Transcript The comparison of the antibacterial effect of various

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).
CULTURE
PREPARATIONS
SUSCEPTIBILITY
TESTS (AST)
MINIMAL
BACTERICIDAL
CONCENTRATION
DETERMINATIONS
(MBC)
MINIMAL INHIBITORY
CONCENTRATION TEST
(MIC)
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
0.144
Take 100ul suspension and
& place on the BHI agar
Mixed 3 suspension
of bacteria.
Using hockey stick spread
the suspension using lawn
method
Bacteria ready to
use
Incubated 24 hrs
at 37°C

The mouth rinses were tested for sensitivity using KirbyBauer susceptibility test with principle of agar disc
diffusion.
Let it
dry at
RT
Prepare disk containing mouth
rinses using whatman sterile
paper disk size 6mm
Let it completely
dry & ready to use
Pipette 20ul mouth
rinse into the disk
Repeat procedure
2 & 3 until total
solution diffused in
a disk 100ul
Fresh bacterial
suspension
Using sterile cotton
swab culture the
bacteria on BHI agar
Inubated
24hrs at
37 oC
Measure & record
the results
2
3
1
+
-
Place the disk
on the agar
using sterile
forcep

Using 96 wells broth micro-dilution assay was
used to determine the minimal inhibition
concentrations (MIC).
Mouth rinse
(500 ul)
Discard
Mouth
Rinse
(X)
1
2
3
4
5
6
Mixed bacteria
suspension
Incubated 37ºc (24 hour)
• The first tube with no bacteria growth (no
turbid suspension) is the dilution containing the
concentration of the mouth rinse which is
refered MIC .

The Minimum Bactericidal Concentration
(MBC) is the lowest concentration of antibiotic
required to kill the germ.
Tube 1
Control ve
Tube 2
Tube 3
Tube 4
Control
+ve
MBC Techniques
Incubated
37º c
( 24 Hour)
Flow chart MIC & MBC
AST
Disc
Inhibition Zones
(cm)
0.12 % CHX
2.73 cm
0.05 % CPC
2.27 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.

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)
MIC & MBC

The MICs and MBCs for CHX and CPC were
determined as low as 0.00375% w/v (tube 3)
and 0.0125% 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).
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: 201-206 (2003).
A.R. Fathilah, Y. Othman and Z.H.A Rahim. J. Oral Sci., 48 (2): 71-75 (2006).
A.R. Fathilah, Z.H.A. Rahim, Y. Othman and M. Yusoff. Pak. J. Biol. Sci., 12(6):
518-521 (2009).
T. Nalina and Z.H.A. Rahim. Pak. J. Biol. Sci., 9: 1470-1475 (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