Team:MIT/Tooth binding assay protocol

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Revision as of 15:38, 27 June 2008

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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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@@ Line 15: / Line 15: @@
-•	Wash 50 mg HA beads 3 times with 1mM phosphate buffer (PB) and equilibrate in PB for 2 hours
+•	Wash 50 mg HA beads 3 times with 1mM phosphate buffer (PB) and equilibrate in PB for 2 hours (or overnight)
-•	HA beads settle for 30 to 60 s, then remove supernatant
+•	HA beads added to 500 microL saliva and stir suspension for 1h at 37 C
-•	'''Control'''
+        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)
-      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
+•	Add 1mL S. Mutans suspension to HA beads, incubate at 37 C at 20 rpm in microfuge tubes
-      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 spot onto plates (10g agar, 15g THB, 500 mL H20)
-•	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
@@ Line 42: / Line 34: @@
 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)