TITLE: Biofilm growth with starch treatment
AUTHORS: Bjørn Peare  Bartholdy, a.g.henry

[DESCRIPTION]
This is the main protocol to grow a calcifying oral biofilm model with starch treatments. It uses a 24 deepwell plate with the accompanying lid as substratum (polypropylene). The protocol takes 25 days to run, with daily tasks.

Modified from protocols by Sissons et al. (1991) and Extercate et al. 2010.

[BEFORE_START]
Surfaces in the lab should be cleaned three times. Once with warm water and starch-free detergent, then with 5% NaOH, followed by a final cleaning with distilled water.

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.

Prerequisites

Required solutions should be prepared beforehand.

- 20% (v/v) sterile glycerol in dH2O
- Artificial saliva (required for day 0)
- Sucrose solution (required for day 0)
- Starch solutions (required for day 9)
- CPMU (required for day 15)
 

 


[GUIDELINES]
The preferred substratum for inoculation is glass or hydroxyapatite. Plastic substrata can be used but are less effective, so surface treatment of the plastic is recommeded (e.g. heated HCl or acetone).

Substrata should be autoclaved prior to use, both before and during the experiment.

[STEPS]
SECTION: Saliva collection
1. Saliva donors rinse their mouth with water for 30 seconds.
SECTION: Saliva collection
2. Stimulate saliva production by chewing tasteless gum or parafilm.
SECTION: Saliva collection
3. Collect the saliva by spitting into 50 ml plastic centrifuge tubes.

 

SECTION: Saliva collection
4. Make a 2-fold dilution of saliva in sterile 20 % (v/v) and vortex the solution.

SECTION: Day 0: Inoculation and feeding
5. Before inoculation, vortex the saliva solution again.
SECTION: Day 0: Inoculation and feeding
6. Pipette the saliva solution into the wells, so approx. 1-2 cm of the substratum is submerged.
SECTION: Day 0: Inoculation and feeding
7. Place the plate in the incubator at 36ºC for 4 hours for static inoculation240 min  

SECTION: Day 0: Inoculation and feeding
8. After inoculation, transfer the samples to a new plate containing the artificial saliva, and place in a shaking incubator at   for  240 min 

 



SECTION: Day 0: Inoculation and feeding
9. Transfer the samples to a plate containing a 5% (m/v)   solution for 6 min , then transfer back to the artificial saliva and leave   .

  

SECTION: Day 1-2: Feeding
10. First thing in the morning, transfer the samples to a new plate containing a 5% (m/v)   solution for 6 min . While in the sucrose solution, add more artificial saliva to the wells on the original plate that have been partially depleted overnight. 

SECTION: Day 1-2: Feeding
11. After the 6 mins. return the samples to the plate with artificial saliva, and cover up the sucrose plate and leave for 480 min .
SECTION: Day 1-2: Feeding
12. After 8 hours, transfer the samples back to the plate with 5% (m/v)   solution for 6 min . Transfer back to the artificial saliva and leave   . Dispose of the sucrose.

SECTION: Day 3: Inoculation and feeding
13. Repeat steps from saliva collection and Day 0: Inoculation and feeding.

 

SECTION: Day 3: Inoculation and feeding
14. Prepare a new plate with artificial saliva. Transfer the samples from the inoculation plate to the artificial saliva.
SECTION: Day 4: Feeding
15. Repeat steps 10 through 12
  

 

SECTION: Day 5: Inoculation
16. Repeat steps 1 through 9
  

SECTION: Day 6-8: Feeding
17. Repeat steps 10 through 12
  
SECTION: Day 9-14: Starch treatment
18. Transfer the samples to a plate with the starch treatment(s) for 6 min at   

Example setup:
 


 

SECTION: Day 9-14: Starch treatment
19. Transfer the samples back to the artificial saliva plate for 480 min  at   

SECTION: Day 9-14: Starch treatment
20. Transfer the samples to a plate with the starch treatment(s) for 6 min at   
SECTION: Day 9-14: Starch treatment
21. Transfer the samples back to the artificial saliva plate at    and leave   

SECTION: Day 15-24: Mineralisation
22. Transfer the samples to a plate containing the calcium phosphate monofluorophosphate urea (CPMU) solution for 6 min  at   

 

SECTION: Day 15-24: Mineralisation
23. Transfer the samples back to the artificial saliva for 120 min  at    

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.
SECTION: Day 15-24: Mineralisation
24. Transfer the samples to a plate with the starch treatment(s) for 6 min at   
SECTION: Day 15-24: Mineralisation
25. Transfer the samples back to the artificial saliva plate at    and leave   

  

SECTION: Analysis
26. Samples should be dried before sampling.

Transfer to a new plate with no liquid and leave in the incubator.
SECTION: Analysis
27. Once dried, samples are processed like archaeological samples.