints were not merely technical. They also helped expose ambiguities in commonly used descriptions of the biofilm life cycle, particularly when terms such as maturation, stability, or dispersal are treated as if they refer to sharply delimited, universally ordered stages. More broadly, they reinforced the need for visual models able to accommodate heterogeneity, coexistence, and context dependence rather than rigid developmental scripts.

A central challenge concerned the representation of heterogeneity. Biofilms are composed of dense microbial populations embedded in extracellular material and shaped by local gradients of nutrients, oxygen, hydration, signalling molecules, and mechanical constraints. Their architecture is irregular, and their physical appearance can vary markedly according to species composition, matrix composition, hydration regime, and substrate properties [4,5,10,21]. A strictly realistic depiction would often result in visually overloaded scenes that are difficult to interpret, whereas excessive simplification would produce misleadingly uniform structures. The illustrations, therefore, rely on selective simplification, highlighting microcolonies, matrix-rich zones, voids, dispersal events, and contrasting textures while preserving enough variability to reflect the non-uniform character of real communities. This was especially important in the domestic scenes, where variation in matrix appearance and the inclusion of bacterial-fungal assemblages helped avoid a falsely homogeneous visual language.

The question of scale was equally important. Biofilm development involves processes operating from the cellular and matrix levels to surfaces, objects, and environmental systems. Instead of separating these dimensions into isolated figures, the approach adopted here combines multiple scales within coherent scenes. Cells, extracellular material, interfaces, residues, droplets, humid recesses, and everyday objects are represented together so that local microbial organisation remains visibly linked to its environmental setting. This multiscale articulation is one of the key contributions of the framework, because it allows the reader to move continuously from microscopic mechanisms to familiar habitats without abandoning spatial coherence.

The consistent use of isometric projection supported this multiscale articulation by providing a stable geometry in which cells, matrices, surfaces, cavities, fluids, and domestic objects could be integrated into a single navigable composition. In this sense, geometry contributed not only to stylistic coherence but also to the conceptual function of the figures by making comparisons across scales easier to follow. Within this visual system, colour and graphical hierarchy also played a conceptual role. In the biofilm life cycle scenes, colour was used to render the classical trajectory visually traceable within a more complex polymicrobial and structurally heterogeneous environment. This choice did not imply that a single canonical sequence exhausts the system's biology. Instead, it provided a guided path through a dense image, allowing the classical cycle to remain pedagogically readable while being re-situated in a richer, more realistic context. More broadly, the figures rely on hierarchy to distinguish what is foregrounded for explanatory purposes from what remains part of the biological and environmental background. Illustration, therefore, functions here not simply by showing more, but by organising visibility. This modelling role has practical value because biofilm research spans medical, food, industrial, environmental, and domestic microbiology, yet these fields often use distinct visual conventions. A coherent graphical language can help make comparable organisational principles visible across systems that differ in species composition, function, and applied relevance. For example, matrix production, surface attachment, spatial gradients, local dispersal, and multispecies organisation may be relevant both in undesirable biofilms associated with contamination or persistence and in beneficial biofilm-like communities involved in fermentation or protective microbial consortia, including deliberately assembled multispecies biofilms designed to constrain undesirable bacteria [22]. By representing these features within a common visual grammar, the framework supports comparison between biofilms that differ in outcome while sharing underlying spatial and ecological principles. This is particularly important for avoiding an exclusively negative representation of biofilms and for showing that their significance depends on context, management, and function.

The approach also has pedagogical significance. Biofilms are intrinsically difficult to teach because they involve invisible structures, emergent collective properties, and interactions across scales. Conceptual illustrations can help address this difficulty by providing visual anchors for processes such as adhesion, matrix protection, physiological heterogeneity, local remodelling, multispecies coexistence, and environmental persistence. They do not replace microscopy or experimentation, but they can help bridge the gap between local observation and synthetic understanding. In the present study, this pedagogical function was reinforced by the use of complementary media, including static figures, extracted panels, animated slide sequences, a simplified video, and a large-format poster (Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5 and Supplementary Materials S1–S3). Each medium contributes differently to explanation, but all rely on the same underlying visual logic.

More broadly, the process of constructing such illustrations contributes to conceptual clarification. Each attempt to build a coherent figure forces explicit decision about what is observed directly, what is inferred from current knowledge, what is generalised across systems, and what remains uncertain. In this respect, scientific illustration acts not only as a communication device but also as a heuristic tool. It can reveal hidden assumptions in established representations, expose tensions between readability and realism, and suggest alternative ways of framing biological questions. For biofilms, this includes asking how to represent coexistence rather than strict succession, spatial embedding rather than isolated stages, and matrix diversity rather than a single generic texture.

Scientific illustration should therefore be considered here as a hybrid framework that is simultaneously conceptual, visual, technical, and pedagogical. In the case of biofilms, it offers a useful means of representing a complex, dynamic, and largely invisible mode of microbial life while remaining grounded in real observations and contemporary biological understanding. The value of the approach lies not in claiming to replace experimental imaging, but in providing a coherent model through which observed forms, known processes, and environmental contexts can be assembled into a more readable and transferable representation of structured microbial life.

5. Conclusion

Representing biofilms in a way that is both scientifically accurate and visually readable remains challenging because these systems are heterogeneous, dynamic, and organised across multiple spatial scales. Simplified diagrams can be pedagogically useful, but they may also suggest overly uniform or rigid developmental patterns. In this work, scientific illustration was used as an intermediate approach to construct conceptual representations grounded in experimental knowledge while preserving clarity and coherence. Rather than proposing a new biological model, the framework presented here reinterprets the graphical use of the classical biofilm life cycle. The classical sequence is retained as a readable explanatory guide, but it is embedded within more complex microbial and environmental scenes. This allows preserving the pedagogical value of the canonical cycle while demonstrating that real biofilms are heterogeneous, polymicrobial, matrix-rich, and strongly context-dependent systems. By linking microbial organisation to familiar domestic environments and grounding selected illustrations in real microscopy observations, the framework connects microscopic processes to recognisable habitats and surfaces. More broadly, this work highlights scientific illustration not only as a communication device but also as a conceptual tool for representing and discussing structured microbial life across disciplines.

CRediT authorship contribution statement

Antoine Carlioz: Conceptualization, Investigation, Methodology, Resources, Validation, Visualization, Writing – original draft, Writing – review & editing. Romain Briandet: Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Writing – original draft, Writing – review & editing.

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Antoine Carlioz reports financial support was provided by Diagonale Paris-Saclay. The corresponding author Romain Briandet is part of Biofilm journal editorial board If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

Data availability

No data was used for the research described in the article.