Team:MIT/Tooth binding assay protocol

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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 (or overnight)

• HA beads added to 500 microL saliva and stir suspension for 1h at 37 C 

       Aspirate saliva and wash s. mutans with 10x PBS
       
       Dilute bacteria with 1x PBS to get the final concentration of 1-2 *10^7 CFU (.075 to .16 OD)


• Add 1mL S. Mutans suspension to HA beads, incubate at 37 C at 20 rpm in microfuge tubes

• Extract 100 μL supernatant after 5 minutes and spot onto plates (10g agar, 15g THB, 500 mL H20)

• 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

OD-CFU conversion factor: optical density at 600 nm of 0.75 to 0.8: 1 × 108 CFU/ml)




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