May 04, 2023

Public workspaceBiofilm growth with starch treatment V.2

DOI
dx.doi.org/10.17504/protocols.io.eq2lypqzelx9/v2
  • 1Leiden University
Bjørn Peare Bartholdy
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.eq2lypqzelx9/v2
Protocol Citation: Bjørn Peare Bartholdy, a.g.henry 2023. Biofilm growth with starch treatment. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lypqzelx9/v2Version created by Bjørn Peare Bartholdy
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 29, 2023
Last Modified: May 04, 2023
Protocol Integer ID: 79636
Funders Acknowledgement:
ERC European Union’s Horizon 2020
Grant ID: STG–677576
Abstract
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.
Image Attribution
Image created by Bjørn Peare Bartholdy using BioRender.com
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.
Materials
Solutions
Artificial saliva
Protocol
Artificial saliva
NAME

Artificial saliva

CREATED BY
Bjørn Peare Bartholdy
CPMU
Protocol
CPMU
NAME

CPMU

CREATED BY
Bjørn Peare Bartholdy

20% (v/v) sterile glycerol in dH2O
5% (w/v) sucrose in dH2O
0.25% (w/v) potato starch in dH2O
0.25% (w/v) wheat starch in dH2O
0.50% (w/v) equal parts wheat (0.25%) + potato (0.25%) in dH2O

Equipment
24 deepwell polypropylene plates (w. lid containing pegs suspended from the lid)
Shaking incubator
Powder free nitrile gloves

Protocol materials
ReagentSucrose
In 3 steps
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)
Protocol
Artificial saliva
NAME
Artificial saliva
CREATED BY
Bjørn Peare Bartholdy

Protocol
CPMU
NAME
CPMU
CREATED BY
Bjørn Peare Bartholdy

Saliva collection
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.

Note
Make sure donors wear gloves to avoid contamination with non-oral bacteria.

Make a 2-fold dilution of saliva in sterile Concentration20 % (v/v) glycerol and vortex the solution.

Day 0: Inoculation and feeding
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 inoculationDuration04:00:00

4h
After inoculation, transfer the samples to a new plate containing the artificial saliva, and place in a shaking incubator at Shaker30 rpm, 36°C for Duration04:00:00

Protocol
Artificial saliva
NAME

Artificial saliva

CREATED BY
Bjørn Peare Bartholdy



4h
Add Amount300 mL distilled (or deionized) dH2O to a Amount1000 mL beaker, with stirring and heat Temperature60 °C .

Add:

- Amount2.5 g ReagentMucin from porcine stomach (Type III)Becton Dickinson (BD)Catalog #M1778
- Amount5 g ReagentTrypticase™ PeptoneBecton Dickinson (BD)Catalog #211921
- Amount10 g ReagentOxoid™ Proteose PeptoneBecton Dickinson (BD)Catalog #LP0085B
- Amount5 g ReagentBacto Yeast ExtractBecton Dickinson (BD)

Let the reagents completely dissolve before continuing to the next step

Add:

- Amount2.5 g ReagentKClBecton Dickinson (BD)
- Amount0.35 g ReagentNaClBecton Dickinson (BD)
- Amount0.2 g ReagentCaCl2Becton Dickinson (BD)
- Amount0.74 g ReagentSodium phosphate dibasicBecton Dickinson (BD)Catalog #7558-79-4
- Amount0.54 g ReagentNaHCO3Becton Dickinson (BD)
- Amount2.5 mg ReagentHeminBecton Dickinson (BD)

Add the remaining Amount700 mL distilled (or deionized) dH2O

Adjust to Ph7 with ReagentNaOHBecton Dickinson (BD) and stirring

Transfer to two 1000 ml bottles, so half of each bottle is filled.

Autoclave at Temperature121 °C , Pressure1 Bar for Duration00:15:00 minutes
Safety information
Do NOT screw bottle caps on tightly.

Loosely screw the caps on the bottles or cover the tops with foil

15m
Once the solution has cooled, add:

  • Amount1 mg ReagentMenadioneBecton Dickinson (BD)
  • Amount0.3 g ReagentUreaBecton Dickinson (BD)
  • Amount0.17 g ReagentL-ArginineBecton Dickinson (BD)Catalog #A5006


Store in fridge at ca. Temperature4 °C

Occasionally test the pH to ensure it stays around Ph7

Transfer the samples to a plate containing a 5% (m/v) ReagentSucrose solution for Duration00:06:00 , then transfer back to the artificial saliva and leave DurationOvernight .

Plates with lids (substratum) in the incubator. Sucrose treatment plates covered with sterilised foil at the back.

6m
Day 1-2: Feeding
Day 1-2: Feeding
8h 12m
8h 12m
First thing in the morning, transfer the samples to a new plate containing a 5% (m/v) ReagentSucrose solution for Duration00:06:00 . While in the sucrose solution, add more artificial saliva to the wells on the original plate that have been partially depleted overnight.

6m
After the 6 mins. return the samples to the plate with artificial saliva, and cover up the sucrose plate and leave for Duration08:00:00 .
8h
After 8 hours, transfer the samples back to the plate with 5% (m/v) ReagentSucrose solution for Duration00:06:00 . Transfer back to the artificial saliva and leave DurationOvernight . Dispose of the sucrose.

6m
Day 3: Inoculation and feeding
Day 3: Inoculation and feeding
8h
8h
Repeat steps from saliva collection and Day 0: Inoculation and feeding.

Expected result
A layer of clear plaque should be visible on the substrata

8h
Prepare a new plate with artificial saliva. Transfer the samples from the inoculation plate to the artificial saliva.
Day 4: Feeding
Day 4: Feeding
8h
8h
Repeat steps 10 through 12
Go to

Note
Prepare a new plate of artificial saliva every third day throughout the experiment. Every other morning, top up the wells with artificial saliva (so ca. 1-2 mm of the substratum is submerged).

8h
Day 5: Inoculation
Day 5: Inoculation
Repeat steps 1 through 9
Go to

Day 6-8: Feeding
Day 6-8: Feeding
Repeat steps 10 through 12
Go to
Day 9-14: Starch treatment
Day 9-14: Starch treatment
8h 6m
8h 6m
Transfer the samples to a plate with the starch treatment(s) for Duration00:06:00 at Shaker60 rpm, 36°C

Example setup:
123456
A
Potato
Potato
Potato
Potato
Potato
Potato
B
Wheat
Wheat
Wheat
Wheat
Wheat
Wheat
C
Potato + Wheat
Potato + Wheat
Potato + Wheat
Potato + Wheat
Potato + Wheat
Potato + Wheat
D
Control
Control
Control
Control
Control
Control


Safety information
If you are using multiple starch treatments within a single plate, take care to avoid cross-contamination.

6m
Transfer the samples back to the artificial saliva plate for Duration08:00:00 at Shaker30 rpm, 36°C

8h
Transfer the samples to a plate with the starch treatment(s) for Duration00:06:00 at Shaker60 rpm, 36°C
6m
Transfer the samples back to the artificial saliva plate at Shaker30 rpm, 36°C and leave DurationOvernight

Day 15-24: Mineralisation
Day 15-24: Mineralisation
8h 30m
8h 30m
Transfer the samples to a plate containing the calcium phosphate monofluorophosphate urea (CPMU) solution for Duration00:06:00 at Shaker60 rpm, 36°C

Protocol
CPMU
NAME

CPMU

CREATED BY
Bjørn Peare Bartholdy

6m
Add Amount300 mL distilled (or deionized) dH2O to a Amount1000 mL beaker, with stirring and heat Temperature60 °C
Add:

- Amount1.55 g ReagentCalcium ChlorideSigma Aldrich
- Amount1.44 g ReagentSodium Phosphate monobasicSigma Aldrich
- Amount0.72 g ReagentSodium FluorophosphateSigma AldrichCatalog #344443
- Amount0.08 g ReagentMagnesium ChlorideSigma AldrichCatalog #AC223210010
- Amount30 g ReagentUreaSigma Aldrich

Add the remaining Amount700 mL and keep stirring until precipitate has completely dissolved
Store in fridge at Temperature4 °C

Transfer the samples back to the artificial saliva for Duration02:00:00 at Shaker30 rpm, 36°C

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.
2h
Transfer the samples to a plate with the starch treatment(s) for Duration00:06:00 at Shaker60 rpm, 36°C
6m
Transfer the samples back to the artificial saliva plate at Shaker30 rpm, 36°C and leave DurationOvernight



Analysis
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.
Protocol references
Exterkate, R. A. M., Crielaard, W., & Ten Cate, J. M. (2010). Different Response to Amine Fluoride by Streptococcus mutans and Polymicrobial Biofilms in a Novel High-Throughput Active Attachment Model. Caries Research, 44(4), 372–379. https://doi.org/10.1159/000316541
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. https://doi.org/10.1177/00220345910700110301