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The oral cavity harbors several hundred different bacterial species that colonize both hard (teeth) and soft tissues, forming complex populations known as microbial biofilms. It is widely accepted that the phenotypic characteristics of bacteria grown in biofilms are substantially different from those grown in suspensions. Because biofilms are the natural habitat for the great majority of oral bacteria, including those contributing to oral diseases, a better understanding of the physiology of adherent populations is clearly needed to control oral microbes in health and disease. In this chapter, we use oral streptococci as examples for studying the physiology of oral biofilms.
Oral biofilms normally exist in dynamic equilibrium with host defenses and are important for preventing colonization by undesirable organisms (1, 2). However, changes in the composition and metabolic activities of biofilm communities that lead to increases in the proportion of pathogenic species can lead to oral diseases, including dental caries and periodontitis. Despite the importance of oral biofilms to health and disease, studies on the physiology and genetics of oral bacteria were primarily conducted using planktonic populations of bacteria. In recent years, the development of in vitro and in vivo biofilm methodologies to study sessile populations have demonstrated that there are many physiologic and molecular differences between planktonic and surface-bound bacteria, suggesting that the organisms can acquire a “biofilm phenotype” (2–4).
Starting with the premise that “all models are wrong, but some are useful,” a quote attributed to the British statistician George Box, there are a variety of in vitro systems to study oral streptococci biofilms. These include simple and economical models in which bacteria are cultivated in batch systems using different surfaces such as glass, plastic, or hydroxyapatite (HA), the latter being used as a surrogate of tooth enamel. These systems can give reproducible results and can be scaled up to provide sufficient biomass for physiologic and genetic studies. In addition to batch and static systems, the use of shear force in continuous flow systems is considered ideal for the analysis of the dynamics of cell attachment to surfaces and the initial stages of biofilm development. Yet another commonly used system is the so-called constant depth film fermentor in which a scraper intermittently passes over the grown biofilms in wells to achieve constant biofilm depth. Batch and continuous feed systems can also be used to generate more complex multispecies biofilms of known composition, or microcosms can be formed from starter biofilm samples from the body and subsequent cultivation of biofilms in vitro. Because of the natural heterogeneity of these more complex biofilms, the interpretation of the behavior of these populations is very challenging.
Here, we focus on batch-culture systems that our laboratories have routinely used for studying the physiology of oral biofilms, with a particular emphasis on Streptococcus mutans biofilms. One of the advantages of the models presented in this chapter is that multiple biofilms can be formed simultaneously, which provides significant benefit in establishing reproducibility of the data and reducing variance. In addition, test agents can be applied and removed from the system instantaneously allowing a tightly controlled substance exposure time. Moreover, biofilms formed on glass slides or HA are amenable to confocal and electron microscopy and can yield a quantity of bacterial biomass that is sufficient for enzymatic assays. Finally, these model systems can be easily adapted for studies with non-streptococcal species.
Base medium (5) per liter
|Casamino acids||2 g|
Dissolve all components in deionized (Milli-Q) water. Autoclave (121°C for 20 min) and store at room temperature (seeNote 1, Note 2).
|100× amino acid stock solution||per 100 mL|
|l-glutamate (l-glutamic acid)||5 g|
Dissolve all components in Milli-Q water. Filter-sterilize (0.22 μm pore size) and store wrapped in aluminum foil (components are light-sensitive) at 4°C for up to 4 weeks.
|100× vitamin stock solution||per 100 mL|
|Pyridoxine HCl||240 mg|
|Nicotinic acid||46 mg|
|Pantothenic acid||24 mg|
|Thiamine HCl||1 mg|
Dissolve all components with Milli-Q water. Filter-sterilize (0.22 μm pore size) and store wrapped in aluminum foil at 4°C.
|Final BM medium composition||per liter|
|Base medium||950 mL|
|MgSO4·7H2O (0.1 g/mL stock)||5 mL|
|CaCl2·2H2O (0.03 g/mL stock)||5 mL|
|100× vitamin stock||10 mL|
|100× amino acid stock||10 mL|
|1 M glucose or 0.5 M sucrose (see Note 3)||20 mL|
Adjust mixed solution to pH 7 and filter-sterilize (0.22 μm filter). Use immediately or store wrapped in aluminum foil at 4°C for up to 1 week.
|Bacto tryptone||30 g|
|Bacto yeast extract||5 g|
Dissolve in Milli-Q water, autoclave (121°C, 20 min), and adjust pH to 7 aseptically with NaOH. Add glucose or sucrose (20% stock solution) to a final concentration of 1% after autoclaving to avoid caramelization.
|Bacto tryptone||25 g|
|Bacto yeast extract||15 g|
Filter solution through a Millipore Prep/Scale-TFF cartridge (10 kDa cut-off) using a peristaltic pump. Add KH2PO4 (25 mM final concentration) and MgSO4 (4 mM final concentration) and adjust the pH to 7. Autoclave (121°C, 20 min). Add glucose or sucrose (20% stock solution) to a final concentration of 1%.
50 mM KCl, 1 mM potassium phosphate (0.35 mM K2HPO4 plus 0.65 mM KH2PO4), 1 mM CaCl2, 0.1 mM MgCl2. Adjust pH to 6.5. Store at room temperature.
Collect 50 mL of whole saliva on ice from one donor. Mix saliva with AB buffer (1:1 ratio, v/v). Add 50 μL 0.1 M phenylmethyl-sulfonyl fluoride (PMSF, store at 4°C for up to 9 months). Centrifuge the mixture (5,500g, 4°C, 10 min). Collect supernatant (clarified whole saliva) and filter through a 0.22 μm PES low protein-binding filter.
100 mM Tris–maleate buffer (pH 7). Prepare a 200 mM stock solution of Trizma-maleate. Adjust 50 mL of stock solution to desired pH with 0.1 M NaOH. Make up to 100 mL with Milli-Q water.
100 mM ZnCl2 plus 15 mM ammonium molybdate.
50 mM KCl plus 1 mM MgCl2.
This method is particularly useful to assess the ability of different strains to form biofilms (seeNote 4).
This method is excellent for experiments that normally require a large bacterial biomass, particularly enzymatic assays.
This method uses hydroxyapatite disks coated with saliva (mimicking the presence of salivary pellicle), placed in a vertical position.
Since most microbes in nature grow in biofilms, the use of biofilms for assessing cidal action is generally more appropriate than the use of suspensions. The example presented here is for acid-mediated killing. This assay can be performed with intact biofilms grown on the surface of microtiter plates for 24–48 h, glass slides, or HA disks. Below we describe a standard protocol using biofilms grown on microtiter plates.
We thank Dr. Pedro Rosalen for kindly providing images used in Fig. 7.1.