Team:MIT/Tooth binding assay protocol

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Revision as of 14:31, 8 July 2008

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Culturing and maintaining S. mutans

Note: S. mutans needs to grow at 37C anaerobically. Estimated doubling time is X minutes.

To make a glycerol stock for long-term storage of live S. mutans, grow a 5mL culture until it is turbid (usually takes 16 hours). Add glycerol to a final concentration of 50%. The final 50% glycerol stock of S. mutans can be stored at -20C for 3 months or at -70C for years.

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 1:5 diluted saliva (with PBS) and stir suspension for 1h at 37 C

Aspirate saliva

Treat HA beads with 2 mg/mL BSA in PBS for 30 min. at 37 C

Take OD of overnight S. mutans culture, if initial OD is above 1 dilute with THB so tat the OD is between .1 and .5

Dilute bacteria with 1mMolar PBS with 2 mg/mL BSA 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 transfer into fresh epindorf tube, leave at 37 C (?)

Repeat above after 1h, 2h

(procedure for 96-well based serial dilution and spotting)

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. Seal bottle with cap to reduce evaporation. Keep the liquid THB at 37C so that it is conveniently pre-warmed.

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 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)
+Extract 100 μL supernatant after letting HA beads settle for 5 minutes. and transfer into fresh epindorf tube, leave at 37 C (?)
 Repeat above after 1h, 2h