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Team:MIT/Tooth binding assay protocol
From 2008.igem.org
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Culturing S. mutans
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 10mMolar PBS
Dilute bacteria with 1mMolar PBS to get the final concentration of 1-2 *10^7 CFU (.075 to .16 OD--use .10)
Add 1mL S. Mutans suspension to HA beads, incubate at 37 C at 20 rpm in microfuge tubes
Extract 100 μL supernatant after letting HA beads settle for 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"
OD-CFU conversion factor: optical density at 600 nm of 0.75 to 0.8: 1 × 10^8 CFU/ml)
Reagents and Recipes
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 (Verify using a pH meter)
Todd Hewitt Broth (THB) liquid and agar - 15g THB powder, (10g agar), bring volume up to 500mL using distilled water from white tap. Stir to dissolve THB powder. (agar remains undissolved until autoclaving) Autoclave using liquid cycle for 30 min. Let THB liquid cool to room temp then add X microL of Streptomycin sulfate (thaw and vortex well before pipetting) and X mL of X% glucose.
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