Oct 27, 2021

Public workspaceArtificial saliva V.1

DOI
dx.doi.org/10.17504/protocols.io.3byl4k1jzvo5/v1
  • 1Leiden University
  • BYOC
Bjørn Peare Bartholdy
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DOI: dx.doi.org/10.17504/protocols.io.3byl4k1jzvo5/v1
Protocol Citation: Bjorn Bartholdy, a.g.henry 2021. Artificial saliva. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4k1jzvo5/v1
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: In development
We are still developing and optimizing this protocol
Created: May 26, 2021
Last Modified: October 27, 2021
Protocol Integer ID: 50241
Funders Acknowledgements:
European Union’s Horizon 2020 research and innovation program
Grant ID: STG–677576 (“HARVEST”)
Abstract
Creating an artificial saliva solution for oral biofilm growth.

This is a modified version of Sissons et al. 1991.
Guidelines
This protocol explains how to mix Amount1000 mL of artificial saliva.

Materials
Chemicals
Mucin from porcine stomach (Type III)
Trypticase peptone
Proteose peptone
Yeast Extract
KCl
NaCl
CaCl2
Na2HPO4
NaHCO3
Hemin
Menadione
Urea
L-Arginine

Equipment
1000 mL beaker
2 x 1000 mL bottle
Magnetic stirrer (with heating element)
Autoclave
pH measure

Protocol materials
ReagentMucin from porcine stomach (Type III)Merck MilliporeSigma (Sigma-Aldrich)Catalog #M1778
ReagentTrypticase™ PeptoneThermo FisherCatalog #211921
ReagentOxoid™ Proteose PeptoneThermo FisherCatalog #LP0085B
ReagentBacto Yeast ExtractBecton Dickinson (BD)
ReagentKCl
ReagentHemin
ReagentNaHCO3
ReagentNaCl
ReagentCaCl2
ReagentSodium phosphate dibasicMerck MilliporeSigma (Sigma-Aldrich)Catalog #7558-79-4
ReagentNaOH
ReagentUrea
ReagentL-ArginineCatalog #A5006
ReagentMenadione
Before start
Mix the solution under a fumehood. It can smell pretty bad.
Also have the incubator under a fumehood, if possible. I had no such luxury...
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