This protocol describes a new method to grow and qualitatively analyze bacterial biofilms on fungal hyphae by confocal and electron microscopy.
Bakteriella biofilmer bildar ofta på svamp ytor och kan vara inblandade i ett stort antal bakteriella svampinteraktionsprocesser, såsom metabolisk samarbete, konkurrens eller predation. Studiet av biofilmer är viktigt i många biologiska områden, inklusive miljövetenskap, livsmedelsproduktionen, och medicin. Emellertid har få studier fokuserat på sådana bakteriella biofilmer, delvis på grund av svårigheten att utreda dem. De flesta metoder för kvalitativ och kvantitativ biofilm analyser som beskrivs i litteraturen är endast lämpliga för biofilmer bildas på abiotiska ytor eller homogena och tunna biotiska ytor, såsom ett monoskikt av epitelceller.
Medan laserscanning konfokalmikroskopi (LSCM) används ofta för att analysera in situ och in vivo biofilmer, blir mycket utmanande denna teknik när den tillämpas på bakteriella biofilmer på svamphyfer, på grund av tjockleken och de tre dimensionerna hos hyfal netwOrks. För att övervinna denna brist, har vi utvecklat ett protokoll som kombinerar mikroskopi med en metod för att begränsa ansamling av hyfernas lager i svampkolonier. Med denna metod kunde vi undersöka utvecklingen av bakteriella biofilmer på svamphyfer vid flera skalor med både LSCM och svepelektronmikroskop (SEM). Denna rapport beskriver protokollet, inklusive mikroorganismer kulturer, bakteriella biofilm bildningsförhållanden, biofilm färgning och LSCM och SEM visualiseringar.
Fungi and bacteria have many opportunities to interact with each other because they cohabit in most terrestrial environments. Due to their diversity and their ubiquity, these interactions are important in many biological fields, including biotechnology, agriculture, food processing, and medicine1,2. Molecular interactions require a certain degree of proximity to allow exchanges between the partners, and in some cases, a physical association of the partners is necessary for a functional interaction3. A common physical association between bacteria and fungi is the formation of bacterial biofilms on fungal surfaces4. This direct contact between bacterial cells and fungal hyphae permits intimate interactions that are involved in various biological processes. For example, in medicine, the study of biofilm formation of Pseudomonas aeruginosa on the opportunistic fungal pathogen Candida albicans could provide insights into the link between biofilm formation and virulence5. In agriculture, studies suggest that plant-growth-promoting rhizobacteria and biocontrol bacteria have an increased efficiency when associated with a fungus in a mixed biofilm. For example, Bradyrhizobium elkanii have enhanced N2-fixing activity when associated with Pleurotus ostreatus in a mixed biofilm6. Finally, in bioremediation, bacterial-fungal mixed biofilms have been used for the remediation of polluted sites7,8.
LSCM is particularly suitable to study biofilms since it allows for a three-dimensional observation of living hydrated biofilms with minimum pretreatments, thereby maintaining biofilm structure and organization. Thus, biofilm analysis by LSCM is very informative, especially to determine the time course of the biofilm formation and the detection of characteristic stages9,10, from the adhesion step to the development of a mature biofilm. It is also particularly adapted to visualize the biofilm structure and matrix11,12 or to quantify the biofilm size13,14. Although this method is suitable to study biofilms on abiotic or thin biotic surfaces, studying bacterial biofilm on a fungal filamentous colony is still very challenging. Indeed, most filamentous fungi build thick, complex, tridimensional networks in culture. Even if thick objects can be imaged by confocal microscopy, the attenuation of the laser penetration and the fluorescence emission often decrease the quality of the final images over a depth of 50 µm15. Moreover, because fungal colonies are not rigid, it is difficult to handle the microorganisms without disturbing the biofilms. Due to the thickness of the samples, the few microscopic analyses of bacterial biofilms on fungal hyphae are usually only performed on a small part of the fungal colony, therefore containing only few hyphae16,17,18. All this limits our ability to describe biofilm distribution on the fungal colony and thus can bring biases into the analysis in case of the heterogenic distribution of the biofilm within the fungal colony.
To overcome such difficulties, we report a method for the growth and the analysis of bacterial biofilm on fungal hyphae. This method was applied to study the biofilm formation in Pseudomonas fluorescens BBc6 on the hyphae of the ectomycorrhizal basidiomycete Laccaria bicolor S238N. These two forest-soil microorganisms were previously described to form mixed biofilm-like structures19,20. This method can easily be further adapted to other filamentous fungal/bacterial systems. The method presented here is based on the combination of a fungal culture method, allowing for the growth of very thin fungal colonies, with LSCM and SEM imaging. This permitted us to obtain micro- (µm range) and meso- (mm range) scale views of the interaction between the two microorganisms, allowing for the qualitative characterization of the biofilm. We also showed that the samples can be observed with SEM, permitting structural analysis of the biofilm at the nano-scale level (nm range).
Bacterial biofilms are retrieved in many environments and have been studied since the 1950s, leading to the development of a number of methods to analyze them24. Classical methods to quantify and monitor biofilms include micro-titer assays and, the most widely-used method, crystal violet (CV) staining. These methods are fast, low cost, and easy to handle25 and are particularly useful to quantify total biofilm biomass or to perform viability and matrix quantification assays. On an other hand, "omics" methods are also useful in biofilm studies, allowing for quantitative and functional analyses of biofilms26,27. Despite the advantages of micro-titer plate and "omics" methods, several essential features of biofilms cannot be captured with these techniques, hindering a complete understanding of this process. Such features include matrix structures, bacterial colony architectures, cell/cell interactions, and colonization patterns, which are key data for understanding both the functioning of biofilms and the dynamics of their formation. Despite the capacity of microscopy to capture these features, microscopy analysis of bacterial biofilms on filamentous fungi are still scarce. This is mainly due to the growth of filamentous fungi, which often forms colonies of thick, complex, tridimensional networks. Formation of bacterial biofilms on fungi is common in diverse environments and is significantly involved in various fields4 (e.g., medicine, agriculture, and environment); hence, it is critical to develop new methods to facilitate their investigation. To this end, we combined a method to generate very thin fungal colonies with microscopic imaging of the bacterial biofilms. In addition, we proposed a set of microscopy tools to qualitatively analyze those biofilms. The success of the method relies upon the ability to produce very thin hyphal colonies and to apply the appropriate dyes. These points are discussed below.
Due to the complex structures of the biofilms, understanding their function requires a multi-scale approach28,29. Distribution patterns of the biofilms, bacterial colony architecture, and matrix structure and composition are analyzed at different scales (i.e., meso-scale and micro-scale). Moreover, nanoscale resolution allows access to the cell/cell physical interactions and the nano-structure of the matrix. Thus, the developed method easily enables a multi-scale analysis of the bacterial biofilms formed on the fungal colony.
In most studies, LSCM analyses of biofilms are limited to the micro-scale, the meso-scale usually being performed by optical coherence tomography30,31,32. The method presented here enables both micro- and meso-scale analyses by LSCM. It demonstrates the utility of combining both analyses in the same region of the sample and even on the same image using new-generation confocal microscopes with high resolution (Figure 3). Thus, issues linked to compiled data gathered at different scales with different methods are here avoided.
This combination of analyses gave access concomitantly to the biofilm repartition on the fungal colony, the bacterial colony architecture along the developing biofilm, and the matrix structure. The meso-scale analysis showed a heterogenic distribution of the bacterial biofilms on the fungal colonies (Figures 2 and 3). This observation would not have been possible with protocols that only permit imaging of a small portion of the fungal colony, which is not necessarily representative of the entire colony. Thus, while often neglected, the meso-scale analysis can give precious information about biofilm distribution patterns.
Finally, the developed method can be used to analyze samples with different microscopy techniques, including scanning electron microscopy. Here, SEM was used to reach the nano-scale and to obtain the bacterial spatial organization within the biofilm. It performed very well with the thin fungal colonies, while SEM only permitted surface imaging. In contrast to LSCM, SEM, however, required sample dehydration and, most often, coating with a conductive metal. This dehydration process might alter biological structures when it is not properly executed and may require optimization. Here, sample dehydration using slow lyophilization was used33. Nevertheless, applying both LSCM and SEM to the samples will allow the performance of correlative microscopy at the same location of the sample.
Despite the advantages described above, some limitations exist. Firstly, it may not be applicable to all kind of fungi. Indeed, this culturing method is developed for fungi spreading radially on the surface of solid media. This method may not be suitable for fungi forming mainly aerial hyphae (e.g., Fusarium sp.) or for micro-aerobic fungi spreading mainly inside agar. Moreover, fungi degrading cellophane may be problematic as well (e.g., Trichoderma sp.). Secondly, it is important to note that the staining strategy is a critical point and the choice of the stain must be made carefully, as the stain must not disturb the biofilm. For example, we noticed that Calcofluor White caused partial biofilm disruption (data not shown), likely due to the high pH of this stain. Also, some dyes produced heterogeneous staining (e.g., Congo Red), while others produced homogeneous staining (e.g., cell wall staining with WGA lectin), giving a heterogeneous image quality. Moreover, it is important to be aware that some dyes might not be fully specific. For example, WGA stains not only fungal cell walls but also N-acetylneuraminic acid in gram-positive bacterial cell walls and adhesins produced by gram-positive and -negative bacteria during biofilm formation34,35. Therefore, using fluorescent protein-tagged bacteria and/or fungi is recommended to avoid multiple staining. If multiple dyes are used, they must not chemically interfere, and their emission spectra should not overlap.
Meso-scale analyses require a large scanned area, and therefore, LSCM may be time-consuming (40 min to 1 hr, depending on the sample thickness) and bottleneck the analysis of a large number of samples. Nonetheless, adjustments can be made depending on the type of data required. It is possible to decrease acquisition time and image size by altering the image quality. For example, high resolution is not necessary to analyze the biofilm general repartition.
Finally, some limitations need to be considered when choosing to display Z- stack data as 2D or 3D projections. Two-dimensional projections are a good way to summarize data, but depth information is lost, and overlapped structures become hidden. On the other hand, 3D projections allow the visualization from different points of view, but they often render poorly in case of spatial complexity.
In conclusion, we have reported a method for the characterization of bacterial biofilms on hyphae at the structural level. The methodology can be extended to other applications. Indeed, this method allows the performance of functional or chemical characterization of bacterial biofilms forming on fungal hyphae. Due to the great variety of existing fluorescent reporter systems, LSCM analysis can be used for multiple purposes29. For example, the fluorescence microscopy could be used to monitor pH gradient36 or molecule diffusion in biofilms37. Additionally, the method allows for community analysis in multispecies biofilms. For example, fluorescence in situ hybridization targeting specific bacterial groups is particularly useful to study specific bacterial repartition in multispecies biofilms38,39. Last, numerous fluorescent dyes can be used to characterize the matrix composition of the biofilms21. Here, proteins were targeted using Sypro, which stains a large range of proteins, among them matrix proteins (Figure 4), but other dyes allow for the visualization of other important matrix constituents, such as exopolysaccharides or extracellular DNA. Interestingly, all these analyses could be performed at the meso-scale using the described method. Since LSCM can be performed on living samples, it is also possible to achieve time-lapse imaging using, for example, coverwell chambers, particularly suitable for thin fungal colonies. This option is particularly interesting, as biofilm formation is a complex, dynamic process. Finally, for a quantitative purpose, the reported method may improve the accuracy of automatic quantitative analysis by making this quantification possible on meso-scale images. This may overcome biofilm heterogeneity and statistical issues29.
The authors have nothing to disclose.
This work was supported by the French National Research Agency through the Laboratory of Excellence ARBRE (ANR-11-LABX-0002-01), the Plant-Microbe Interfaces Scientific Focus Area in the Genomic Science Program, and the Office of Biological and Environmental Research in the DOE Office of Science. Oak Ridge National Laboratory is managed by UT-Battelle, LLC, for the United States Department of Energy under contract DE-AC05-00OR22725.
6 well Falcon Tissue Culture Plates | Fisher Scientific | 08-772-33 | Used in 2.2 & 3.1 |
Congo Red | Fisher Scientific | C580-25 | Used in 3.1.4.1 |
FUN 1 Cell Stain | Thermo Fisher Scientific | F7030 | Used in 3.1.4.1 |
Wheat Germ Agglutinin, Alexa Fluor 633 Conjugate | Thermo Fisher Scientific | W21404 | Used in 3.1.4.1 |
DAPI solution | Thermo Fisher Scientific | 62248 | Used in 3.1.4.2 |
Propidium iodide | Thermo Fisher Scientific | P3566 | Used in 3.1.4.3 |
FilmTracer SYPRO Ruby Biofilm Matrix protein Stain | Thermo Fisher Scientific | F10318 | Used in 3.1.4.4 |
Fluoromount-G Slide Mounting Medium | Fisher Scientific | OB100-01 | Used in 3.1.7 |
LSM780 Axio Observer Z1 | Zeiss | Used in 3.2.1 | |
ZEN 2.1 lite black software | Zeiss | Used in 3.2.1 | |
High Vacuum Coater Leica EM ACE600 | Leica | Used in 4 | |
GeminiSEM-FEG | Zeiss | Used in 4 |