IGEM:MIT/2008/Notebook/Yogurt/toothbindingassay

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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

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