Oral and Dental Therapy Test

Oral and Dental Therapy Experiment Purpose

To determine the antimicrobial effect of Nature’s Rite Oral and Dental Therapy tonic on biofilms derived from oral plaque.

Oral and Dental Therapy Testing Methods

An oral plaque sample was collected with a sterile toothpick from the gingival margin of a healthy volunteer with no or very minimal gingivitis. After collection of sample, the toothpick was broken and inserted into a vial containing sterile Ringers salts solution. The vial was refrigerated overnight.

5 ml of saliva was collected from same subject as the plaque sample. The saliva was diluted 10-fold in a 0.1M phosphate buffer and filter sterilized. Hydroxyapatite (HA) disks (0.25 cm dia) were conditioned overnight at room temperature in 10% sterile saliva (this step is necessary for biofilm formation and is similar to the formation of the acquired pellicle following a professional cleaning of teeth).

The conditioned disks were placed in a 12-well tissue culture plate containing 2 ml Trypticase-soy br (TSB) per well and one disk per well (in each of 8 wells).

The plaque sample was sonicated gently to remove plaque from the toothpick and 50 µl of the sonicate was used to inoculated the HA disk in each of 8 wells. One well was left un-inoculated as a negative control for contamination.

The tissue culture plate was incubated anaerobically in an anaerobe chamber containing 10% H2, 10% CO2, and the balance N2 at 37o C.

The disks were transferred to fresh TSB on day 2.

On day 3, the plate was removed from the anaerobic chamber and each disk was transferred to a fresh 12-well plate with each of 8 wells containing 1 ml of the above referenced ODHR undiluted. The biofilms were exposed to the ODHR for 0 (positive control), 20, 30, 40, 60, 90, 120, and 150 minutes.

After exposure, each biofilm disk was transferred to 1-ml of Ringers salts solution containing 0.5% Tween 20 (a mild detergent) and sonicated for 10 secs to remove the biofilm bacteria from the disk support.

(We have found that bacteria grown in biofilm mode tend to rapidly re-aggregate together when removed from the HA support. The addition of Tween 20 at 0.5% has proven sufficient to prevent this aggregation.)

A series of 10-fold dilutions were performed using Ringers w/ Tween 20 as the diluent. Dilutions were performed from 10-1 to 10-8. 100 μl of the dilutions 10-5 to 10-8 were plated onto Trypticase-soy agar plates supplemented with 5% defibrinated sheep blood and hemin (5 μg/ml) and menadione (0.05 μg/ml). This yielded plate dilutions of 10-6to 10-9.

The plates were incubated anaerobically for 4-5 days and then the plates containing from 30–300 colonies were counted. Plates containing between 30 and 300 CFUs were considered to be statistically valid.

Following the dilution series, the vials containing the HA support and the sonicated biofilm were measured with a calibrated spectrometer at OD600 to obtain an estimate of the relative numbers of cells present in each biofilm sample. This provided a total cell number consisting of both live and dead cells.

This was primary done to ensure that the number of cells present in each biofilm were approximately the same prior to the exposure to the ODHR for various lengths of time. Total cell numbers from the spectrometer ranged from 2.65 X 1010 to 6.91 X 1010.

Oral and Dental Therapy Testing Results

Time exposed (minutes) Trial 1 Trial 2 Trial 3 Mean % Relative to control % reduction
0 (control) 7.40x109 1.20 x 1010 8.50x109 9.30x109 100.00% N/A
20 4.40x109 7.40x109 5.30x109 5.47x109 61.29% 38.71%
30 6.20x109 5.30 x 109 4.90x109 5.47x109 58.78% 41.22%
40 2.60x109 3.20 x 109 3.40x109 3.07x109 32.97% 67.03%
60 2.20x109 4.40 x 109 9.00x108 2.50x109 26.88% 73.12%
90 1.80x108 5.20 x 108 3.20x108 3.40x108 3.66% 96.34%
120 5.60x108 2.20 x 108 3.10x108 3.63x108 3.91% 96.09%
150 4.20x108 1.80 x 108 2.40x108 2.80x108 3.01% 96.99%

Based on the results in the table above, inhibition of bacteria in a mixed culture biofilm gradually increases with increased exposure with the maximum effect reached after 90 minutes. It is not surprising that a long exposure is necessary to see the maximum effect. Biofilms are notoriously well known to be highly resistant to antimicrobials for several reasons.

  1. The inability for the antimicrobial to readily penetrate the biofilm
  2. The lower metabolic activity present in biofilms.
  3. Perhaps the most important reason seems to be that bacteria grown in biofilm mode express a different series of genes than do planktonic grown cells in broth and these genes may render the bacteria less tolerant to antimicrobials.

In the experiment reported here, we chose to look at the effect on an early intermediate biofilm. Latter stage mature biofilms likely would not have shown very much effect, not because the antimicrobial was not effective but due to the inactivity of this biofilm stage.

Late stage mature and climax biofilms undergo very little metabolic activity and this is thought to be the major reason that antimicrobials are less effective.

Effect of exposure time on survival