Archs oral Biol. Vol. 41, No. 1, pp. 27- 34.

1996
Copyright ~ 1996 ElsevierScienceLtd. All rights reserved
Pergamon Printed in Great Britain
0003-9969(95)00103-4 0003-9969/96 $15.00 + 0.00

INHIBITION BY ETHANOL OF THE GROWTH OF

BIOFILM A N D DISPERSED MICROCOSM DENTAL
PLAQUES
C. H. SISSONS, L. W O N G and T. W. CUTRESS
Dental Research Unit (HRC), Wellington School of Medicine, P.O. Box 27007, Wellington, New Zealand

(Accepted 22 August 1995)

Summary--Inhibition of microcosm plaque biofilm growth by periodic application of ethanol was

compared with the minimum inhibitory concentration (MIC) and bactericidal effects of ethanol on liquid
cultures of dispersed plaque bacteria. Microcosm plaques were cultured from saliva in a multiplaque
"artificial mouth' and their growth in wet weight measured daily. Nutrient conditions included: a
continuous supply of a medium containing 0.25% mucin, and 8-hourly 5% (w/v) sucrose (1.5 ml over
6 min). Plaque biofilm growth was strongly inhibited by exposure to 40% (v/v) ethanol applied in volumes
of 3.75 ml over 15 min, six times daily. Application of 1.5 ml over 6 min inhibited much less or not at all.
Ethanol concentrations lower than 40% caused less inhibition, with 10% having almost no effect. The
pH response to sucrose was unchanged by prior application of 40% ethanol for 30 min. Some evidence
was obtained for either bacterial adaptation to ethanol or selection of ethanol-resistant bacteria. The MIC
and bactericidal effects of ethanol were assessed by growth of dispersed plaque in liquid culture; the
bactericidal effect was measured as the induced delay in growth. The aerobic and anaerobic M1C of
ethanol for growth was 10% and 8%; 50% inhibition of growth rate occurred at 3.7% and 2.8%. Ethanol
(40%) was bactericidal within I-2 min, but 10% had almost no effect. It was concluded that, despite the
well-known high ethanol sensitivity of dispersed plaque bacteria, prolonged application of ethanol
concentrations in the order of 40% are necessary to inhibit growth of plaque biofilms.

Key words: ethanol, plaque, in vitro, antiplaque agent, minimum inhibitory concentration, bactericidal
effect.

INTRODUCTION bacteria/n vitro, despite the high sensitivity of most

bacteria to ethanol. Oppermann and Gjermo (1980)
Ethanol at a concentration of 70% (w/v) is used also reported that direct application of 70% (w/v)
routinely as a disinfectant (Morton, 1950; Mertz ethanol for 2min to plaque in vivo reduced the
et aL, 1984; Spicher and Peters, 1991). Bacteria vary carbohydrate-induced pH decrease for 24 h, and 50%
in their sensitivity to ethanol but concentrations of ethanol reduced it slightly for 2 4 h.
5-10% are bacteriostatic and 15% is lethal to most Ethanol 70% is needed to kill plaque microflora
bacteria (Koshiro and Oie, 1984; Ingram and Buttke, smeared on glass slides (Nolte, 1982). Listerine is a
1984; Ingram, 1990). The primary target of ethanol is well-documented, effective oral mouthrinse: in ad-
the cell membrane (Ingram, 1990) and it induces the dition to 27% ethanol it contains thymol, menthol,
heat-shock response (Boutibonnes et aL, 1991; Piper eucalyptol, methyl salicylate, borate and benzoate
et al., 1994).
(Kornman, 1985; Mandel, 1988; Brecx et al., 1992;
Dental plaque is exposed to ethanol at concen- Ross et al., 1993). An in vivo study of 27% ethanol
trations of about 40% in persons who drink un- used twice daily as mouthrinse for 9 months showed
diluted spirits. Ethanol is also commonly present no evidence of reduced plaque growth or gingivitis
as a solvent and preservative in mouthrinses; for (Gordon, Lamster and Seiger, 1985).
example, Listerine (Warner-Lambert Co., Morris The apparent ineffectiveness of ethanol as an an-
Plains, N.J.), used since the late 19th century (Miller, tiplaque agent and inhibitor of plaque bacteria confl-
1890), contains 27% (v/v) ethanol. Ingested ethanol icts with the well-established high sensitivity of
exposes dental plaque to apparently bacteriostatic bacteria to ethanol and its use as an effective preser-
and bactericidal concentrations but there has been vative at 10-12%. One possible explanation is that
almost no study of the consequences for plaque bacterial biofilms have greater resistance than dis-
growth and metabolism. In the most detailed report persed bacteria (Anwar, Strap and Costerton, 1992;
of the effects of ethanol on plaque (Oppermann and ten Cate and Marsh, 1994). Another is the limited
Gjermo, 1980), a 5-min treatment with 35% but not relevance of using monocultures of selected species
17.5% ethanol inhibited growth of dispersed plaque for determining MIC and bactericidal effects. Because
the mouth is exposed to high concentrations of
Abbreviations: BMM, basal medium mucin; MIC, minimum ethanol, its effects on plaque and plaque bacteria
inhibitory concentration. need clarification.

27
28 C. H. Sissons et al.

We have developed a multiplaque 'artificial growth. Plaque wet weight was calculated by subtrac-
mouth' system and cultured microcosm dental tion of the coverslip tare weight. The coefficient of
plaques (Sissons et al., 1991, 1992). Microbial micro- variation for the weighing technique was less than
cosms are laboratory-evolved subsets of the natural 10% for plaques heavier than 30 mg wet wt. For
flora (Wimpenny, 1988). In vitro microcosm plaques growth of replicate plaques within the one exper-
are similar in structure, composition, growth rate and iment, the coefficient of variation was less than 30%
behaviour to natural plaques but are in a more above 250 mg and 20% above 600 mg (Sissons et at.,
consistent environment than the mouth and can be 1995).
better controlled and manipulated than plaques
in vivo (Sissons et al., 1991, 1992, 1994a, b; Wong, (A)
Sissons and Cutress, 1994). Plaque growth and
consequently the action of antiplaque agents can also 400
be studied in detail (Sissons, Wong and Cutress,
1995).
Our general objectives now were to establish an -- Cont.~ t
effective protocol for short applications of antiplaque
agents to microcosm plaque biofilms in vitro, and to 300 -

develop an analysis of the corresponding inhibition of

growth of the same mixed plaque flora dispersed in
homogeneous liquid culture. Our more specific aim
was to investigate ethanol effects on plaque in detail
by (i) examining inhibition of microcosm plaque
biofilm growth by periodic applications of ethanol,
200
including effects on the sucrose-induced pH response;
and (ii) measuring overall MIC and bactericidal
effects of ethanol on the growth of total dispersed
microcosm plaque in liquid culture.
1oo

MATERIALSANDMETHODS
Procedure for growth o f microcosm plaques
Microcosm plaques were cultured at 35°C in a o I
five-plaque 'artificial mouth' (Sissons et al., 1991,
0" 2 4 6 8 10
T. (B)
1992). Each plaque was initiated with a 5 ml portion
of stimulated whole saliva. Oral hygiene procedures
were omitted by the saliva donor for 24-30 h before 801-- Con~ol
the collection to increase the proportion of plaque
bacteria in the saliva. The plaque growth stations
were inoculated once only. A medium (BMM),
modified from Glenister et al. (1988), was supplied
continuously at approx. 3.6 ml/h ,to each plaque. 60
This contained: 0.5% trypticase peptone (BBL,
Cockeysville, MD), 1.0% proteose peptone (Oxoid
Unipath Ltd, Basingstoke, U.K.), 0.5% yeast extract
(Difco Laboratories, Detroit, MI), 0.25% KC1,
5mg/1 haemin, 1 mg/1 menadione, 0.25% partially 40
purified pig gastric mucin (Type III, Sigma Chemical
Co., St Louis, MO), 1 mmol/1 arginine and 1 mmol/1
urea. Every 8h, 1.5ml of 5% w/v sucrose was
supplied to each plaque over 6 min in the absence
of a simultaneous BMM flow. 5% CO2 in N 2 was 20
supplied for 1 min, four times daily. 40%
A
Measurement o f plaque growth by increase in wet
weight z x / A
Plaques were grown on plastic, 25-mm dia, Lux I I
Thermanox TMcoverslips on supports (Sissons et al., 1 2 3 4 5 6
1992). Growth was measured by weighing the whole Days
plaque daily and analysing the accumulating wet
weight. The BMM flow to the plaques was stopped Fig. 1. Inhibition of plaque biofilm growth by periodic
application of 40% (v/v) ethanol. The ethanol was applied
and the plaques angled in situ at about 40 ° for 15 min under the standard regimen. Control plaques, mean and SE
to drain excess fluid. The support, coverslip and ( 0 - - 0 ) ; ethanol-treated plaque ( A - - A ) . The experiments
plaque were then removed from the culture chamber, in (A) with three control plaques and in (B) with four
and the coverslip with plaque weighed to 0.1 mg and control plaques were inoculated with saliva from different
then replaced in the culture chamber for further persons to give different growth-rate plaques.
 Effects of ethanol on in vitro plaques 29

600 F
Periodic ethanol application to microcosm biofilm
(A) plaques

/
Control ,e
All ethanol concentrations used in this study were
500 -
% (v/v).
Effect of 40 % ethanol applied periodically on plaque
biofilm growth. Ethanol at 40% was applied to each
plaque in a standard regimen of 3.75 ml over 15 min,
400 six times a day, with the BMM flow stopped to avoid
dilution. Delivery was timed to end 0.5 h before and

///J
3.5 h after each 8-hourly sucrose delivery. In the first
experiment an ethanol-treated plaque was compared
300 to three control plaques, weighed twice daily during
the 9 days of growth. In a second experiment the
ethanol-treated plaque was similarly compared with
four control plaques.
200
Effect of varying the duration of 40% ethanol
applications. Application periods of 0, 6, 15 and
30 rain delivered 0, 1.5, 3.75 and 7.5 ml of ethanol,
100 respectively. These were timed to end 0.5 h before and
3.5 h after a sucrose delivery and applied six times
daily. The plaques were weighed daily for 8 days. In
a second experiment, plaque wet wt was measured
after 5 days of growth.
0 2 4 6 8 Effect of varying ethanol concentration. Ethanol
concentrations of 0, 10, 20, 30 and 40% were applied
o 100 D

(B) for 15 min (3.75 ml) using the standard regimen. In

t~
the first experiment, plaques were grown for 6 days
and the control plaque dispersed for MIC estimation.
The 10% ethanol-treated plaque was cultured for
75 m

another 2 days, then dispersed and used for exper-

iments on bactericidal effects.

50
200 10%

25

150

0 Control Control
I I Con~ol
6 min 15 rain 30 min
EtOH EtOH EtOH

Fig. 2. Effect of variation in the duration of application of i00

40% (v/v) ethanol. (A) Growth curves with ethanol appli-
cations for 0 (O--O), 6 (O--O), 15 ( A - - A ) and 30min
(O--O). (B) Effect of 6-, 15-, and 30-min 40% ethanol
applications on plaque growth for 5 days.
50 w/m-'-'--= 20%
30%
40%
Measurement of plaque pH
Plaque pH was continuously measured using a I I
micro-oesophageal glass pH electrode (1.6 mm dia; 2 4 6
Model 814, Diamond General Co., Ann Arbor, MI)
and a micro-reference electrode (Model 401, Days
Diamond General) (Sissons et al., 1992). The elec-
trodes were connected through a custom-built signal Fig, 3. Effect of ethanol concentration applied under the
standard regimen on the inhibition of plaque growth. Appli-
conditioner to a Macintosh Ilci computer running
cation of 0 (O--Q), I0 (O--O), 20 ( m - - n ) , 30 ( o - o )
LabVIEW software (National Instruments Corp., and 40 (A--A) % (v/v) ethanol. The control and 10%
Austin, TX) (Wong et al., 1994). The electrodes were ethanol-treated plaques were dispersed on day 5 and day 7,
calibrated against pH 7 and 4 buffers before insertion respectively, for the MIC experiment shown in Fig. 6 and
into, and after removal from, the plaque. bactericidal effect shown in Fig. 7, respectively.
30 C.H. Sissons et al.

Anaerobiosis was confirmed by using indicator tubes

I0%
500 of medium that also contained 0.0001% resazurin.
Triplicate cultures were grown under both air and
anaerobic gas phases in a shaking incubator
(180rev/min) at 35°C. The absorbance at 650nm
(A6S°), measured directly on the sealed culture
400
tubes with a spectrophotometer (Bausch and Lomb,
ontrol
Spectronic 20), was used to follow bacterial growth at
approx. 30-min intervals up to 8 h, and measured at
24 and 48 h of growth. The growth rate was measured
300
as the rate of increase of A 65° from a logarithmic plot
during exponential phase. The experiment was
6 min ~ 3 0 % repeated under aerobic conditions only and the
o
cultures incubated for 125 h.
200 _

Bactericidal effect of ethanol on dispersed plaque

bacteria
The bactericidal effects of 10, 20, 30 and 40%
100 ethanol were determined using a dispersed microcosm
(1% plaque cultured for 7 days with 10% ethanol applied
using the standard application regimen. The exper-
iment was repeated with 40% ethanol only using a
~ i i i I I I
2 4 6 8 10 12 14
plaque that had not been pre-exposed to ethanol
solutions. The plaque was dispersed by vortex mixing
Days with glass beads into 10 ml of BHY broth to give a
3% suspension. One ml of suspended cells was placed
Fig. 4. Effect of reducing the duration of different ethanol in BHY broth at the appropriate ethanol concen-
concentrations for I day during plaque growth. The plaques tration and incubated at 35°C. At pre-determined
were grown with 15-rain ethanol applications but this was times up to 150 rain, 200/~1 of cell/ethanol mixture
reduced to 6rain on day 5 of growth; 15-min ethanol was placed in fresh culture tubes containing 10ml
applications were then continued for another 7 days. Con- BHY broth, cultured aerobically at 35°C in a shaking
trol plaque with no ethanol (O--O); ethanol at 10 (O--O),
incubator and growth measured by A 65°. Control
30 (O--O) and 40 ( A - - A ) % (v/v).
cultures comprised an untreated plaque suspension of
diluted cells inoculated into BHY broth containing
Effect of a short-term reduction in duration of the ethanol concentration corresponding to the 50-
ethanol application. Ethanol at 0, 10, 20, 30 and 40% fold dilution of the ethanol-treated cells (e.g. 0.8%
was applied to a set of plaques. Initially, 15 min for the 40% ethanol treatment).
(3.75ml) of ethanol was applied until day 5 of The bactericidal effect was estimated as the delay
growth, then the duration of applications was re- in growth caused by exposure to ethanol, expressed
duced to 6 min (1.5 ml). On day 6, the 15-min regimen in units of doubling time (D,) using the equation:
was restored for 7 days.
Bactericidal
Effect of ethanol on the plaque p H response to
sucrose. The pH of two plaques was measured simul- effect = [D....... j × 100]/[Dtco,trol+ (tethanol- tcontro0]
taneously after, the application of 7.5 ml of 40% Where: t is the time for the A 65° to reach a value of
ethanol for 30 min (timed to finish 0.5 h before and 0.5, and (tetha,o~-- t~o,tro~) is the delay due to ethanol
3.5 h after a 6-rain application of 10% (w/v) sucrose). exposure. Cultures with no growth after 48 h were
regarded as showing 100% kill.
Measurement of the MIC of ethanol on dispersed
plaque bacteria
The MIC for ethanol was determined with a 5-day RESULTS
microcosm plaque not previously exposed to ethanol.
The whole plaque was dispersed into 28 ml of thiogly- Effects of ethanol on the growth of plaque biofilms
collate broth (Difco) by vortex mixing with glass 40% ethanol applied Jor 15rain six times daily.
beads to give a 0.5% (w/v) suspension. Volumes of Applications under the standard regimen strongly
suspended cells (100/tl) were removed to inoculate a inhibited plaque growth (Fig. IA and B). Plaques in
series of 15 x 160mm screw-capped culture tubes different experimental runs differed in growth rate,
containing 10ml BHY broth supplemented with especially when grown from the saliva of different
ethanol concentrations up to 20%. BHY contains persons, but the inhibition of growth by ethanol was
brain-heart infusion broth and 0.5% yeast extract similar.
(Difco), 5/~g/ml haemin and 0.5/~g/ml menadione. Duration of 40% ethanol applications. Doubling the
These procedures were done in an anaerobic hood duration of ethanol application to 30 min (7.5 ml)
(COY Laboratory Products, Grass Lake, MI) with a increased the growth inhibition (Fig. 2A); decreasing
gas phase of 10% CO2/10% H 2 in N 2. The broths it to 6min (1.5 ml) substantially reduced the inhi-
were made anaerobic by placing the tubes with bition. In another experiment (Fig. 2B), there was no
loosened caps in the anaerobic hood overnight. inhibition by 6-min ethanol after 5 days of growth.
 Effects of ethanol on in vitro plaques 31

Ethanol concentration. Plaque growth was increas- estimating the delay in growth (with 30% ethanol) is
ingly inhibited by ethanol at concentrations from 10 shown in Fig. 7(A).
to 40% (Fig. 3). At 10%, no effect was observed. Ethanol at 40% caused a virtually complete kill,
Short-term reduction in duration o f ethanol appli- within a few minutes, of bacteria from a plaque that
cation at different concentrations. Reducing ethanol had been grown exposed to periodic 10% ethanol
applications to 6 from 15 min during day 5 of growth applications (Fig. 7B). Ethanol at 10% had little
(Fig. 4) removed the growth inhibition at all concen- effect. Ethanol concentrations of 20 and 30% caused
trations. Reapplication of 15-min, 40% ethanol a rapid then slower rate of inactivation until a
stopped growth. However, the plaques treated with relatively resistant portion of the population re-
other ethanol concentrations were no longer inhib- mained. The bactericidal effects increased with
ited, giving growth rates similar to the control. ethanol concentration. In a further experiment with
Ethanol and sucrose-induced p H responses. Ethanol plaque grown in the absence of ethanol, 40% ethanol
40% applied for 30min affected the pH during caused a complete kill inside 30 s.
application but had no effect on the pH response to
10% sucrose applied 30 rain later (Fig. 5).
DISCUSSION

Sensitivity to ethanol o f dispersed plaque bacteria in Ethanol effects on microcosm plaque

culture
The MIC of ethanol for the aerobic and anaerobic
MIC. Mixed plaque bacteria cultured in BHY cultivable plaque flora of 10% or less is typical of
broth yielded typical batch-culture growth curves, ethanol-sensitive bacteria (Ingrain and Buttke, 1984;
exponential between absorbance values of about Ingram, 1990)• There was no sign of bacteria resistant
0.03~).7 with little difference between aerobic and to ethanol concentrations higher than 10%. Although
anaerobic growth (Fig. 6A). Growth rates were inhib- bacteriostatic, 10% ethanol was not significantly
ited by 50% in 2.8 and 3.7% ethanol under anaerobic bactericidal; higher concentrations were and 40%
and aerobic conditions, respectively (Fig. 6B). The ethanol was rapidly lethal• This is consistent with the
MIC after 48h was about 10% ethanol for the findings of Oppermann and Gjermo (1980), who
aerobically grown bacteria, and 8% ethanol for previously reported that 5-min treatment with 35%
anaerobically grown bacteria (Fig. 6C). Aerobic (but not 17.5%) ethanol completely killed plaque
culture for up to 125 h in a further experiment gave bacteria. The lack of effect on any aspect of the
the same result. sucrose-induced pH response by 40% ethanol pre-
Bactericidal effect o f different ethanol concen- treatment was also consistent with their finding of a
trations. The bactericidal effect estimated as the dur- slight effect with 50% ethanol.
ation of the delay in growth caused by ethanol was Much greater exposure to higher ethanol concen-
expressed in units of the exponential growth rate trations was needed to inhibit the plaque biofilm than
(l/D,) of the control cultures. The procedure for its component bacteria. For example, despite being

70t
6.5

6.0

5.5

5.0 : . .• ;

: . : . . :
• . ;. :.. '.
.o
4.5
I I I I
90 95 I00 105 110 115 120

Hours

Fig. 5. Effect of 40% ethanol on the plaque pH response to sucrose. Ethanol 40% (7.5 ml) was applied
over 30 min (l~) and sucrose (am) applied 30 min later. The pH of two plaques with different resting pH
values was measured simultaneously.
 32 C. H. Sissons et al.

1.000 _- (A) Or.

One possible mechanism for ethanol resistance is the
0.500
/ induction of the heat-shock adaptive response to
stress induced by ethanol concentrations above 4%
(Boutibonnes et al., 1991; Piper et al., 1994). Diffu-

/
o
sion limitations in 1-2 m m thick biofilms (Dibdin,
o.too 1990; Sissons et al., 1992, 1994b) may slow the rise in
e~
8 o.oso / ethanol concentration inside the biofilm sufficiently to
.8 allow the heat-shock response to occur.
<
Periodic 15-min exposure of plaque to 40% ethanol
was required to achieve growth inhibition and such
O.OIO
conditions probably do not often occur in vivo. The
0.005 I I I I I I I I 27% ethanol concentration of the original Listerine
2 4 6 8 I0 12 14 16 18 20 22 24 formulation is one of the highest concentrations in
Hours mouthrinses but had no effect when regularly used
(B) over 9 months in a clinical study of plaque growth
100
(Gordon et al., 1985). Indeed, increased plaque
growth in vivo was reported after short-term rinsing
80 with 50% ethanol, twice a day for 4 days (Gjermo
et al., 1970). Some suggestion of ethanol-induced
~ 60 resistance or adaptation was observed here (Fig. 4)
e~
O and if either occurs, increased plaque accumulation
o 40

20 - (A)

Ethanol o.~o - o - o -
2I 4 6 8 l0 12 14 16 1.o - 30% ore__/
: /3
t35.C//

1.2 (C)
20

0.8 1 -~

~ 0.I
~ 0.6

< o.4 0.05 I I I

l 2 3 4 5 8 9 to
0.2 Hours

2 4 6 8 I0 12 14 16
I
Ethanol (%) 100',~ID-~--'--.--..-.______O__.__.___..___..~IO%
Fig. 6. MIC of ethanol on growth in culture of dispersed
microcosm plaque bacteria. (A) Logarithmic plot of growth ~, 50'
of dispersed plaque bacteria in BHY broth, measured as A 65°
I
in aerobic ( O - - O ) and anaerobic conditions ( O - - O ) . (B)
Effect of ethanol concentration (% v/v) on the rate of '-" I0 20%
change in A 65° for aerobic ( O - - O ) and anaerobic ( O - - O ) +
growth expressed as a percentage of the control rate. (C) ~ 5'.
A 650 after 8 h of aerobic ( 0 - - 0 ) and anaerobic ( ~ - - ~ ) 30%
growth, and 48 h of aerobic ( O - - O ) and anaerobic growth
(o-o). 40%
I I I I I I I
20 40 60 80 100 120 140

lethal to dispersed plaque bacteria, 40% ethanol Time of ethanol treatment (min)
applied over 6 m i n had little impact on biofilm
growth. Further, although 10% ethanol was bacterio- Fig. 7. Bactericidal effect of ethanol on dispersed microcosm
static, applied over 15 min it either had no effect on plaque bacteria. (A) Illustration of delay in growth induced
plaque biofiim growth or stimulated it. Hence, plaque by exposure to 30% (v/v) ethanol for 0 ( O - - O ) , 35
bacteria in a biofilm are much more resistant to, or (©--©), 48 ( A - - / ~ ) and 120 ([S]--I-q) s. The time to reach
an A 65° of 0.5 is shown as t,, where n is seconds of ethanol
protected from, ethanol than the same bacteria when treatment. The delay in growth is t , - t ~ . (B) Bactericidal
dispersed. Bacteria in biofilms may physiologically effect {% Dt/[D,+ (tetha.oI --to)]} of treatment at 35°C for
adapt and become more resistant to stress, including different times with ethanol at 10 ( O - - O ) , 20 ( A - - A ) , 30
that from antimicrobial agents (Anwar et al., 1992). ( O - - O ) , and 40 ( 0 - - 0 ) % (v/v).
 Effects of ethanol on in vitro plaques 33

could result from regular use of ethanol-containing bactericidal effect. Therefore, rate changes apparent
products. within the inactivation curves obtained (Fig. 6B)
could include both heterogeneity in resistance and
Testing o f antiplaque agents by periodic application to effects on lag phases.
microcosm plaque biofilms The determination of overall MICs and bacteri-
A major purpose of this study was to establish a cidal effects on dispersed microcosm plaques has the
regimen for testing periodically applied antiplaque advantages of using a flora with a direct relation to
agents on microcosm plaque biofilms. The adopted the flora of plaque in vivo (Sissons et al., 1991, 1992,
regimen combined periodic, short-term exposure of 1994a, b, 1995) and of producing large amounts of
plaque with probable saturation by excess reagent, replicable material; it therefore provides a useful
allowed experiments on variation in duration and supplement to studies of individual organisms.
concentration of reagent, and on the interaction with
carbohydrate-induced pH changes. Microcosm bio-
film plaques, which evolve from the whole mixed oral Acknowledgement~We thank L'Or6al Laboratoires de
flora, have a high species diversity--indicated by the Recherche Appliqu6e et D6veloppement for partial support.
typical plaque flora present (Sissons et al., 1991), the
regular finding of motile organisms after 2-5 weeks of
culture, and the observation of spirochaetes in a
REFERENCES
2-week-old plaque (C. H. Sissons, unpublished). They
provide a powerful system for examining plaque Anwar H., Strap J. L. and Costerton W. (1992) Establish-
susceptibility to antiplaque agents. ment of aging biofilms: possible mechanism of bacterial
resistance to antimicrobial therapy. Antimicroh. Agents
M I C and bacterial tests by culture o f the dispersed Chemother. 36, 1347-1351.
microcosm plaque flora Boutibonnes P., Gillot B., Auffray Y. and Thammavongs
In the evaluation of antiplaque agents, MIC and B. (1991) Heat shock induces thermotolerance and
inhibition of lysis in a lysogenic strain of Lactococcus
bactericidal tests are usually done on specific strains lactis. Int. J. food Microbiol. 14, 1 10.
of organisms to define their physiological response to Brecx M., Brownstone E., MacDonald L., Gelskey S. and
an inhibitor. Extrapolating results from a few species Cheang M. (1992) Efficiency of Listerine, Meridol and
to the 300 or more in dental plaque (Tanner et al., chlorhexidine mouthrinses as supplements to regular
1994) has limited validity. Using cultures of dispersed tooth-cleaning measures. J. clin. Periodont. 19, 202 207.
microcosm plaque flora bypasses these difficulties, Dibdin G. H. (1990)Plaque fluid and diffusion: study of the
but with limitations. cariogenic challenge by computer modelling. J. dent. Res.
Growth conditions and inhibitory interactions 69, 1324-1331.
among bacteria inevitably prevent the growth of Gjermo P., Lyche K., Baastad K. L. and Rolla G. (1970)
The plaque-inhibiting capacity of 11 antibacterial com-
some. These could include bacteria with high ethanol
pounds. J. perio. Res. 5, 102 109.
resistance (Ingrain and Buttke, 1984; Ingram, 1990) Glenister D. A., Salamon K. E., Smith K., Beighton D. and
but this seems unlikely given the high sensitivity of Keevil C. W. (1988) Enhanced growth of complex
the flora that was cultivable. The dose-response communities of dental plaque bacteria in mucin-limited
curves in mixed-flora cultures are different from those continuous culture. Microbial Ecol. Hlth Dis. l, 31 38.
in cultures of cloned single species. With a single Gordon J. M., Lamster I. B. and Sieger M. C. (1985)
organism, an increasing concentration causes an in- Efficacy of Listerine antiseptic in inhibiting the develop-
creasing physiological response. In mixed-flora cul- ment of plaque and gingivitis. J. clin. Periodont. 12,
tures, there will be a range of bacterial sensitivities 697 704.
Ingram L. O. and Buttke T. M. (1984) Effects of alcohols
and growth rates, and hence an increasing proportion on micro-organisms. Adv. micro. Phys. 25, 253 300.
of the bacteria will respond to increasing inhibitor. lngram L. O. (1990) Ethanol tolerance in bacteria. Crit. Rev.
Nevertheless, we found that the inhibition of growth Biotechnol. 9, 305 319.
rate followed the pattern expected for a single organ- Kornman K. S. (1985) Antimicrobial Agents. State-of-the-
ism, suggesting similar ethanol sensitivities and science review. In Dental Plaque Control Measures and
growth rates for the flora cultivable under the con- Oral Hygiene Practices (Eds Loe H. and Kleinman D.),
ditions used. Further developments might include pp. 121--142. IRL Press, Oxford.
identification of this flora. Koshiro A. and Oie S. (1984) Bactericidal activity of ethanol
The bactericidal effect was estimated indirectly against glucose nonfermentative Gram-negative bacilli.
Microbios. 40, 33-40.
from the duration of delay in growth. The A 65°
Mandel 1. D. (1988) Chemotherapeutic agents for con-
doubling time (D,) of the culture approximates to a trolling plaque and gingivitis. J. olin. Periodont. 15,
doubling of cell biomass; hence a delay in exponential 488-498.
growth of one D, compared to the control is equival- Mertz P. M., Alvarez O. M., Smerbeck R. V. and Eaglstein
ent to a halving of the original cells present and, W. H. (1984) A new in vivo model for the evaluation
therefore, a 50% kill. This assumes that there was no of topical antiseptics on superficial wounds. The effect
induced lag phase or change in growth rate. In of 70% alcohol and povidone-iodine solution. Arch.
practice, the observed growth rate was not greatly Dermatol. 120, 58~52.
affected by increased time of exposure to ethanol Miller W. D. (1890) The Micro-Organisms of the Human
Mouth. The SS White Dental Mfg. Co, Philadelphia.
(Fig. 6A), While the use of absorbance rather than (Unaltered reprint with an introductory essay. K. G.
cultivable units to measure growth has the advantage Konig (1973). S. Karger, Basel.)
of speed and simplicity, it does not allow for the Morton H. E. (1950) The relationship of concentration and
detection of any reagent-induced lag phase, which is germicidal efficiency of ethyl alcohol. Ann. N.Y. Acad.
therefore an unknown component of the calculated Sci. 53, 191-196.
34 C . H . Sissons et al.

Nolte W. A. (1982) Sterilization and disinfection. In Oral Sissons C. H., Wong L., Hancock E. M. and Cutress T. W.
Microbiol (Ed. Nolte W. A.), 4th edn, pp. 55-87. CV (1994b) pH gradients induced by urea metabolism in
Mosby, St Louis, MO, 'artificial mouth' microcosm plaques. Archs oral Biol. 39,
Oppermann R. V. and Gjermo P. (1980) In vivo effect of four 507 511.
antibacterial agents upon the acidogenicity of dental Sissons C. H., Wong L. and Cutress T. W. (1995) Patterns
plaque. Scand. J. dent. Res. 88, 34-39. and rates of growth of microcosm dental plaque biofilms.
Piper P. W., Talreja K., Panaretou B., Moradas-Ferreira P., Oral Microbiol. lmmunol. 10, 160-167.
Byrne K., Praekelt U. M., Meacock P., R6nacq M. and Spicher G. and Peters J. (1991) Efficacy of formaldehyde,
Boucherie H. (1994) Induction of major heat-shock glutardialdehyde, peracetic acid, chloramine T (N-chloro-
proteins of Saccharomyces cerevisiae, including plasma 4-toluenesulphonamide), m-cresol, ethanol, and ben-
membrane Hsp30, by ethanol levels above a critical zyldimetbyldodecylammoniumbromide on bacteria in
threshold. Microbiology 140, 3031-3038. coagulated blood (Model Experiments for Chemical
Ross N. M., Mankodi S. M., Mostler K. L., Charles C. H. Disinfection of Instruments). Zbl. Hyg. 191, 457~477.
and Bartels L. L. (1993) Effect of rinsing time on Tanner A., Maiden M. F. J., Paster B. J. and Dewhirst F. E.
antiplaque-antigingivitis efficacy of Listerine. J. clin. (1994) The impact of 16S ribosomal RNA-based phy-
Periodont. 20, 279-281. logeny on the taxonomy of oral bacteria. Periodont. 2000
Sissons C. H., Cutress T. W., Hoffman M. P. and Wakefield 5, 26-5 I.
J. St. J. (1991) A multi-station dental plaque microcosm ten Cate J. M. and Marsh P. D. (1994) Procedures for
(artificial mouth) for the study of plaque growth, establishing efficacy of antimicrobial agents for
metabolism, pH and mineralization. J. dent. Res. 70, chemotherapeutic caries prevention. J. dent. Res. 73,
1409-1416. 695 703,
Sissons C. H., Cutress T. W., Faulds G. and Wong L. (1992) Wimpenny J. W. T. (1988) Introduction. In CRC Handbook
pH responses to sucrose and the formation of pH gradi- of Laboratory Model Systems for Microbiol Ecosystems
ents in thick 'artificial mouth' microcosm plaques. Archs (Ed. Wimpenny J. W. T.), Vol. I, pp. I 17. CRC Press,
oral Biol. 37, 913422. Boca Raton, FL.
Sissons C. H., Wong L., Hancock E. M. and Cutress T. W. Wong L., Sissons C. H. and Cutress T. W. (1994) Control
(1994a) The pH response to urea and the effect of liquid of a multiple dental plaque culture system and long-term,
flow in 'artificial mouth' microcosm plaques. Archs oral continuous, plaque pH measurement using LabVIEW,
Biol. 39, 497 505. Binary 6, 173-180.