Journal of Dental Research http://jdr.sagepub.

com/

Mechanism of Action of an Antiseptic, Anti-odor Mouthwash

G. Pitts, C. Brogdon, L. Hu, T. Masurat, R. Pianotti and P. Schumann J DENT RES 1983 62: 738 DOI: 10.1177/00220345830620061001 The online version of this article can be found at: http://jdr.sagepub.com/content/62/6/738

Published by:
http://www.sagepublications.com

On behalf of:
International and American Associations for Dental Research

Additional services and information for Journal of Dental Research can be found at: Email Alerts: http://jdr.sagepub.com/cgi/alerts Subscriptions: http://jdr.sagepub.com/subscriptions Reprints: http://www.sagepub.com/journalsReprints.nav Permissions: http://www.sagepub.com/journalsPermissions.nav Citations: http://jdr.sagepub.com/content/62/6/738.refs.html

Downloaded from jdr.sagepub.com by guest on August 7, 2011 For personal use only. No other uses without permission.

Mechanism of Action of an Antiseptic, Anti-odor Mouthwash

G. PITT$, C. BROGDON, L. HU,* T. MASURAT, R. PIANOTTI, and P. SCHUMANN**
Consumer Products Group, Warner-Lambert Company, Morris Plains, New Jersey 0 7950

Inter-related determinants of oral malodor were measured over a three-hour period in 30 human subjects after mouthwash treatments. Re-odoration was important to mouthwash activity for 30 min. At
post-treatment times of 60-180 min, the anti-odor activity of the product is due solely to its anti-microbial action.

J Dent Res 62(6):738-742, June 1983

Introduction.
Most oral malodor originates in the mouth.1'2 The substances which are principally responsible for oral malodor are methanethiol, hydrogen sulfide, and related volatile sulfur compounds3-16 which are produced in the mouth primarily by gram-negative anaerobes such as Fusobacterium spp. and Bacteroides spp.17-19 acting on available substrates. A mouthwash which is cidal to odorigenic micro-organisms, such as the one studied here,20-26 could be expected to depress oral malodor through an anti-microbial mechanism of action. However, the subject mouthwash contains ingredients which are both anti-bacterial and capable of masking malodors. Thus, in order to determine the relative contributions of masking and antisepsis to overall anti-odor effectiveness of the product, it was necessary to simultaneously measure the odor of the mouth, the concentrations of malodorous microbial metabolites in mouth air, and populations of oral odorigenic micro-organisms in each subject while holding other factors constant.

Materials and methods.

Test materials and controls. -The three formulations which were compared were "Mouthwash" (a commercial product),*** "Placebo" (Mouthwash without the essential oils which give the product its anti-microbial and odoxr masking activities), and "Water" (distilled water, pH unadjusted). The compositions of these materials are given in Table 1. Both Placebo and Water controls were included in order to clearly distinguish the effects of the essential oils from those of the solvent, alcohol. It was found, in two preliminary in vivo studies, that the sialogogic activities of Mouthwash and Placebo were similar to but greater than that of water. However, the sialogogic activity of neither Mouthwash nor Placebo was significant for more than five min after treatment. One of these studies was carried out by volumetric quantitation of post-rinse expectorates, and the other used "Curby Cups" to follow parotid salivary flow for up to 30 min after rinsing. Also, through spectrophotometric determination of erythrosin-labeled intrinsic oral debris, it was found that all three treatments were approximately equal in oral detergency. Thus, neither debris removal nor stimulation of salivary flow should be considered to be variables in the study. Received for publication May 17, 1982

Accepted for publication December 29, 1982 *Present address: 226 East Hunting Ridge Road, Stamford, CT 06903 **Present address: 24 Essex Drive, Mendham, NJ 07945 ***Listerine Antiseptic, Warner-Lambert Co., Morris Plains, NJ 07950 Downloaded from jdr.sagepub.com by guest on August 7, 2011 For personal use only. No other uses without permission. 738

Design of the study.- Al subjects were adult volunteers who had no obvious oral pathology. Each subject received Mouthwash, Placebo, and Water in random order on different test days. The subjects were not informed as to which treatment they received on any given day. Subjects reported to the laboratory between 7:00 and 8:00 a.m. on their assigned test days, having abstained from any eating, drinking, or oral hygiene since arising. They had also abstained from eating garlic- or onion-containing foods on the evening prior to the test day, from wearing perfume or cologne on the morning of the test day, and from smoking for one hr prior to the start of the test. Subjects were screened for methanethiol in the mouth air, and those with less than 30 ppb of this odorant were rejected for that test session. Qualified individuals were then subjected to pre-treatment sampling for all test parameters, immediately after which they were given the treatment assigned for that day. They rinsed, under supervision, with 20 ml of treatment for 30 s and expectorated. They were then sampled according to the schedule given in Table 2. Methods of assay. -A. Populations of odorigenic microorganisms. Five samples of tongue odorigens and seven samples of crevicular odorigens from each subject at each sampling time were drawn and analyzed by previously described methods.24 Tongue samples were taken by the use of sterile toothbrushes, and gingival crevices were sampled with sterile, endodontic paper points. Relative quantitation of odorigens was obtained by dilution to extinction in a specific indicator medium. The identities of treatments used by subjects were not known to the microbiologists at either sampling or tube evaluation times. Recent work in our laboratories26 has shown that this method provides results essentially identical to platecounting. However, since the plate-counting technique is tedious and slow, dilution to extinction was employed in the present work. B. Oral malodor. A direct nose-to-mouth, organoleptic technique was used. Five judges independently evaluated the hedonic quality of each oral odor sample using a verbal scale anchored with nine category descriptors: extremely pleasant, very pleasant, pleasant, slightly pleasant, neutral, slightly unpleasant, unpleasant, very unpleasant, and extremely unpleasant. The verbal scale judgments were converted by the investigators to numerical scores (extremely pleasant = 1 . . . neutral = 5 . . extremely unpleasant = 9). The judges were selected from a pool having approximately equal distribution between sexes and technical/ non-technical job functions. All were experienced insofar as they had participated in prior studies which involved odor intensity and hedonic evaluations; however, they received no special training. Judges were required to refrain from: (1) wearing cologne, aftershave, or perfume on the test day, (2) smoking for five min before evaluations, and (3) chewing gum, using a mouthwash, eating, or drinking for five min before evaluations. Judges were not informed as to the identity of the mouthwashes under test; blanks (extra donors who had been given no treatment) were randomly interspersed with subjects and were presented to the judges as a check on their reliability.

Vol. 62 No. 6

MOUTHWASH MECHANISM

739

An evaluation booth was used to maintain judge and donor anonymity and to allow the study to be conducted in a double-blind manner. The booth was constructed of plywood and cloth. It was painted with high gloss enamel to eliminate odor interference from the wood and to minimize subsequent odor retention. Cloth panels were used to isolate the judges during the evaluations. Prior to use, the cloth was washed, the paint was thoroughly dried, and the entire apparatus was judged to be odor-free. The booth was located in an environmentally-controlled, wellventilated room during the study. Glass tubes, 35 mm x 60 mm (one per subject per sampling time), were used to permit the judges to make a direct evaluation of subject mouth air without physical contact. Tubes were washed and dried after each use to eliminate odor carry-over between measurements. A glass tube was placed through the hole (sniffing port) in the partition, and the donor placed his mouth over the tube, held his breath and signaled the judge when ready. The judge placed his nose to the glass tube, sniffed, and recorded his hedonic judgment for each subject on individual evaluation forms provided by the investigators. C. Volatile sulfur compounds in mouth air. 1. Instrumentation. A gas chromatographt equipped with a flame photometric detector was used. A low intensity hydrogen flame in the detector was produced by an air/hydrogen gas stream consisting of 50 ml/min hydrogent and 40 ml/min air.t Oxygen was omitted because it produces an intense flame with a resulting high background noise level which interferes with the detection of low levels of volatile sulfur compounds. Two additional modifications were made in order to increase the sensitivity of the flame photometric detector and to decrease baseline noise. The 392-nm filter in the photomultiplier assembly was replaced with a wide bandpass filter; this decreased the sulfur specificity slightly, but substantially increased the overall sensitivity of the detection system. A multiple-stage electronic filter was interfaced between the electrometer output of the gas chromatograph and the integrator/recorder; a cutoff frequency of 0.01 Hz was used to block the high-frequency noise components of the photomultiplier. The filter provided a smooth baseline with minimal reduction of the peak signal. The attenuation of the electrometer was set at 64 x 10-10 amps full scale/mv. The electrometer signal was fed into an integrating computer11 for data processing. The volatile sulfur compounds were separated on a 36 foot x 1/8 inch fluorinated ethylene propylene column packed with 5% polyphenyl ether and 0.05% phosphoric acid on a 40/60 mesh absorbant.O Air' at a flow rate of 60 ml/min was used as the carrier gas. The column was kept isothermal at 50C, and the sampling oven containing the sampling valve and sampling loop was kept isothermal at 400C. 2. Calibration. Known quantities of methanethiol were obtained from a gravimetrically calibrated permeation tube' as described by O'Keefe and Ortman.27 The permeation tube was placed in a temperature-controlled calibration
tTracor Model 550, Tracor, Inc., Austin, TX 78721 tZero-Hydrogen( and Zero-Air, Liquid Carbonic Corporation, Chicago, IL 60603 Model 921, Spectrum Scientific Corp., Newark, DE 19711 //Sigma 10 Chromatography Data Station, Perkin-Elmer Corp., Norwalk, CT 06856 Chromosorb T, Supelco, Inc., Bellefonte, PA 16823 Zero-Air, Liquid Carbonic Corporation, Chicago, IL 60603 *AID, Inc., Avondale, PA 19311

assembly with flow rates set to provide precise methanethiol levels ranging from 30 to 600 parts per billion (vol/vol). Hydrogen sulfide concentrations were estimated from methanethiol standards using the relationship:

[H2S]

mol [CH3SHJ mol wtwt H2S CH3SH

3. Mouth-air sampling procedure. A constant volume of mouth air was injected into the gas chromatograph by means of a gas sampling valve fitted with Teflon inlet and outlet tubing and calibrated sampling loops. An interchangeable two-inch Teflon probe attached to the inlet portion of the tube was placed directly into the subject's mouth, and, using a 20-ml gas syringe attached to the end of the outlet tube, approximately 20 ml of mouth air were slowly aspirated through the sampling loop. The contents of the sampling loop (10.0 ml) were then injected onto the column. The subjects kept their mouths closed and breathed normally through the nose for one min prior to sampling. Duplicate mouth air samples were taken at each of the sampling periods, and data were averaged. The operator was not informed as to the identity of the treatment used by the subject.

Results.
Chemical, microbiological, and psychophysical observations of the effects of an antiseptic mouthwash on 30 human subjects are reported. Figs. 1-5 compare the treatment means of observations in each parameter. The effect of Mouthwash was to significantly depress (p < 0.05) the level of each of these determinants of oral malodor at all post-treatment sampling times. At all but one post-treatment sampling time (Mouthwash vs. Placebo, methylmercaptan level at 30 min, p = 0.066) the effect of Mouthwash was significantly greater (p q 0.05) than that of Water and Placebo. Two-tailed t tests were used for all probability estimates. A digest of the data is given in Table 3.
TABLE 1 COMPOSITION OF TREATMENTS

Per 1000 ml Per 1000 ml Ingredient Mouthwash Placebo Ethanol 4.78 mol 4.78 mol Benzoic Acid 12.3 mmol 12.3 mmol Essential Oils# 16.4 mmol 0 mmol Caramel, Acid-proof 231.4 mg 231.4 mg Non-ionic Surfactant* 1.0 g 1.0 g Hydrochloric Acid or Sodium Hydroxide Adjust pH 4.2+0.1 Adjust pH 4.20.1 Water, Potable Adjust to volume Adjust to volume #Thymol, Eucalyptol, Menthol, and Methyl Salicylate *Pluronic F-127, BASF Wyandotte, Wyandotte, MI 48192
TABLE 2

SAMPLING SCHEDULE
Minutes After Treatment (Nominal Sampling Period) (1) (2) (3) (4) (5) 15 45 75 105 135 15 45 75 105 135 20 50 80 110 140 20 50 80 110 140 30 60 90 120 150

Parameter (Pre) Crevicular Odorigens Pre Pre Tongue Odorigens Methanethiol Pre Hydrogen Sulfide Pre Oral Odor Pre

(6) 165 165 170 170 180

Downloaded from jdr.sagepub.com by guest on August 7, 2011 For personal use only. No other uses without permission.

740

PITTS ETAL.

JDent Res June 1983

r:t
D
0

E .2
70
m
0
11

* MOUTHWASH

0I
m

0
"I
I

* PLACEBO -- WATER

2
n

0C

(5
z

t
r_ m

:E
Post Treatment

Time (Minutes)

50

80

110

i4O

170

Fig. 1 - Effect of treatment on oral malodor; mouthwash (*), placebo (I), water (o).

Elaposed

Time After Treatment Minutes

Fig. 4 - Effect of treatment on volatile sulfur odorants; mouthwash (*), placebo (a), water (o).
c

.2
co

CL

a,I

IL
c

0
m

0ru

u4,
v

0r
uQ

t0
x

* MOUTHWASH

*PLACEBO
:-WATER

135

165

Elapsed Time After Treatment (Minutes'

Elapsed Time After Treatmnent (Minutes'

Effect of treatment on crevicular odorigenic bacteria; mouthwash (o*j, placebo (@), water (o).

Fig. 2

Fig. 5 - Effect of treatment on volatile sulfur odorants; mouthwash (*), placebo (), water (o).

Ell----.-----------

4
ID

45

75

105

Elapsed Time After Treatment(Minutes)

Fig. 3 - Effect of treatment on lingual odorigenic bacteria; mouthwash (*), placebo (-), water (E).

Discussion.
The fact that the subject mouthwash kills substantial numbers of odorigenic micro-organisms has been amply demonstrated through prior research.20-25 These decreases in odorigenic bacteria, which occur after mouthrinsing, have been shown to be well-correlated with decreases in oral malodor22-24 and oral volatile sulfur malodorants.23

However, it has been pointed out24 that the definitive experiment to determine the relative contributions of this product's anti-microbial activity and its odor-masking activity to its overall anti-odor effect must entail simultaneous determination of odorigenic bacteria, oral malodor, and oral malodorants in each subject. These previously established criteria for separating the masking and anti-microbial activities of this product have been met in the study reported herein. Upon plotting logarithms of bacterial populations against oral malodor (Fig. 6) and concentrations of odorous bacterial metabolites against oral malodor (Fig. 7), it can clearly be seen that there is, within limits reasonably anticipated in in vivo experiments, a direct relation between odor and bacteria and between odor and odorous bacterial metabolites for all treatments at all times except for Mouthwash at 30 min. The perceived odor at the Mouthwash 30minute observation (Fig. 6 coordinates 2.4 x 5.5, Tongue and 1.7 x 5.5, Crevicular; Fig. 7 coordinates 115 ppb x 5.5, H2S and 82 ppb x 5.5, MSH) is much more pleasant than should be predicted from concentrations of odorants present, thus indicating that masking is an important influence up to 30 min, but not at or beyond 60 min after using the mouthwash. Conversely, the observation that perceived malodor bears essentially the same relation to odorigenic bacteria (Fig. 6) and their malodorous metab-

Downloaded from jdr.sagepub.com by guest on August 7, 2011 For personal use only. No other uses without permission.

Vol. 62 No. 6

MOUTHWASH MECHANISM
TABLE 3 DIGEST OF DATA

741

OBS TIME TRT CREV TONG 1 0 3.12423 3.77279 H2O 2 0 LIS 3.37707 3.73224 3 0 PLA 3.62454 3.92262 4 1 3.15459 3.79853 H20 5 1 LIS 2.05112 2.59953 6 1 PLA 2.86383 3.61221 7 2 3.19886 3.82302 H20 8 2 LIS 2.14514 2.91151 9 2 PLA 3.20445 3.71483 10 3 3.20370 3.99133 H20 11 3 LIS 2.31640 2.89161 12 3 PLA 2.90789 3.62856 13 4 3.18139 3.97000 H20 14 4 LIS 2.38331 3.08100 15 4 PLA 2.92535 3.78524 16 5 3.02531 3.81981 H20 17 5 LIS 2.34133 3.04289 18 5 PLA 3.04101 3.80355 19 6 3.23672 4.09658 H20 20 6 LIS 2.49500 3.11312 21 6 PLA 3.01373 4.06029 OBS = Observation number. TIME = Nominal sampling time (see Table 2). TRT = Treatment identity. CREV = Crevicular bacterial population, mean loglo reciprocal dilution. TONG = Tongue bacterial population, mean log reciprocal dilution. HED = Hedonic Score. H2S Concentration of hydrogen sulfide in mouth air, parts-per-billion (v/v). MSH Concentration of Methanethiol in mouth air, parts-per-billion (v/v).

HED 6.75167 6.73833 6.57667 6.64500 5.52000 6.47333 6.67222 6.29167 6.72333 6.68944 6.22000 6.70517 6.68667 6.29167 6.73333 6.80667 6.48000 6.75833 6.65167 6.34500 6.67500

H2S
164.490 158.704 171.347 155.644 111.014 151.815 175.848 110.483 147.483 180.085 105.919 166.050 178.448

MSH
134.973 152.165 160.128 111.792 83.462 121.787 139.740 84.533 123.970 141.483 83.980 139.738 139.105 88.546 160.083 156.179 77.455 140.647 148.942 93.682 150.452

116.641
177.117 174.632 100.690 172.807 171.758 119.828 177.498

c
0

-4.0
0

_ 2.8
i.

.
.
0
o 00

_3.8 .2
._

CL
._

2.6- _3.6
0

CC
0

2.5-

W4
0

_3.4
0

o 0
0

-1

23

_3.2

U
0
vn

'Z 2.2_ _3.0

0

c
0

ua

2.1_

_2.8 "

j
._

2.01.9

O
0

*Mouthwash,Crevicular

_2.6 ,
C
*

*Mouthwash, Tongue
*

1.8_
o

1.7 _
I 5.5
I
I

Placebo,. Crevicular Placebo, Tongue Water, Crevicular Water, Tongue

6.8

5.6

5.7

5.8

5.9

6.0

6.1

6.2

I 6.3

I 6.4

6.5

Oral

Malodor

(Hedonic

6.6 Units )

6.7

6.9

7.0

Fig. 6 - Relation of odorigenic bacterial populations to oral malodor; mouthwash, crevicular lar (-), placebo, tongue (o), water, crevicular (m), water, tongue (o).

(*), mouthwash, tongue

(*), placebo, crevicu-

Downloaded from jdr.sagepub.com by guest on August 7, 2011 For personal use only. No other uses without permission.

742
a

PITTS ETAL.

J Dent Res June 1983

cco
Co 1 90- 160,

I180-~

L1 70m

1 50

_'
CL

140

160-

r_

130 't
c

15040
140-

n
,

C
10:

-il

110

_
._

(AI1320t 11

s5

a~
90 E
100
80t

*Placebo H2S
_

Mouthwash H2S *Mouthwash MSH

Placebo MSH

Water H2S
608

Water MSH
69
3.0

5.5

5.6

5.7

5.8

5.9

6.0

6.1

62

6.3

64

6.5

0.6

6.7

Oral

Malodor IHedonic

Units

Fig. 7 - Relation of odorant (volatile sulfur) concentration to oral malodor; mouthwash H2S (*), mouthwash MSH (*), placebo H2S (.), placebo MSH (o), water H2S (a), water MSH (-).

olites (Fig. 7) for all treatments (except Mouthwash at 30 min) confirms that the decrease in malodor effected by Mouthwash treatment is due solely to its anti-microbial activity.

7. TONZETICH, J. and NG, S.K.: Reduction of Oral Malodor by Oral Cleansing Procedures, Oral Surg 42:172-181, 1976. 8. TONZETICH, J. and KESTENBAUM, R.: Odor Production by Human Salivary Fractions and Plaque, Arch Oral Biol 14:815827, 1969. 9. SCHMIDT, N.F.; MISSAN, S.R.; TARBET, W.J.; and COOPER, A.D.: The Correlation Between Organoleptic Mouth Ratings and Levels of Volatile Sulfur Compounds, Oral Surg 45:560567, 1978. 10. TONZETICH, J.: Direct Gas Chromatographic Analysis of Sulfur Compounds in Mouth Air in Man, Arch Oral Biol 16:587597, 1971. 11. SCHMIDT, N.F. and TARBET, W.J.: The Effect of Oral Rinses on Organoleptic Mouth Odor Ratings and Levels of Volatile Sulfur Compounds, Oral Surg 45:876-880, 1978. 12. TONZETICH, J. and RICHTER, V.J: Evaluation of Volatile Odoriferous Components of Saliva, Arch Oral Biol 9:39-45, 1964. 13. BLANCHETTE, A.R.; COOPER, A.D.; and TARBET, W.J.: Quantification and Evaluation of Volatile Sulfur Compounds in Mouth Air, IADR Progr & Abst 53:No. 142, 1974. 14. SOLIS, M.C. and VOLPE, A.R.: Determination of Sulfur Volatiles in Putrefied Saliva by a Gas Chromatography-Microcoulometric Titrating System,JPeriodontol 44:775-778, 1973. 15. SOLIS-GAFFAR, M.C.; NILES, H.P.; RAINIERI, W.C.; and KESTENBAUM, R.C.: Instrumental Evaluation of Mouth Odor in a Human Clinical Study, JDent Res 54:352-357, 1975. 16. BLANCHETTE, A.R. and COOPER, A.D.: Determination of Hydrogen Sulfide and Methyl Mercaptan in Mouth Air at the Parts-Per-Billion Level by Gas Chromatography, Anal Chem

Conclusions.
Thirty human subjects who had oral malodor used Mouthwash, Placebo, and Water to rinse their mouths in a randomized, double-blind, complete cross-over study. At intervals before and up to three hr after treatment, evaluations of overall oral odor, specific malodorants, and specific odorigenic bacteria were made in each subject. Mouthwash was highly effective in depressing all determinants of oral malodor; controls were relatively ineffective. While the effects of the treatments differed in magnitude, the malodor determinants were well-correlated for all treatments at all times with one important exception: In the first sample taken after antiseptic mouthwash use, oral malodor was substantially less than predicted from volatile sulfur or bacterial levels. Analysis of these data demonstrates that re-odoration is important to the overall activity of the product only for about 30 min after treatment and, at posttreatment times of 60-180 min, the anti-odor activity of the product is due to its anti-microbial action.
REFERENCES 1. PRINZ, H.: Offensive Breath, Its Causes and Its Prevention, Dent Comos 72:700-707, 1930. 2. SULSER, G.E.; LESNEY, T.A.; and FOSDICK, L.S.: The Reduction of Breath and Mouth Odors by Means of Brushing the Teeth, JDent Res 19:173-177, 1940. 3. RICHTER, V.J. and TONZETICH, J.: Evaluation of Volatile Odoriferous Components of Saliva, Arch Oral Biol 9:47-53, 1964. 4. TONZETICH, J.; EIGEN, E.; KING, W.J.; and WEISS, S.A.: Volatility as a Factor in the Inability of Certain Amines and Indole to Increase the Odor of Saliva, Arch OralBiol 12:11671175, 1967. 5. TONZETICH, J.: Production and Origin of Oral Malodor: A Review of Mechanisms and Methods of Analysis, J Periodontol 48:13-20, 1977. 6. TSUNODA, M.: Analysis of Fetor Ex Ore by Gas Chromatography,JJpnAssnPeriodontol 17:1-13, 1975.

48:729-731, 1976.
17. McNAMARA, T.F.; ALEXANDER, J.F.; and LEE, M.: The Role of Microorganisms in the Production of Oral Malodor, Oral Surg 34:4148, 1972. 18. SOLIS-GAFFAR, M.C.; FISCHER, T.J.; and GAFFAR, A.: Instrumental Evaluation of Odor Produced by Specific Oral Microorganisms, JSoc Cosmet Chem 30:241-247, 1979. 19. TONZETICH, J. and McBRIDE, B.C.: Characterization of Volatile Sulfur Production by Pathogenic and Non-Pathogenic Strains of Oral Bacteroides, Arch Oral Biol 26:963-969, 1981. 20. PIANOTTI, R. and PITTS, G.: Effect of an Antiseptic Mouth Rinse on Crevicular Anaerobes, IADR Progr & Abst 57:No. 560, 1977. 21. PIANOTTI, R. and PITTS, G.: A Method for the in situ Evaluation of Antiseptic Mouthrinses, IADR Progr & Abst 57:No. 651, 1978. 22. PIANOTTI, R. and PITTS, G.: Effects of an Antiseptic Mouthwash on Odorigenic Microbes in the Human Gingival Crevice, JDent Res 57:175-179, 1978. 23. PIANOTTI, R.; PITTS, G.; MASURAT, T.; BROGDON, C.; and THAKKER S.: Correlation Between Oral Malodor and Populations of 6dorigenic Bacteria, IADR Progr & Abst 58: No. 1160, 1979. 24. PITTS, G.; PIANOTTI, R.; FEARY, T.W.; McGUINESS, J.; and MASURAT, T.: The in vivo Effects of an Antiseptic Mouthwash on Odor-producing Microorganisms, J Dent Res 60:1891-1896, 1981. 25. PITTS, G.; PIANOTTI, R.; MASURAT, T.; and SCHUMANN, P.: Effectiveness of an Antiseptic Mouthwash on Determinants of Oral Malodor, IADRProgr & Abst 60:No. 276, 1981. 26. PIANOTTI, R. and PITTS, G.: A Simplified Agar Medium for Cultivation of Oral Odorigenic Bacteria, IADR Progr & Abst 60: No. 75, 1981. 27. O'KEEFE, A.E. and ORTMAN, G.C.: Primary Standards for Trace Gas Analysis, Anal Chem 3 8:760-763, 1966. 28. KAIZU, T.; TSUNODA, M.; AOKI, H.; and KIMURA, K.: Analysis of Volatile Sulfur Compounds in Mouth Air by Gas Chromatography, Bull Tokyo Dent Coll 19(1):43-52, 1978. 29. KAIZU, T.; TSUNODA, M.; SATO, H.; and SATO, T.: Reduction of Bad Breath from Periodontal Patients by Dilute Hydrogen Peroxide Solution, Bull Tokyo Dent Coll 19(4):209-216, 1978.

Downloaded from jdr.sagepub.com by guest on August 7, 2011 For personal use only. No other uses without permission.