Three-dimensional visualization of mixed species biofilm formation together with its substratum



Nobuhiko Nomura, Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan. Tel: +81 29 853 6627; Fax: +81 29 853 6627; email:


Biofilms, such as dental plaque, are aggregates of microorganisms attached to a surface. Thus, visualization of biofilms together with their attached substrata is important in order to understand details of the interaction between them. However, so far there is limited availability of such techniques. Here, non-invasive visualization of biofilm formation with its attached substratum by applying the previously reported technique of continuous-optimizing confocal reflection microscopy (COCRM) is reported. The process of development of oral biofilm together with its substratum was sequentially visualized with COCRM. This study describes a convenient method for visualizing biofilm and its attached surface.

List of Abbreviations

continuous-optimizing confocal reflection microscopy


confocal reflection microscopy


fluorescent in situ hybridization




tryptic soy broth

In nature, microorganisms form complexes called biofilms, which are attached to the surfaces of various substrata. Most microorganisms on Earth live in these complexes [1]. It has been demonstrated that biofilm-formed microorganisms exhibit different gene expression patterns than do those in the planktonic state [2, 3]. Therefore, many researchers are currently focusing on biofilms. In the study of biofilms, observation of their structure is important and interesting; various observation techniques have been developed. Recently, researchers have commonly used fluorescence imaging using confocal laser-scanning microscopy and fluorescent proteins to observe biofilms [4-7]. This technique makes it possible to sequentially observe biofilm development. However, because fluorescence imaging requires microorganisms, which have established transformation methods, to be in conditions where fluorescent protein functions normally, the range of applications for this technique is greatly restricted. COCMR, which was developed by our laboratory [8], is based on confocal reflection microscopy [9]. Unlike fluorescence microscopy, COMCR can visualize everything that reflects light. Previously, we used the COCRM technique to visualize Streptococcus mutans biofilm and Pseudomonas aeruginosa growing as planktonic cells [10, 11], as well as mixed species of activated sludge [8, 12]. However, in our previous study, we only visualized the biofilm-forming process on substrates other than glass at fixed time points. In principle, the COCRM technique would allow visualization of the substratum to which biofilm is attached as well as the biofilm; we examined the latter in our previous study. Therefore, in this study, we visualized the process of development of mixed species biofilm at its attached substratum from the same perspective.

Oral biofilms, also known as dental plaques, cause dental caries and periodontal disease. These resemble the mixed species biofilm model [13]. Researchers have previously estimated that more than 500 bacterial species exist in the human oral cavity [13, 14]. Due to the complexity of the oral environment, these biofilms have complex community and spatial structures. In previous studies, oral biofilms were observed mainly using electron microscopy and FISH [15-17]. However, because these methods require fixation of specimens, they cannot be used for sequential visualization of development of intact oral biofilm. Although researchers have used the CRM technique to observe dental plaque, they have not performed fixed point and continuous observation of the process of biofilm development together with substratum [18]. Therefore, we used COCRM to sequentially visualize the process of oral biofilm development with substratum from the same viewpoint.

First, we visualized biofilms that imitated dental plaque. We formed imitation dental plaque on a hydroxyapatite disk in TSB with dextrose (Becton Dickinson, Sparks, MD, USA). We used human saliva (diluted to 1% with TSB medium) as inoculum. We simultaneously performed cultivation and microscopic observation. We kept the culture temperature at 37°C using a thermoplate (TOKAI HIT, Shizuoka-ken, Japan). We collected early morning saliva samples from laboratory members aged over 20 years. The collected samples were mixed together to preserve anonymity. The formed biofilm was visualized by COCRM using an uplight confocal microscope (LSM5 PASCAL; Carl Zeiss, Oberkochen, Germany) and a water immersion lens (Carl Zeiss). The COCRM images not only demonstrated that this method can visualize intact biofilms but also revealed their basal materials (in this case, on HA disks; Fig. 1a–c). For biomass quantification, we analyzed images using the COMSTAT computer program [19], which functions under MATLAB (Mathworks, Natick, MA, USA).

Figure 1.

3D images of the HA disk and mixed-species oral biofilm visualized by COCRM and Gram staining of oral biofilm. (a) Image of an intact HA disk. (b) Image of an oral biofilm cultured with TSB medium (1% saliva) on an HA disk. The biomass of biofilm is 5.82 µm3/µm2. Each projection shows fields of 140 by 140 µm (xy), as indicated. (c) Orthometric view of (b) acquired using COCRM. The bar represents 40 µm. (d) Gram staining of oral biofilm formed on an HA disk. The bar represents 10 µm. Experiments were repeated at least three times and representative images are shown.

We briefly examined the diversity of the imitated biofilms by Gram staining (Fig. 1d). The results of Gram staining indicated that this procedure accurately imitated dental plaque and at least partially reflected the complex oral environment. In the COCRM images, we observed filamentous microorganisms with similar Gram staining. In addition, the COCRM images showed details of the biofilm, including single cells and streptococci-chained cells. These findings indicated that COCRM accurately visualized the mixed-species biofilm and its attached substrate; thus, this method is applicable to visualization of mixed-species biofilms.

We next utilized COCRM to sequentially visualize the process of biofilm development. We used diluted 10% human saliva in saline and 1% sucrose to form biofilm on HA disks. This culture medium was heated to 37°C on a thermoplate under microscopic observation. Using a water immersion lens, we performed observations every 10 mins from 0 to 15 hrs. In addition to microscopic observation, we clearly visualized the process of biofilm development on HA disks by the COCRM technique (Fig. 2). These observations were performed continuously, generating a sequential biofilm development process movie (Supplemental Movie 1). In formation of oral biofilm, attachment of pellicle to a clean tooth surface is the first step [20]. Pellicle is a thin protein-containing film derived from salivary glycoproteins. Second, early colonizers recognize binding protein in the pellicle and adhere to it. After the early colonizers have attached, subsequent colonizing bacteria recognize polysaccharide or protein receptors on the early colonizer's cell surfaces and attach or coaggregate with them. The continuous visualization showed the early colonization (Supplemental Movie 1; 0–4 s) and later colonization and coaggregation (Supplemental Movie 1; 4–11 s). These findings indicate that COCRM is an efficient tool for visualizing mixed-species biofilms and their developmental processes.

Figure 2.

Process of developmental of oral biofilm cultured with 10% saliva diluted with saline (containing 1% sucrose) visualized by COCRM technique. Images were acquired at 0, 3, 6, 9, 12, and 15 hrs after inoculation and were taken at the same point of the HA disk during cultivation. The values indicate the biofilm biomass. Each projection shows fields of 140 by 140 µm (xy) as indicated. Experiments were repeated at least three times and representative images are shown.

In this study, we used COCRM to visualize oral mixed-species biofilms and their attached substrata. Nonetheless, COCRM has a much broader range of applications. Because COCRM does not require fluorescence, it can be used in conditions that are unsuitable for fluorescent protein, such as high or low temperatures and anaerobic conditions. Thus, COCRM allows observation of the biofilm formation process at near natural conditions in the laboratory. We expect that this means of visualization of biofilm will be adapted for a variety of environmental conditions. In addition, being a non-fluorescent visualization method, the COCRM technique may allow visualization of interactions between biofilms and their attached substrata. In fact, we visualized caries, which was caused by Streptococcus mutans UA159 biofilm, on an HA disk (Fig. 3). We acquired this image after removing the biofilm 7 days post-inoculation. We replaced the culture medium with fresh TSB medium every 12 hrs over the duration of the culture. Because the pH of the culture medium of the biofilms was approximately 5.0, whereas that of the control medium was 7.0, demineralization of the HA disk was likely due to low pH, presumably caused by lactic acid production in biofilm. This image shows that COCRM is potentially applicable to visualizing interactions between biofilms and their substrata, which often results in dental caries. This technique requires no fixation such as is required for scanning electron microscopy and allows for quantification of the volume loss caused by caries. In addition, fixed-point sequential observation can be used to visualize the process of development of caries. Therefore, the COCRM technique is a very useful method for visualizing the process of development of biofilm, environmental adaptation and biofilm–substratum interactions.

Figure 3.

Visualization of dental caries caused by Streptococcus mutans UA159 biofilm on HA disk. Overnight cultures of UA159 diluted 1:100 with fresh TSB medium were inoculated on HA disks. (a) TSB medium control. (b) 7 days after inoculation. The projection shows fields of 140 by 140 µm (xy) as indicated. Experiments were repeated at least three times and representative images are shown.


This study was supported in part by a Grant-in-aid for Scientific Research to Nobuhiko Nomura from the Ministry of Education, Culture, Sports, and Technology of Japan. The Japan Science and Technology Agency, CREST, and ALCA also supported this research financially. We thank Dr. Hidenobu Senpuku for providing Streptococcus mutans strain UA159 and Dr. Yukihiro Kaneko for helpful comments.


The authors have no relationship or financial support that may result in conflict of interest with any company.