Team:MIT/Tooth binding assay protocol

Culturing and maintaining S. mutans
Note: S. mutans needs to grow at 37C anaerobically. Estimated doubling time is 90 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.

Night before binding assay--Inoculation--set up 3 tubes with 5 mL THB and add 10 (should be about OD 1.8 after 15 hours), 25 and 50 microL of Glycerol stock to the tubes.

Tooth binding assay (reference: PMID 9062560)
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 that 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

Aspirate and wash beads twice with 1 mL PBS (with or without BSA), rotate tube vertically to mix instead of vortex

Add 1 mL PBS with 1 mM EDTA to beads, rotate vertically at 37 C for 10 min

Let beads settle and take 100 microL of supernatant

(proceed to serial dilution and spotting)

Count colonies (approximately CFUs) on the three samples 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)

Why is it novel?
A couple of things are novel in the protocol. Firstly, we modified the protocol by including a dechaining step to prevent S. mutans from sticking to each other (We read an article which stated that S. mutans form long chains in solution). We dechanined them by passing them through a syringe 15 times and also vortexing them with glass beads. Secondly, we are coating the beads with BSA to prevent non specific binding. We increased the saliva coating time than what is reported in the literature after we got results indicating a correlation between time beads remain in saliva and binding. Thirdly, we washed the beads in EDTA and plated both the supernatant and the beads to confirm there is bacteria on the beads (the paper with the original protocol failed  to prove that there was bacteria on the beads using the indirect assay). Finally, our most significant addition to the protocol has been the way we plate after incubation with HA beads. We are using the spotting method where we take some supernatant from each tube and serially dilute it in a 96 well plate. Then we spot, drawing about 5microliters from each microwell, onto a rectangular plate.

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 500 mL 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 2 mL of 50 mg/mL Streptomycin sulfate (thaw and vortex well before pipetting; our S. mutans strain is resistant to streptomycin so we use the antibiotic to reduce contamination) and 5 mL of 10% glucose. Seal bottle with cap to reduce evaporation. Keep the liquid THB at 37C so that it is conveniently pre-warmed.