ORAL MICROBES  The microorganisms that form the plaque/biofilm on the surfaces of the teeth are mainly Streptococcus mutans.  Other species :- › Streptococcus mitis › Streptococcus sanguis.  If not taken care of, via brushing or flossing and rinsing, the plaque can turn into tartar (its hardened form) and lead to gingivitis or periodontal disease.

Various streptococci in a biofilm in the oral cavity (electron microscope)

Gram staining of Streptococcus mutan

MOUTH RINSES  Mouth rinse is a product used to enhance oral hygiene.  Antibacterial agent in the mouth rinse can kill the bacterial plaque causing caries, gingivitis, and bad breath.  Active ingredients :-  Chlorhexidine gluconate (CHX),  Cetylpyridinium chloride (CPC),  Thymol (THY).

 To compare the antibacterial effect of the commercially available mouth rinses towards mixed three oral bacteria commonly associated with dental caries. › Streptococcus mutans › Streptococcus mitis › Streptococcus sanguinis).

SUSCEPTIBILITY TESTS (AST) SUSCEPTIBILITY TESTS (AST) CULTURE PREPARATIONS MINIMAL INHIBITORY CONCENTRATION TEST (MIC) MINIMAL BACTERICIDAL CONCENTRATION DETERMINATIONS (MBC)

3 Streptococcus spp. from ATTC were used in this study. BHI media was used to revived & culture the microorganisms. Take the bacteria using sterile cotton swab & make a suspension. Read OD Mixed 3 suspension of bacteria. Take 100ul suspension and & place on the BHI agar

Using hockey stick spread the suspension using lawn method Incubated 24 hrs at 37°C Bacteria ready to use

 The mouth rinses were tested for sensitivity using Kirby- Bauer susceptibility test with principle of agar disc diffusion. Let it dry at RT Repeat procedure 2 & 3 until total solution diffused in a disk 100ul Prepare disk containing mouth rinses using whatman sterile paper disk size 6mm Pipette 20ul mouth rinse into the disk Let it completely dry & ready to use

Place the disk on the agar using sterile forcep Fresh bacterial suspension Using sterile cotton swab culture the bacteria on BHI agar Inubated 24hrs at 37 oC Measure & record the results

 Using 96 wells broth micro-dilution assay was used to determine the minimal inhibition concentrations (MIC).

Mouth rinse (500 ul) Discard Mixed bacteria suspension Mouth Rinse (X) Mouth Rinse (X) The first tube with no bacteria growth (no turbid suspension) is the dilution containing the concentration of the mouth rinse which is refered MIC. Incubated 37ºc (24 hour)

 The Minimum Bactericidal Concentration (MBC) is the lowest concentration of antibiotic required to kill the germ. MBC Techniques

Flow chart MIC & MBC

DiscInhibition Zones (cm) 0.12 % CHX2.73 cm 0.05 % CPC2.27 cm THY Resistant 0.12 % CHX - Standard(+ve control) 2.53 cm D.H2O (-ve controll) Resistant Table 1: Sizes of inhibition zones that were present after 24 hour. The susceptibility of active compound was shown by the formation of clear zone of growth inhibition around the paper discs. AST

 It was clearly shows that CHX and CPC containing mouth rinse are able to prevent the growth of the mixed oral bacteria at the tested concentrations. THY showed less susceptibility effect when compared with blank control and positive control (fig.AST).  The greater the size of inhibition zones, the greater the antibacterial potency of the mouth rinse.

MIC & MBC HIGHER CONCENTRATION LOWER CONCENTRATION TURBID -  HIGH BACTERIA

MIC & MBC Figure MBC: Minimal bactericidal concentration (MBC) Test. The concentration on the agar that shows no bacteria growth was recorded as the MBC of the mouth rinse – agar label as tube 3 shown NO GROWTH (MBC)

The MICs and MBCs for CHX and CPC were determined as low as % w/v (tube 3) and % w/w respectively. This showed that CHX and CPC exhibited profound antibacterial activity on selected common oral bacteria as demonstrated by the very low MIC and MBC values. THY showed its antibacterial effect only at higher concentration (>50 % v/v). MIC & MBC

 The present study showed that mouth rinses vary significantly in their capability to inhibits and kill oral bacteria.  The CHX containing mouth rinse demonstrated superior antibacterial activity toward selected mixed tested bacteria followed by CPC containing mouth rinse.  This implies that the incorporation of CHX and CPC in mouth rinses are better than that containing THY.

 A.R. Fathilah and Z.H.A Rahim. J. Oral Sci., 45 : (2003).  A.R. Fathilah, Y. Othman and Z.H.A Rahim. J. Oral Sci., 48 (2) : (2006).  A.R. Fathilah, Z.H.A. Rahim, Y. Othman and M. Yusoff. Pak. J. Biol. Sci., 12(6) : (2009).  T. Nalina and Z.H.A. Rahim. Pak. J. Biol. Sci., 9 : (2007).  Pan P. C., Harper S., Ricci-Nittel D., Lux R. and Shi W. J. Den 38, S1 S16-S20 (2010) This study was financially supported by the Department of Oral Biology, University of Malaya Grant (BM271) All Staff & student Oral biology Department