CLINICAL AND MATERIALS SCIENCES Microbial Characterization of an Experimental Cariogenic Plaque in Man CARL A. OSTROM, THEODORE KOULOURIDES, FRANCES HICKMAN, and JERRY R. MCGHEE

Institute of Dental Research and Department of Oral Biology, School of Dentistry, University of Alabama in Birmingham, Birmingham, Alabama 35294, USA Experimentally induced plaque seemed to originate by direct contact inoculation from the vestibular mucosa and saliva. During the next seven days, this plaque developed its own characteristics. Populations of Streptococcus mutans, usually less than 2% of total streptococci population in plaques that were less than three days old, increased between days 3 and 7. Proportions of S sanguis, high in early samples, decreased after day 3. Populations of S salivarius, which usually outnumbered other streptococci, fluctuated widely through day 3, and then increased in proportion in subjects who were more productive of experimental caries, but decreased in subjects who were less productive. Proportions of plaque flora comprising lactobacilli paralleled those of S salivarius. J Dent Res 56(6): 550-558 June 1977. The intraoral cariogenicity test (ICT) provides an experimental model for studies of current caries activity in man. The ICT appliance (Fig 1) is used to carry specimens of test enamel that are implanted in pockets on acrylic buccal flanges subjacent to the permanent mandibular first molars. Flat polished slabs of bovine enamel are covered with Dacron* mesh to enhance colonization of the experimental plaque by the subject's normal oral flora. Expressed in terms of subsurface demineralization of the test enamel slabs, the experiThis investigation was supported, in part, by USPHS Contract NIH-NIDR-72-2030 from the National Institutes of Health, Bethesda, Md, and Grant DE-02670 from the National Institute of Dental Research, Department of Health, Education and Welfare. Received for publication September 9, 1975. Accepted for publication July 9, 1976. * Dacron (Registered US trademark), No. 61021, 0.015 mm thickness, Unted States Catheter and Instrument Co., Glenn Falls, NY. t Vortex Jr., Scientific Industries, Inc., Queens Village, NY.

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mental caries can be measured in a standard indenter test by changes of penetration (in micrometers) or by determining the depth of the subsurface radiolucency in microradicgraph of thin sections.1-3 Exposure to the oral environment for seven days has produced measurable ICT carious lesions that varied between individuals, between the right and left sides of a subject's mouth, or over a period of months on the same side of a subject's mouth. A schedule for four trials in each of four subjects within a short period of time was shown to yield reproducible data. Extraoral immersion of a test slab in 3% sucrose solution for four daily, widely spaced, ten-minute intervals approximately doubled the depth of the experimental demineralized layer.3-5 Comparative studies in children and older adults have shown that the resultant degree of ICT caries was characteristic for each subject and was not related to the numbers of carious lesions in the subject's natural teeth.6 The present report describes studies that were designed to compare the microbial flora of the ICT plaque with the flora of oral sources that may have contributed to it, and to observe any changes in the microbial composition of this plaque at intervals that varied from two hours to seven days after the ICT appliance had first been placed in the test subject's mouth.

Materials and Methods MICROBIOLOGICAL PROCEDURES.-In preliminary studies, the total viable counts and the proportions consisting of streptococci were comparable after dispersal of ICT plaques either by sonication or by the use of a mechanical shakert with the aid of eight to ten 3-mm diameter glass beads in a 1OX75-mm test tube,

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551 MICROBIOLOGY OF EXPERIMENTAL PLAQUE choscii for this stiidy. Both had experienccd recent caries in their natural teeth and, in previolu.s tests, hadl shoxswn a moderate degree of ICT (carics.6 In the same studies, these participants usually had S mutans in a range from zero to less than 1% of the total streptococci in plaqiie samples collected on dental floss froin the clinically and radiographically cariesfree interproximal surfaces of the perinanent left maxillarv first anid second premolars; FIG 1.- ICT appliance. This photograph whereas sample's froin the interproximail-1 sufaces shows Crozat orthodontic appliance adapted to of the similarly cariesfiee permanent mandibucarry test enamel slabs in a fully dentulous test lhr first and second molars usuallv conitained S subject. In test subjects who are bilaterally rnutais in iumibers that approximated 20% edentulois, denitures miay be used in similar of the total streptococci recovered on MS agar. manner. Fully dentulous, thcse participants xwere fitted wsithi a modified orthodontic appliaince that for fixve minuites. Recognizing that the total via- carried the ICT slabis. ble counrt might be influenced hv the sonic riupElach pair of trials consisted of a two-hour ture of somle sensitive species, the present studs ancl a seven-day ICT. Immediately preceding xwas based on use of the mechanical mixer. each pair of trials, the participants liad a Samples xsere collected in 2 nil of 0.067 M thorotigh prophylaxis that inicluded puimice potassium3li phoslhate buffer (pl-h 7.2). Within drawn by dental floss across each interproximal two hlours, the samples xsere dispersed atid sirface. The ICJT appliance, xv ith Dacronserial diluitionis in tubes of the samie collectinrg covered test slabs in place, was inserted. The mnediumiii xcre plated on thrce diffTrent culture w as requiested to eat and driiik niothing, media. A culture medium containing bl:ood, subject in two houirs. At this time, microand return to cyxsteine, and xeast extract in a,ar (BCY) as xweie taken, as shown in the flowv bial sanmples used to CLeLnimerale the total rccoxerable microDacron-covere d slabs in fresh With clhart. aerophilic bactelia.7 Streptococci were isolated place, the ICT appliance vas again i;nserted. 011 Miti.s-Slixi'riuls (MS agar.t Lactobacilli The particil)ants were instruc ted to x-e-ar the were isolated oni Rogosa (SL) agar.I The MS appliance for seven daxs, to continue th-eir noaragar plates were incubated at 37 C for 18 to 21 houlzrs in 95'-%. N., and 5S% CO., after xThich nial dietary habits, and to iilixei-c the test they xx.ere exposed to air at roonm temperature for 2-4 honirs to enhance clifferential colonial SrTIDY I FOW CHXCRT morphliology.5 Vl other plates were inlcsubated at 37 C in 95% N,o and 5cc CO., for three 1. Pt.imice all trculh days. At X 10 magnification, colonies on MS 2. Wear ICT appliance for two houirs agar were identified Iy morplhology as StrepTake niicrobial samples 3. tococcus mutans, S soalivirius, S scanguis, or a. ICT Dacrori m'sh (body of ICT plaque) 'other"' species.'.. "achi timre MIS agar plates b. Washed ICT slab surface (adherent ICT we re couinted, the icleititv of representative plaque) colonies xxas (cn0firmed ly standard physiologc. Saliva ical t(ests.10 d. Vestibular mrucosa e. Tonguie mucosa STunvY 1.-RELxTIoN or ICT PIAQtE f. lBuccogingival suirface of termnanent left POTFNTIAL INOCULATION SOURCES. FLORA rradibul:ilar second molar A scries of fouir paired trials xNas designed to C'revicular sample the flora in compare the comiposition of the h. Inuterproximal surfaccs of permanent left ICT plaqlue with' that in selected natuiral denmandibular first and second molars tal plaques anid with the flora recovered from i. Interproximnal surfaces of permanent left that might have contributed other oral imaxillary first and s5e(nd prennolars to the bacterial inoculation of the ICT plaque. 4. Wear ICT appliance for seven days Several factors goverined the selection of 5. Take microbial samples the test suibjects, aged 13 and 15, who were a. ICT Dacron mesh b. Washed ICT slab surface

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OSTROM ET AL

J Dent Res June 1977

slabs in 3% sucrose solution for ten minutes at experienced no caries in their remaining natfour widely spaced intervals daily. To conclude ural teeth for more than five years In previous the seven-day test, microbial samples were trials, two subjects (No. 1 and No. 2) demontaken from the Dacron mesh covers and from strated a high degree, and two (No. 3 and No. the ICT slab surfaces. 4) had a low degree of ICT caries.3'5 In each The samples containing the body of the participant, four complete ICTs were perICT plaque were obtained by scalpel excision formed for each of the five test periods: 2, 6, of the Dacron mesh from the slab surface. and 24 hours and three and seven days. After this, the slab was held under vigorously Throughout this study, the subjects were reflowing tap water for 15 seconds to wash off quested to continue their normal dietary and nonadhering bacteria. The slab was then dried oral hygiene habits. In order to enhance rapid with compressed air and the adherent fraction plaque formation and cariogenesis as well as of the plaque was scraped from the surface potential adhesion of S mutans to the test with a 1.5 mm-wide, sharp, plexiglass chisel enamel surfaces, the ICT slabs were immersed blade. Saliva samples consisted of that quan- extraorally in 3% sucrose solution at frequent tity of unstimulated saliva that collected under intervals. All the tests (two hours to seven the tongue in 15 seconds and was absorbed by days) followed the immersion schedule given a one-eighth-inch cotton pellet. Before each in Table 1 for the first 24 hours. In the threesampling, the sites to be described later were and seven-day tests, the participants continued freed of excess saliva by spraying with com- to immerse the test enamel slabs in the sucrose pressed air. The vestibular mucosa sample was solution for ten minutes at each of four widely taken from the area that was normally in con- spaced intervals daily for the remainder of tact with the ICT slab. The tongue mucosa each test period. sample was taken from the dorsal surface near In order to study the microbial distributhe permanent left mandibular second molar. tion at different depth, the ICT plaque that The vestibular and tongue mucosa samples accumulated on the slabs on the right and left were collected on a sharp discoid scaler drawn sides of the ICT appliance were analyzed (in once across approximately 1 cm of the mucosal alternate sequence) as the total or as fractions surfaces. The buccogingival surface of the of the total plaque as follows: The total plaque.-This included the enpermanent left mandibular second molar was sampled with a sharp discoid scaler drawn tire excised Dacron mesh and all the material once across the surface, with care being taken that could be scraped from one half of the to avoid contact with the gingivae and the surface of the test enamel slab with a 1.5-mm adjacent interproximal plaque. The crevicular wide, sharp, plexiglass chisel blade. The Dacsample was collected on a blunt periodontal ron mesh and the chisel blade, each with its probe drawn once through the buccogingival crevice of the permanent left mandibular secTABLE 1 ond molar. Each collecting instrument was SCHEDULES FOR TEN-MINUTE EXTRAORAL twirled vigorously in a tube of collecting soluIMMERSION OF THE ICT SLABS IN 3% tion to free its sample; other samples were SUCROSE SOLUTION dropped into a tube of collecting solution. The interproximal plaque samples were collected Hr at Which Test Slabs Were Immersed on sterile, unwaxed dental floss drawn with a 1 0* 10 21 23 9 5 7 3 Test sawing motion three times against each surface. Sections of the floss containing the sam2hr x x x x x 6 hr ples were cut with scissors and dropped into x x x x x x x 24hr tubes of collecting solution. x x x x x 3 dayt STUDY 2.-SERIAL DEVELOPMENT OF THE x x x x x 7 dayt MICROBIAL FLORA IN THE ICT PLAQUE.-This study was designed to analyze the microbial * For the two-hour tests, the slabs were immersed composition of ICT plaque after selected pe- immediately before the first intraoral insertion of the ap riods of time following initial oral insertion pliance. Any excess sucrose solution was shaken off, but not rinsed away. After all other immersions, the appliof the appliance. ance was rinsed with tap water to minimize exposure of The four mature adults selected as sub- the natural teeth to sucrose. t In the three- and seven-day tests, after the first jects for this study wore partial dentures that 24 hours, the slabs were immersed daily at four widely carried the ICT slabs.3 These participants had spaced, convenient intervals. Downloaded from jdr.sagepub.com at NORTH DAKOTA STATE UNIV LIB on June 13, 2015 For personal use only. No other uses without permission.

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TABLE 2 PROPORTIONS OF STREPTOCOCCI AND OF LACTOBACILLI IN Two-HOUR AND IN SEVEN-DAY ICT PLAQUE AND THAT FROM PROBABLE SOURCES, EXPRESSED IN PERCENT Streptococci % of Total Streptococci

Sample Permanent left maxillary first and second premolars* Permanent left mandibulart first and second molars Permanent left mandibular: second molar Crevice Tongue Saliva Vestibule 2-hour D ICT§ Dacron slab 7-day t Dacron slab ICT§ 5

% of Total Count

S mutans

S sanguis

25-35

Microbial characterization of an experimental cariogenic plaque in man.

CLINICAL AND MATERIALS SCIENCES Microbial Characterization of an Experimental Cariogenic Plaque in Man CARL A. OSTROM, THEODORE KOULOURIDES, FRANCES H...
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