Biofilm growth with starch treatment
Bjorn Bartholdy, a.g.henry
Abstract
Protocol to grow mature, mineralised oral biofilm with starches.
Modified from protocol by Sissons et al.:
Sissons, C. H., Cutress, T. W., Hoffman, M. P., & Wakefield, J. S. J. (1991). A Multi-station Dental Plaque Microcosm (Artificial Mouth) for the Study of Plaque Growth, Metabolism, pH, and Mineralization: Journal of Dental Research . http://doi.org/10.1177/00220345910700110301
Before start
Saliva donor criteria
- Must have no/limited history of dental caries
- Must not have used antibiotics in the past 6 months
- Abstain from oral hygiene 24 hours prior to donation
- Refrain from eating and drinking (except water) 2 hours before donation
For experiments involving starches, donors should avoid eating starch-containing foods on the day of donation. To make this more bearable, saliva donation should take place in the morning before breakfast.
Steps
Saliva collection
Saliva donors rinse their mouth with water for 30 seconds.
Stimulate saliva production by chewing tasteless gum or parafilm.
Collect the saliva by spitting into 50 ml plastic centrifuge tubes.
Make a 2-fold dilution of saliva in sterile 20% (v/v) and vortex the solution.
Day 0: Inoculation and feeding
Before inoculation, vortex the saliva solution again.
Pipette the saliva solution into the wells, so approx. 1-2 cm of the substratum is submerged.
Place the plate in the incubator at 36ºC for 4 hours for static inoculation4h 0m 0s
After inoculation, transfer the samples to a new plate containing the artificial saliva, and place in a shaking incubator at 30rpm for 4h 0m 0s
Artificial saliva
Transfer the samples to a plate containing a 5% (m/v) 0h 6m 0s , then transfer back to the artificial saliva and leave .
Day 1-2: Feeding
First thing in the morning, transfer the samples to a new plate containing a 5% (m/v) 0h 6m 0s . While in the sucrose solution, add more artificial saliva to the wells on the original plate that have been partially depleted overnight.
After the 6 mins. return the samples to the plate with artificial saliva , and cover up the sucrose plate and leave for 8h 0m 0s .
After 8 hours, transfer the samples back to the plate with 5% (m/v) 0h 6m 0s . Transfer back to the artificial saliva and leave . Dispose of the sucrose.
Day 3: Inoculation and feeding
Repeat steps from saliva collection and Day 0: Inoculation and feeding.
Prepare a new plate with artificial saliva. Transfer the samples from the inoculation plate to the artificial saliva .
Day 4: Feeding
Repeat steps 10 through 12
Day 5: Inoculation
Repeat steps 1 through 9
Day 6-8: Feeding
Repeat steps 10 through 12
Day 9-14: Starch treatment
Transfer the samples to a plate with the starch treatment(s) for 0h 6m 0s at 60rpm
Transfer the samples back to the artificial saliva plate for 8h 0m 0s at 30rpm
Transfer the samples to a plate with the starch treatment(s) for 0h 6m 0s at 60rpm
Transfer the samples back to the artificial saliva plate at 30rpm and leave
Day 15-24: Mineralisation
Transfer the samples to a plate containing the calcium phosphate monofluorophosphate urea (CPMU) solution for 0h 6m 0s at 60rpm
CPMU
Transfer the samples back to the artificial saliva for 2h 0m 0s at 30rpm
Put a lid on the plate with CPMU (or cover with foil) to prevent evaporation.
Repeat step 22 and 23, four more times every two hours.
Transfer the samples to a plate with the starch treatment(s) for 0h 6m 0s at 60rpm
Transfer the samples back to the artificial saliva plate at 30rpm and leave
Analysis
Samples should be dried before sampling.
Transfer to a new plate with no liquid and leave in the incubator.
Once dried, samples are processed like archaeological samples.
