Oral Probiotic Competition in Dental Biofilms

ISEF Category: Microbiology

Subcategory: Bacteriology  ·  Difficulty: Advanced  ·  Setup: University Lab  ·  Time: Full Year

The Hook

Your mouth is a battlefield. Some bacteria help keep the peace, and others push the system toward tooth decay. A probiotic strain called Streptococcus salivarius K12 may help crowd out more harmful species, but that effect depends on who gets there first, and what surface they stick to. You can test that idea with a dental-like surface, a pH probe, and a biofilm stain.

What Is It?

This project studies competition between bacteria on a surface that acts a little like a tooth. Hydroxyapatite is the mineral that makes up most of tooth enamel, so chips or beads made from calcium phosphate give you a place for bacteria to attach. Once bacteria form a biofilm, they build a sticky layer that helps them stay put and share resources. Think of it like a crowded apartment building, where the first tenants can make it hard for new ones to move in.

Streptococcus salivarius K12 is sold as an oral probiotic. In simple terms, a probiotic is a microbe that may help the body by competing with less helpful microbes. In this project, you would compare how well K12 limits biofilm formation and acid production by a substitute oral Streptococcus, such as S. mitis, on hydroxyapatite chips. You can measure acid production with a pH probe, since more acid usually means a bigger drop in pH. You can also stain the biofilm with crystal violet, which binds to the biomass and gives you a rough measure of how much stuck material formed.

Why This Is a Good Topic

This is a strong science fair topic because you can ask real questions about competition, attachment, and acid production without needing a giant lab setup for every part of the analysis. It connects to dental health, which makes the biology feel real instead of abstract. You can vary who arrives first, which strain is present, or what surface conditions favor one species over another. That gives you clear variables, measurable outcomes, and room to build a project that looks like actual research.

Research Questions

• How does precolonization by Streptococcus salivarius K12 change the biofilm coverage of S. mitis on hydroxyapatite chips?
• What is the effect of adding S. salivarius K12 at the same time as S. mitis on final pH compared with growing S. mitis alone?
• Does the order of inoculation change how much crystal violet staining remains on hydroxyapatite chips?
• To what extent does increasing K12 abundance reduce acidification by the mixed biofilm?
• Which surface condition, smooth or rough hydroxyapatite, leads to stronger competitive exclusion by K12?
• How does nutrient availability change the balance between biofilm mass and pH drop in the mixed culture?

Basic Materials

• Hydroxyapatite chips or aquarium-grade calcium-phosphate beads.
• Streptococcus salivarius K12 probiotic source.
• Streptococcus mitis culture from a teaching or research supplier, or an approved lab strain.
• Sterile broth or oral microbiology growth medium.
• pH probe with meter or handheld pH tester.
• Crystal violet stain.
• Microcentrifuge tubes or sterile culture tubes.
• Petri dishes or multiwell plates.
• Micropipettes and sterile tips.
• Digital scale with 0.01 g or better precision.
• Disposable gloves, lab coat, and eye protection.
• Biohazard waste container and disinfectant approved by your lab.

Advanced Materials

• Hydroxyapatite disks or standardized enamel-mimetic coupons.
• Validated Streptococcus salivarius K12 strain.
• Validated Streptococcus mitis strain, or a closely matched oral isolate approved by your lab.
• Anaerobic jars or chamber access.
• Spectrophotometer or plate reader for optical density and stain quantification.
• Real-time pH microelectrode or benchtop pH meter with microprobe.
• Confocal microscope or fluorescent imaging system for biofilm architecture.
• Sonicator for controlled biofilm recovery.
• Sterile flow cell or drip-flow system for surface attachment studies.
• ATP or lactic acid assay kit for added metabolic readouts.
• ImageJ for coverage analysis and thresholding.
• R for statistical analysis and mixed-effects modeling.

Software & Tools

• ImageJ: Measures stained biofilm area on hydroxyapatite chips and compares coverage across groups.
• R: Runs statistical tests, plots pH change, and models competition effects.
• Google Sheets: Organizes sample labels, raw readings, and replicate tracking.
• GraphPad Prism: Fits growth or response curves if your school or lab already has access.
• PubMed: Finds review articles and primary papers on oral probiotics, Streptococcus biofilms, and dental plaque.

Experiment Steps

1. Define the competition question you want to answer first, such as timing, dose ratio, or surface type.
2. Choose a measurement plan that gives you one signal for acidity and one signal for attached biomass.
3. Set up controls that separate single-species growth from mixed-species competition and from surface-only background staining.
4. Build a repeatable scoring system for biofilm coverage so your image or stain data stay comparable across trials.
5. Plan your analysis before collecting data, including how you will compare replicate groups and handle outliers.
6. Pilot the system on a small scale, then refine the protocol until your readouts stay stable from run to run.

Common Pitfalls

• Using an unverified probiotic product instead of a known strain, which makes your results hard to interpret.
• Letting pH probes drift between samples, which makes acidification look bigger or smaller than it really is.
• Skipping single-strain controls, which makes it impossible to tell whether K12 or S. mitis caused the effect.
• Counting crystal violet stain without normalizing to chip size or surface area, which confuses coverage with sample shape.
• Changing light, imaging distance, or stain wash consistency between trials, which breaks your biofilm comparisons.

What Makes This Competitive

A strong version of this project does more than compare two tubes. You can push it by testing multiple competition scenarios, such as timing of colonization, starting ratio, and surface texture. You can also pair stain-based biomass data with pH data and use statistics that compare whole response patterns, not just averages. That kind of design shows you understand mechanism, not just outcome.

Project Variations

• Test whether K12 blocks biofilm formation better on smooth hydroxyapatite disks than on rough beads.
• Compare K12 against another oral Streptococcus species to see whether competitive exclusion depends on the rival strain.
• Add a saliva-like coating step before inoculation to see whether surface conditioning changes attachment and acid production.

Learn More

• PubMed: Search for review articles on Streptococcus salivarius K12, oral probiotics, and dental biofilms.
• NIH NIDCR: Read public pages on tooth decay, plaque formation, and oral microbiology on the National Institute of Dental and Craniofacial Research site.
• NCBI Bookshelf: Find free textbook chapters on bacterial biofilms and microbial competition.
• ASM Microbe Library: Search educational articles and methods notes on biofilm staining, oral bacteria, and Streptococcus species.
• MIT OpenCourseWare: Look for free microbiology or microbiology lab materials that cover bacterial growth, biofilms, and data analysis.