This material was originally published in the Purdue Cytometry CD-ROM Series

Flow Cytometry and Microbiology

Activity of common oral antiseptics against bacteria
assessed using the oxonol DIBAC4(3).

F. Paul, R. Jepras, D. Hynes, A.Smith and B. Marken
Analytical Sciences
SmithKline Beecham Pharmaceuticals
Brockham Park, Betchworth
Surrey RH3 7AJ, UK


The activity of triclosan (TRI), chlorohexidine (CHX) and cetylpyridinium chloride (CPC), commonly found in consumer healthcare products, against some strains of oral bacteria were compared. A flow cytometry technique using the membrane potential probe bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] was performed in parallel with conventional CFU assays. By monitoring the mean fluorescence of Streptococcus mutans, Streptococcus oralis and Streptococcus sanguis the activity of these compounds was demonstrated. Comparison of flow cytometry viable counts showed a close correlation with the corresponding CFU assays and on no occasion was an effect shown by CFU assay and not by flow cytometry. Results were obtained in a few minutes using flow cytometry whereas information from standard CFU assays took at least 24-48h. Flow cytometry gave information on the immediate effects of the compounds, that is, their activity in the first few minutes which is more appropriate for assessing the effectiveness of orally active compounds. The flow cytometry results indicated that the activity of the compounds can be determined in most instances within the first 2 minutes of treating the bacteria.

There is an obvious requirement for the active ingredients used in oral care product such as mouth wash and toothpaste to rapidly kill the bacteria that cause tooth decay and gum disease. This is mainly because of the short exposure times and rapid dilution of active ingredients by saliva. It follows, therefore, that methods that can quickly detect this early activity is highly appropriate and beneficial in evaluating new compounds.

In this study some common biocides were compared for their activity against oral bacteria using flow cytometry and standard CFU assays. They all show similarities in their activity by acting on the cytoplasmic membrane. Consequently the membrane potential sensitive oxonol DiBAC4(3) was used as an appropriate fluorescent viability probe(1).

Cetylpyridinium chloride (CPC) is a cationic quaternary ammonium compound and acts on the cell membrane resulting in a generalized loss in its function as a permeability barrier(2). Chlorhexidine (CHX) is a biguanide and causes the collapse of membrane potential via the disruption of the membrane rather than ATPase activity(3). Triclosan [5-chloro-2-(2,4-dichlrophenoxy phenol] (TRI) acts on the cytoplasmic membrane and is bacteriostatic at low concentrations interfering with the uptake of nutrients(4).

Materials and methods
Culture samples were removed from their growth medium by centrifugation and washed 3 times in 0.22 um filtered PBS. The concentration was adjusted to approximately 1x107 organisms/ml. DiBAC(4)3 (Molecular Probes. Inc.) was added to give a final concentration of 10 ug/ml. The biocides were added to give a 5 x MIC for each of the microorganisms (see figure legends for further details). Samples were removed at various times for CFU assays and flow cytometry studies using a Bryte HS (Bio-Rad) flow cytometer. Excitation and emission wavelengths were 470-490 nm and 520-550 nm respectively.

Results and discussion
The typical biocide effects on bacteria are shown in Fig.1. It represents the flow cytometry dot plot depicting DiBAC(4)3 fluorescence against forward angle light scattering from S. mutans before and after 15 mins treatment with CHX. There are two distinct populations of cells following treatment The denser of the two has a higher fluorescence intensity and represents the dead or dyeing cells. The dead cell population has a much broader light scattering distribution. Thus, indicating that the cell membrane or wall has been perturbed causing a loss in membrane potential and a change in cell morphology. Viable counts can be determined directly by placing regions of interest around the clusters of live cells.

Figure 1. DiBAC4(3) fluorescence (FL1) versus forward light scattering (LS1) dot plot of S. mutans before (top) and after (bottom) treatment with 9 mg/ml chlorohexidine for 15 min.

Figs. 2,3 and 4 show the effects increasing biocide concentration after 30s treatment on the mean fluorescence intensity of the microorganisms. There is very little difference in the effects of each compound at lower concentrations, however, at concentrations above 12.5 ug/ml CPC has the highest activity against all strains whereas TRI has the least. S. sanguis is the least affected of the strains tested by all the compounds.

Figure 2. Effects of triclosan, chlorohexidine and cetylpyridinium chloride on DiBAC4(3) fluorescence from S. mutans after 30s of treatment.

Figure 3. Effects of triclosan, chlorohexidine and cetylpyridininum chloride (CPC) on DiBAC4(3) fluorescence from S. oralis after 30s treatment.

Figure 4. Effects of triclosan, chlorohexidine and cetylpyridininum chloride on DiBAC4(3) fluorescence from S. sanguis after 30s treatment.

Flow cytometry used in combination with the membrane potential probe DiBAC4(3) offers several advantages over standard CFU assays for determining the effectiveness of antibacterial compounds used in oral care products. The immediate effects on bacteria can be detected, i.e., the actions within 30s of exposure which may be a more relevant indicator of efficacy. At the same time results are obtained the same day as the experiment. The acute effect of antibacterial compounds is difficult to obtain using conventional microbiological methods which involve, serial dilution's, plating and a 24-48h incubation period. The ability to obtain results quickly is important particularly when screening new compounds and during QA tests. The use of the Flow Cytometric assay will have particular usefulness during formulation experiments where a large range and combination of active materials needs to be tested. The effect of other constituents of oral care products could also be determined.

1) Jepras. R.I., Carter, J.H., Pearson, S.C., Paul, F.E. and Wilkinson, M.J. (1995). Development of a robust flow cytometric assay for determining viable numbers of bacteria, Applied and Environmental Microbiology, 61, 2696-2701.

2) Hugo,W.B. (1992). Disinfection mechanisms, In: (Russell, A.D., Hugo,W.B. and Ayliffe, G.A.J. eds.), Principles and Practice of Disinfection, Preservation and Sterilisation. 2nd edn, pp. 180-186. Oxford: Blackwell Scientific Publications.

3) Russell, A.D. (1995). Biocides: Activity, Action and Resistance. In ( Hunter, P.A., Darby, G.K. and Russell, N.J. eds). Fifty years of antimicrobials: Past Perspectives and Future Trends. pp. 327 - 365. Cambridge University Press.

4) Regos, J. (1974). Investigations on the mode of action triclosan, a broad spectrum antimicrobial. Zbl. Bakt. Hyg. I.Abt.Orig. A 226, 390-401.

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