Tooth binding assay protocol

Tooth binding assay
•	Wash 50 mg HA beads 3 times with 1mM phosphate buffer (PB) and equilibrate in PB for 2 hours

•	HA beads settle for 30 to 60 s, then remove supernatant

•	Control

o	  add 1mM phosphate-phosphate buffered saline (PBS) for 1h o	  wash with 10mM PBS o	  add saliva for 1h o	  wash with 10mM PBS •	Variable

o	  Add saliva (amount?) for 1h o	  Wash with 10mM PBS o	  Add p1025 (amount?) extract o	  Wash with 10mM PBS

•	Add 1mL S. Mutans suspension (1-2*10^7 colony-forming units (CFU))

•	Extract 100 μL supernatant after 5 minutes and plate onto Todd Hewitt Broth (THB), or whatever media the S. Mutans comes in

•	Repeat above after 1h, 2h

•	Count colonies (approximately CFUs) on the three plates and calculate the amount attached to HA beads through the relation “CFU supernatant time 0 – CFU supernatant time 1h, 2h = CFU on HA beads"

PBS—naCL 8.0 g L-1, KCl 2.0 g L-1, Na2HPO4, 2H20 2.0 g L-1, KH2PO4 2.0 g L-1; pH 7.2