244 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS l, H2S 2. CH3 SH :5. {CH3}2 S COLUMN: FEP TEFLON COLUMN PACKED WITH 5 % POLYPHENYL ETHER AND 0.05 % H3PO 4 ON 30/60 MESH CHROMSORB T ATTENUATION = 1.28 x I0 -•ø AFS, TEACOR MT 220 TEMPERATURE = 60 øC CARRIER GAS: AIR Figure 1. GC-FPD chromatogram of headspace of incubated system containing gram-negative microor- ganism. peaks were shown to be that of hydrogen sulfide and methyl mercaptan as major components and dimethyl sulfide as a minor component. The gram-staining character of the microorganisms and the ability to produce VSC from the sterile saliva system with L-cysteine as substrate, in the presence of human blood is shown in Table I. From the table it is clear that only the gram-negatives were capable of producing the volatile sulfur compounds from the sterile incubated system. Veillonella alcalescens, Fusobacterium nucleatum, Bacteroides melaninogenicus and Kleb- siella pneumoniae produced VSC. However, the gram-positives failed to produce VSC. An unpleasant odor was detected organoleptically when VSC was present in the incubated system. Although it is possible to quantitate the amounts of VSC produced, at this time the evaluations were qualitative to determine if indeed the VSC produced Table I Odor and VSC Production by Organisms and Gram Staining Characteristics VSC Odor Gram Organism Production Produced Stain Streptococcus sanguis No None Streptococcus salivarius No None Streptococcus mutans No None Actinomyces naeslundii No None Lactobacillus acidophilus No None Staphylococcus aureus No None Candida albicans No None Bacteroides melaninogenicus Yes Unpleasant Fusobacterium nucleatum Yes Unpleasant Veillonella alcalescens Yes Unpleasant Klebsiella pneumoniae Yes Unpleasant Positive Positive Positive Positive Positive Positive Positive Negative Neganve Negauve Negative Incubation period: 3 hr or 24 hr, 37øC

INSTRUMENTAL EVALUATION OF ODOR 245 Table II Controls VSC Odor Production Produced Sterile saliva No None Sterile saliva + L-cysteine No None Cysteine + H20 No None Non-sterile saliva + L-cysteine Yes Unpleasant Incubation period: 3 hr or 24 hr, 37qC by microorganism was associated with the malodor present in the incubated system. Among t'he gram-negatives tested, K/ebsie//a pneumoniae was observed to produce the most abundant amounts of VSC after 3 hr or 24 hr incubation. The controls for this study are shown in Table II. It is clear that the sterile systems failed to produce the characteristic and distinctive malodor observed with incubated non-sterile saliva. Moreover, VSC was not detected in the headspace of the sterile controls. DISCUSSION The results of this study confirm the earlier findings by Berg and Fosdick (4) and later by McNamara and co-workers (1) that oral microorganisms are involved in producing mouth malodors. Significantly, our results indicate that this odor is primarily caused by the formation and presence of VSC. Moreover, the ability to produce malodor resides on the gram-negative microorganisms. It has been pointed out by Socransky and Manganiello (5) that different microorganisms colonize in different sites of the oral cavity, that is, the organisms have a primary ecologic niche. The ecologic niche of the four gram-negatives that produced VSC in this study seems to be those areas where saliva readily stagnates. In general, these areas include the gingival crevice, interdental spaces, papillary crypts of the tongue, tonsillar folds and accumulations of dental plaque. Among the four gram-negatives studied, Bacteroides me/aninogenicas and Fusobacterium nucleatum are predominantly found in the gingival crevice, Veillonella alcalescens in plaque and Klebsiella pneumoniae in the interdental spaces. Possibly these areas provide the environmental conditions favorable to the growth of gram-negative anaerobes. According to McNamara and co-workers (1) saliva that was allowed to stagnate resulted in a shift in the ratio of gram-positive to gram-negative organisms favoring the Table III pH Changes Before and After Incubation at 370C t Microorganisms pH Before pH After Incubation Incubation 3 hr 24 hr Klebsiella pneumoniae 7.6 10.0 10.5 Fusobacterium nucleatum 7.2 7.2 7.9 Bacteroides melaninogenicus 7.0 7.7 8.9 Veillonella alcalescens 7.2 7.3 7.5 •Sterile saliva + L-cysteine + sterile human blood.