Method of quantitating sorption of stannous by microbial cells of a biofilm

1. A method of quantitating sorption of stannous by microbial cells of a biofilm comprising the steps:

a. wearing an oral appliance for 6 hours to 4 days wherein at least a portion of the oral appliance comprises hydroxylapatite (HA) as a surface for attachment of the biofilm;

b. treating the biofilm with a stannous containing oral care product;

c. labeling the biofilm with a microbial fluorescent probe;

d. labeling the biofilm with a stannous fluorescent probe;

e. providing excitation light to labeled biofilm;

f. quantitating labeled cells by measuring fluorescence light emitted from the microbial labeled cells by confocal laser scanning microscopy (CLSM);

g. measuring fluorescence light emitted from cells having labeled co-localized stannous ions by CLSM;

h. quantitating co-localized stannous by quantifying the overlap of measured fluorescence light from microbial fluorescent labeled cells and measured fluorescent light from stannous fluorescent labeled cells;

wherein the portion of the oral appliance comprising HA further comprises a plurality of grooves, wherein an inside surface of the groove is said surface of the biofilm; and

wherein at least one grove is about 250 microns to about 350 microns deep and from about 250 microns to about 350 microns wide.

2. The method of claim 1 , wherein the steps of labeling the biofilm with the microbial fluorescent probe and the stannous fluorescent probe are before the step of treating the biofilm with the stannous containing oral care product.

3. The method of claim 1 , wherein treating the in situ plaque biofilm with the stannous containing oral care product is treated in vivo.

4. The method of claim 1 , wherein treating the in situ plaque biofilm with the stannous containing oral care product is treated ex vivo.

5. The method of claim 1 , further comprising the step of having human subjects wear the oral appliance for 1-3 days wherein at least a portion of the oral appliance comprises hydroxylapatite (HA) as a surface for attachment of the biofilm wherein the HA is on the buccal side of the oral appliance.

6. The method of claim 1 , wherein the biofilm is an in situ plaque biofilm.

7. The method of claim 1 , wherein the HA surface of the biofilm is treated with the stannous containing oral care product ex vivo.

8. The method of claim 1 , wherein the microbial fluorescent probe is a fluorescently labeled rRNA-directed oligonucleotide.

9. The method of claim 1 , wherein the stannous fluorescent probe is selected from the group consisting of:

a. tert-butyl (3′,6′-diamino-3-oxospiro[isoindoline-1,9′-xanthen]-2-yl)carbamate;

b. tert-butyl (3′,6′-bis(dimethylamino)-3-oxospiro[isoindoline-1,9′-xanthen]-2-yl)carbamate;

c. tert-butyl (3′,6′-bis(diethylamino)-3-oxospiro[isoindoline-1,9′-xanthen]-2-yl)carbamate;

d. tert-butyl (3′,6′-bis(ethylamino)-2′,7′-dimethyl-3-oxospiro[isoindoline-1,9′-xanthen]-2-yl)carbamate;

e. tert-butyl (3′,6′-diamino-2′,7′-dimethyl-3-oxospiro[isoindoline-1,9′-xanthen]-2-yl)carbamate;

f. tert-butyl (3-oxo-3′,6′-di(pyrrolidin-1-yl)spiro[isoindoline-1,9′-xanthen]-2-yl)carbamate;

g. tert-butyl (3-oxo-3′,6′-bis(phenylamino)spiro[isoindoline-1,9′-xanthen]-2-yl)carbamate;

h. tert-butyl (3-oxo-3′,6′-di(piperidin-1-yl)spiro[isoindoline-1,9′-xanthen]-2-yl)carbamate;

i. tert-butyl (3′,6′-dimorpholino-3-oxospiro[isoindoline-1,9′-xanthen]-2-yl)carbamate;

j. tert-butyl(2′,7′-dibutyl-3′,6′-bis(diethylamino)-3-oxospiro[isoindoline-1,9′-xanthen]-2-yl)carbamate;

k. tert-butyl (2′,7′-dimethyl-3-oxo-3′,6′-di(piperidin-1-yl)spiro[isoindoline-1,9′-xanthen]-2-yl)carbamate;

l. tert-butyl (3-oxo-1′,2′,3′,4′,10′,11′,12′,13′-octahydrospiro[isoindoline-1,7′-pyrano[2,3-f:6,5-f]diquinolin]-2-yl)carbamate;

m. tert-butyl (3-oxo-1′,2′,3′,4′,8′,9′,10′,11′-octahydrospiro[isoindoline-1,6′-pyrano[3,2-g:5,6-g′]diquinolin]-2-yl)carbamate;

n. N-(3′,6′-bis(diethylamino)-3-oxospiro[isoindoline-1,9′-xanthen]-2-yl)propionamide;

o. N-(3′,6′-bis(diethylamino)-3-oxospiro[isoindoline-1,9′-xanthen]-2-yl)butyramide; and

p. N-(3′,6′-bis(diethylamino)-3-oxospiro[isoindoline-1,9′-xanthen]-2-yl)pentanamide;

wherein emitted light of the stannous fluorescent probe is collected at a wavelength of 560-660 nm when the stannous fluorescent probe is exposed to excitation light at a wavelength of 543 nm.

10. The method of claim 1 , further comprising the step of dividing said quantified overlap of stannous labeled bacteria with all quantified measured fluorescent light in the biofilm to determine a co-localization percentage of stannous in bacteria.