Association between clinical and salivary microbial parameters during orthodontic treatment with removable appliances with or without use of fluoride mouth rinse.

AIM: To evaluate the correlation between clinical and salivary microbial parameters during treatment with removable orthodontic appliances with or without use of fluoride mouth rinse. MATERIALS AND METHODS: A group of 48 patients completed this randomized, controlled, parallel-group, clinical pilot study. 24 patients of the test group (TG) rinsed after tooth brushing with a fluoride mouth rinse (100 ppm AmF/150 ppm NaF) while 21 patients of the control group (CG) did not. Clinical parameters [Approximal Plaque Index (API), Papillary Bleeding Index (PBI)] as well as levels of salivary mutans streptococci (MS) and lactobacilli (LB) were assessed at baseline and after 3, 6, 9 and 12 months. Compliance was documented by participants. Appliance wear-times were estimated retrospectively by the patients. All participants received the same brushing instructions and were supplied with the same toothbrush/toothpaste. RESULTS: A significant correlation of API values with MS levels (p=0.0003) or with LB levels (p=0.001) was observed. Furthermore, appliance wear-times were significantly associated with API values (p=0.02). The changes of MS or LB levels between beginning and end of the study did not differ significantly between study groups. The changes of the API scores showed slightly different median values (-3.5 in the TG vs. 0 in the CG), the difference, however, was not significant. CONCLUSIONS: The results emphasise the need for a careful monitoring of the oral hygiene status in patients with removable orthodontic appliances. Larger controlled clinical trials are necessary to investigate treatment options such as use of supplementary fluoride mouth rinse.

Cariogenic effects of probiotic Lactobacillus rhamnosus GG in a dental biofilm model.

Probiotic bacteria have been suggested to inhibit Streptococcus mutans (SM) and thus prevent dental caries. However, supporting evidence is weak and probiotic species might be cariogenic themselves. Thus, we compared and combined the probiotic Lactobacillus rhamnosus GG (LGG) with SM and analysed the resulting mineral loss (ΔZ) in dental tissues. We simulated three biofilm compositions (SM, LGG, SM × LGG), two lesion sites (smooth enamel, dentin cavity) and two nutrition supply frequencies (twice/day, 6 times/day) in a multi-station, continuous-culture biofilm model. A total of 240 bovine enamel and dentin samples were cut, polished and embedded. All experimental procedures were performed in independent duplicates, with 10 samples being allocated to each group for each experiment (final sample size n = 20/group). Biofilms were cultured on the specimens and supplied with 2% sucrose medium and artificial saliva in consecutive pulses. After 10 days, ΔZ and bacterial numbers were assessed. SM × LGG biofilms caused significantly increased ΔZ compared with SM or LGG biofilms (p < 0.01, Mann-Whitney test), and ΔZ was significantly increased in dentin cavities compared with smooth enamel lesions (p < 0.01). Bacterial numbers did not significantly differ between biofilms of different species (p > 0.05, ANOVA). Frequent nutrition supply significantly increased bacterial numbers (p < 0.01). Biofilms in dentin cavities compared to smooth enamel harboured significantly more bacteria (p < 0.05). LGG induced mineral loss especially in dentin cavities and under highly cariogenic conditions. LGG did not have inhibitory effects on SM, but rather contributed to the caries process in vitro.

Antibacterial action of Chlorhexidine/thymol containing varnishes in vitro and in vivo.

OBJECTIVES: The antibacterial activity of two different formulations of a chlorhexidine/thymol varnish should be elucidated in vitro and in vivo. METHODS: The agar diffusion assay with Cervitec(®) and CervitecPlus(®) and three reference strains each of streptococci, lactobacilli, actinomyces and periodontal pathogens was performed. In a split-mouth study, 40 volunteers applied the test (CervitecPlus(®), solvent water and ethanol) and control (Cervitec(®), solvent ethyl acetate) varnish at buccal recessions of premolar teeth at baseline as well as after two, four and seven days. Supra- and subgingival plaques were collected 2 weeks before baseline and at the screening appointments. Supragingival plaque was analysed for mutans streptococci and lactobacilli and subgingival samples for Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis and Porphyromonas intermedia. Friedman/Wilcoxon tests and U-test were used for statistical analysis (P < 0.05). RESULTS: Most reference strains were susceptible with inhibition zones (mm) as follows: Cervitec(®)/CervitecPlus(®) streptococci 27 ± 1.7/21.3 ± 2.5, lactobacilli 26 ± 9.2/23.7 ± 4.9, actinomyces 36.3 ± 6.6/27.3 ± 1.5, periodontal pathogens 18.7 ± 7.6/18 ± 1.7. Both varnishes reduced significantly the counts of mutans streptococci and lactobacilli in the patients. However, no significant differences were found between test and control sides at any time. The total counts of periodontal pathogens were low. A tendency to higher counts of A. actinomycetemcomitans at the control side could be shown; the test side did not harbour significantly higher counts. CONCLUSION: Both varnishes may influence the plaque formation and reduce mutans streptococci in supragingival plaque.

[Oral health of pregnant women and their awareness of oral hygiene]. / Die Mundgesundheit von Schwangeren und deren Mundgesundheitsbewusstsein.

AIM: Pregnancy is related with changes in oral health. While the medical care of pregnant women in Germany is well-regulated, there are no structured recommendations for dental examinations. During an "Oral Health Awareness Week" the periodontal conditions of pregnant women in the Federal State of Thuringia (Germany) were examined. METHODS: 85 pregnant women participated in this study. Oral health indices (PSI, BoP and DMFT) were recorded. 61 of the 85 women followed the invitation to repeat the investigations after parturition. Additionally, birth weight and height of the newborn were recorded. RESULTS: There were no correlations between periodontal condition and birth weight. However, pregnant women demonstrated in high levels periodontal inflammation (only 1.2% showed periodontal health). Pregnant woman with periodontitis (PSI 3 and 4) demonstrated clear deficiencies in health education regarding oral health and adverse pregnancy outcomes. CONCLUSIONS: These findings implicate the importance of periodontal care in pregnant women. An association between periodontitis and reduced birth weight was not observed in the studied Thuringian cohort.

Acid production by oral strains of Candida albicans and lactobacilli.

Both Candida albicans and lactobacilli are common colonizers of carious lesions in children and adolescents. The purpose of this study is to compare the velocity of acid production between C. albicans and several Lactobacillus species at different pH levels and concentrations of glucose. Washed, pure resting-cell suspensions were obtained by culturing a total of 28 oral isolates comprising the species C. albicans, Lactobacillus rhamnosus, Lactobacillus paracasei paracasei, Lactobacillus paracasei tolerans and Lactobacillus delbrueckii lactis. Acid production from glucose was determined at a constant pH of 7.0, 5.5, 5.0 and 4.0 by repeated titrations with NaOH in an automated pH-stat system. Acid formation rates of yeast and lactobacilli proved to be similar at both neutral and low pH, while in a moderately acidic environment C. albicans produced less acid than the lactobacilli. Ion chromatographic analysis of the cell-free medium after titration revealed pyruvate to be the predominant organic acid anion secreted by C. albicans. The proportion of organic acids to overall acid production by the yeast was below 10% at neutral conditions, in contrast to 42-66% at pH 4.0. Compared to lactobacilli, yeast required a concentration of glucose that was about 50 times higher to allow acid production at half the maximum speed. Considering the clinical data in the literature about the frequency and proportions of microorganisms present in early childhood caries lesions, the contribution of oral lactobacilli as well as C. albicans to overall microbial acid formation appears to be important.

Attitudes of general dental practitioners in Europe to the microbial risk associated with dental unit water systems.

UNLABELLED: Dental Unit Water Systems (DUWS) are used in dental practices to provide water for cooling of dental equipment and irrigation of the oral cavity. However, they have been demonstrated to be contaminated with micro-organisms. There are currently no European Union (EU) Commission guidelines for the microbial quality of water discharged by DUWS. This study was part of an EU research programme to investigate the microbial contamination of DUWS in general dental practice (GDP) in the UK, Denmark, Germany, The Netherlands, Ireland, Greece and Spain. OBJECTIVE: To undertake a questionnaire survey on the type of DUWS in use and determine the attitude of GDPs to the risk of microbial infection from DUWS. MATERIALS AND METHODS: The questionnaire was written and translated into the language of each country before being posted to each participating dentist. Dentists were asked to complete the questionnaire survey and return it by post. RESULTS AND CONCLUSIONS: The major findings were that the majority of dentists did not clean, disinfect or determine the microbial load of their DUWS, and that dentists would welcome regular monitoring and advice on maintaining their DUWS; the introduction of guidelines; and recommendations on controlling the microbial load of DUWS.

Efficacy of chlorhexidine varnish applications in the prevention of early childhood caries.

AIM: A prospective clinical study was conducted to evaluate the influence of 3-monthly applications of the chlorhexidine- containing varnish Cervitec on the colonisation of a child's oral cavity by mutans streptococci (MS) and on caries prevalence. METHODS: 200 children aged 11.7 +/- 0.7 months were examined. Children's caries risk was assessed on the basis of their MS scores in saliva. Out of the 48 children in whom a high caries risk (> or =10(5) CFU/ml) was expected, 23 were treated with Cervitec at 3-month intervals (CHX group). The remaining 25 children of the high-risk group received no treatment and served as controls (group C). All parents received detailed information on the prevention of early childhood caries. 172 children had completed the study after one year. RESULTS: During the course of the study the percentage of children with visible plaque on their maxillary incisors increased from 17.8% to 40.1% and the percentage of children given sweetened drinks in nursing bottles for the night rose from 16.3% to 18.0%. At the closure of the study 26.2% of the two-year-olds had salivary scores of MS > or =10(5) CFU/ml of saliva. The mean d(1-4)mft value increased from 0.05 -/+ 0.4 to 0.8 -/+ 2.9 and the mean d(1-4)mfs value rose from 0.08 -/+ 0.8 to 1.8 -/+ 5.9. No significant differences were demonstrable between the two-year-olds in groups CHX and C for colonisation of the oral cavity by MS or for d(1-4)mft values. In contrast the d(1-4)mfs values were significant lower in the CHX group as in the group C. CONCLUSION: Poor feeding habits and deficits in oral hygiene cannot be compensated by the application of Cervitec.

First-time isolation of Candida dubliniensis from plaque and carious dentine of primary teeth.

AIM: To determine those organisms of the genus Candida associated with dental caries by investigating samples from active carious lesions. Within the genus Candida, the species Candida albicans and Candida dubliniensis are capable of forming chlamydospores and germ tubes. Until it became possible in 1995 to differentiate between the two species taxonomically, C. dubliniensis was falsely identified as C. albicans. Whilst the importance of C. albicans for rapidly progressing early childhood caries (ECC) has been recognised, so far there have been only reports about C. dubliniensis in connection with children/mothers who have been infected with HIV or already developed AIDS. In the present study, C. dubliniensis was for the first time isolated from plaque and carious dentine of a healthy five-year-old boy. METHODS: As part of the investigation, a number of samples were collected from individual children affected by active dental caries. Amongst the samples, one in particular indicated that Candida species might be involved. The patient was a five-year-old boy with ECC of the primary dentition, scheduled for restorative treatment under general anaesthesia. Before treatment, a salivary, plaque (region of 54/55) and soft carious dentine sample from the tooth 51 was taken before extraction. The counts of yeasts, lactobacilli (LB) and mutans streptococci were determined in the samples. RESULTS: The boy's dmft was 11, which was dominated by the d component. In the saliva of the boy, LB and mutans streptococci (MS) were detected. In plaque and carious dentine, MS and most interestingly C. dubliniensis were present. The yeasts were visualised in carious dentine by means of scanning electron micrographs. CONCLUSIONS: Plaque and carious dentine may be a further habitat of C. dubliniensis.

Activity of citrinin metabolized by rat and human microsome fractions in clastogenicity and SCE assays on Chinese hamster V79-E cells.

The mycotoxin citrinin is a potent inducer of chromosomal aberrations in the clastogenicity assay on V79-E cells when metabolized by rat and human liver microsomes. Rat and human liver microsomes, standardized on protein content, activate citrinin at equal levels. 5 X 10(-4) M citrinin induces complex translocations in a high frequency as well as defects of chromosomal coiling. Higher concentrations are cytotoxic, lower ones are almost inactive. After metabolization of mycotoxin by rat-kidney microsomes or an S9 mix fraction containing rat liver and kidney microsomes, toxic effects predominate and chromosomal aberrations are diminished. Clastogenic citrinin concentrations do not induce an increase of SCE frequency. Although the mode of action of this mycotoxin on chromosomal structure remains obscure, possible explanations are discussed.

Patulin, a further clastogenic mycotoxin, is negative in the SCE assay in Chinese hamster V79-E cells in vitro.

Patulin is a potent inducer of chromatid-type aberrations in Chinese hamster V79-E cells, but loses its activity when 9000 g supernatant of rat-liver homogenate is added. The narrow dose range of patulin clastogenicity shows a quantitative relationship between absolute amount of mycotoxin applied and the number of indicator cells treated. Within a dose range permitting survival of V79-E, patulin does not induce an increase of the SCE rate. It is suggested that patulin clastogenicity is caused by interaction with chromosomal proteins and that DNA is not the virtual target of this mycotoxin.

Diversity of Lactobacillus species in deep carious lesions of primary molars.

AIM: This was to determine the prevalence of Lactobacilli (LB) species in different stages of caries progression and are considered as secondary invaders of existing carious lesions and specialists for caries progression. METHODS: Carious dentine samples were collected from 70 primary molars (M) during step-wise (S1, S2: n = 35 M) or one-step (O1: n = 35 M) caries treatment and after 11 months of temporary restorations (S3, O2). LB were identified by selected physiological and biochemical characteristics, ratio of lactic acid isomers, electrophoretic mobilities of lactic acid dehydrogenases, and shotgun mass mapping by MALDI mass spectrometry. RESULTS: LB were isolated from 46% of soft dentine samples (S1). The prevalence of LB from hard dentine collected during caries excavation (O1) reached 34%, after 8 weeks of temporary filling (S2) 11%, and 9% each after 11 months of temporary restoration (S3, O2). The mean total bacterial counts (cfu) of soft dentine (S1) were 3.6 x 105. From hard dentine during caries excavation (O1) 4.4x104 cfu were calculated, at S2 3.7 x 10³ cfu, at S3 0.1 x 10³ cfu, and at O2 1.8 x 10³ cfu. The percentages of LB in the cfu for LB positive dentine samples were for S1 / S2 / S3 / O1 / O2: 60% (16 M)/34% (4 M)/54% (3 M)/57% (9 M), and 64% (3 M). Five LB species were identified from carious dentine: L. paracasei subsp. paracasei, L. paracasei subsp. tolerans, L. rhamnosus, L. gasseri, and L. alimentarius. CONCLUSIONS: While L. rhamnosus and L. paracasei subsp. paracasei occurred in all caries progression stages, the other species were found only sporadically. L. paracasei subsp. paracasei and L. rhamnosus might be the specialists of the LB in carious progression.

Phenotypic heterogeneity of Streptococcus mutans in dentin.

Information concerning phenotypic heterogeneity of Streptococcus mutans in carious dentin is sparse. Matrix-assisted laser-desorption/ionization-time-of-flight mass-spectrometry (MALDI-TOF-MS) facilitates the phenotypic differentiation of bacteria to the subspecies level. To verify a supposed influence of restorative treatment on the phenotypic heterogeneity of S. mutans, we isolated and compared a total of 222 S. mutans strains from dentin samples of 21 human deciduous molars during caries excavation (T(1)) and 8 wks (T(2)) after removal of the temporary restoration. Phenotypic heterogeneity was determined by MALDI-TOF-MS and hierarchical clustering. Thirty-six distinct S. mutans phenotypes could be identified. Although indistinguishable phenotypes were found in the same teeth at T(1) and T(2), as well as in different teeth of individual participants, the phenotypic heterogeneity increased significantly, from 1.4 phenotypes per S. mutans-positive dentin sample at T(1) to 2.2 phenotypes at T(2). We attribute this to an adaptation of S. mutans to the modified environment under the restoration following caries excavation.

Streptococcus sobrinus in children and its influence on caries activity.

AIM: This was to study the longitudinal assessment of caries activity of Streptococcus sobrinus (SS) positive children during their mixed dentition. METHODS: The occurrence of mutans streptococci (MS) in plaque and saliva was determined in a representative sample of 55 children aged 8 to 12 years over a period of 4 years. A total of 708 bacterial strains was isolated which were identified as MS or SS. Caries activity (DeltaD(1-4)MFS) as well as plaque and gingival inflammation were recorded. RESULTS: During the period of observation 52 of the 55 children harboured MS; 12 of these children were SS positive. SS was not permanently detectable and 3 of the children were MS and SS negative. SS was not found without the presence of MS. Children that were infected with both SS and MS showed a slightly higher increase in caries compared with children that were infected exclusively by MS (DeltaD(1,2)MFS 6.2 vs. 3.0 and DeltaD(3,4)MFS 5.3 vs. 3.8) over the period of 4 years. An SS infection accelerated the increase of DeltaD(3,4)MFS significantly by a factor of 4 one year after its detection, whereas the DeltaD(1,2)MFS was 3 times as high during the period of infection. CONCLUSION: The findings suggest that an SS infection represents an important additional risk factor for dental caries due to its obvious aggravating of caries activity.

Comparison of the efficacies of disinfectants to control microbial contamination in dental unit water systems in general dental practices across the European Union.

Water delivered by dental unit water systems (DUWS) in general dental practices can harbor high numbers of bacteria, including opportunistic pathogens. Biofilms on tubing within DUWS provide a reservoir for microorganisms and should be controlled. This study compared disinfection products for their ability to meet the American Dental Association's guideline of <200 CFU x ml(-1) for DUWS water. Alpron, BioBlue, Dentosept, Oxygenal, Sanosil, Sterilex Ultra, and Ster4Spray were tested in DUWS (n = 134) in Denmark, Germany, Greece, Ireland, The Netherlands, Spain, and the United Kingdom. Weekly water samples were tested for total viable counts (TVCs) on yeast extract agar, and, where possible, the effects of products on established biofilm (TVCs) were measured. A 4- to 5-week baseline measurement period was followed by 6 to 8 weeks of disinfection (intermittent or continuous product application). DUWS water TVCs before disinfection ranged from 0 to 5.41 log CFU x ml(-1). Disinfectants achieved reductions in the median water TVC ranging from 0.69 (Ster4Spray) to 3.11 (Dentosept) log CFU x ml(-1), although occasional high values (up to 4.88 log CFU x ml(-1)) occurred with all products. Before treatment, 64% of all baseline samples exceeded American Dental Association guidelines, compared to only 17% following commencement of treatment; where tested, biofilm TVCs were reduced to below detectable levels. The antimicrobial efficacies of products varied (e.g., 91% of water samples from DUWS treated with Dentosept or Oxygenal met American Dental Association guidelines, compared to 60% of those treated with Ster4Spray). Overall, the continuously applied products performed better than those applied intermittently. The most effective products were Dentosept and Oxygenal, although Dentosept gave the most consistent and sustained antimicrobial effect over time.

Differentiation of mutans streptococci by intact cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

It is difficult to distinguish mutans streptococci on the species level, and even more so on the subspecies level. Intact cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) (ICM) was applied to reference strains of five of the species of the mutans group (Streptococcus criceti, Streptococcus downei, Streptococcus mutans, Streptococcus ratti, Streptococcus sobrinus), nonmutans streptococci (Streptococcus oralis, Streptococcus mitis, Streptococcus salivarius, and Streptococcus sanguinis), and 177 mutans streptococci isolated from saliva of 10 children. From the analysis of the reference strains, readily distinguishable ICM mass spectra were obtained for the different species. Based on multivariate statistical analysis, a correct and unambiguous assignment was made of the spectra of 159 isolated mutans streptococci to S. mutans and 16 isolates to S. sobrinus. Two isolates were sorted out and were identified by sequencing of their 16S rRNA genes as Streptococcus anginosus. In addition, ICM indicated a misclassification for some reference strains (AHT, V 100 and E 49) and re-classified AHT and E 49 as S. ratti and V 100 as S. sobrinus. This was confirmed by 16S rDNA sequencing. Based on a statistical similarity analysis of the spectra of reference strains and a quantitative assessment of the reproducibility of ICM, the isolates identified as either S. mutans or S. sobrinus were phenotyped on the subspecies level. In the population of the clinical isolates, 14 unambiguously different S. mutans and three different S. sobrinus phenotypes were detected. ICM proved to be a powerful tool for a differentiation of mutans streptococci down to the subspecies level.

[Oral Streptococci - a scheme for the identification of Streptococcus mutans]. / Orale Streptokokken - Ein Beitrag zur Identifizierung von Streptococcus mutans.

A reliable simple scheme for the rapid identification of certain species of oral streptococci has been developed and compared with biochemical and physiological results of 45 well known clinical isolates and stock strains. Moreover, a method for the determination of H2O2 production was tested. With selected reactions as for instance acid formation in mannitol and raffinose broth, hydrolysis of arginine and esculin, the production of peroxidase, and the resistance of bacitracin the species S. rattus, S. sobrinus, S. mutans, S. cricetus, S. ferus, S. milleri, S. mitis, S. sanguis, S. mitior, and S. salivarius were differentiated.

[The establishment efficiency of S. sobrinus OMZ 176 in a model caries experiment on the Osborne-Mendel rat]. / Etablierungseffizienz von S. sobrinus OMZ 176 im Kariesmodellversuch an der Osborne-Mendel Ratte.

S. sobrinus OMZ 176 was inoculated in young OM rats during tooth eruption in two experiments and the microbial composition of dental plaque as well as the caries decay was estimated after 25 days on cariogenic sucrose rich diet. Germs were reproducibly established in the oral cavity by such way. The resulting caries decay correlated with the streptococci in the dental plaque. Due to this fact it seems to be necessary to identify not only the inoculated microorganisms at the end of each animal experiment but also to estimate the qualitative composition of the whole dental flora which may have an essential cariogenic effect.

[Cotton roll or rubber dam in endodontic treatment?]. / Watterolle oder Kofferdam bei endodontischen Behandlungsmassnahmen?

In a clinical-microbiological study of the bacterial flora on deep areas of carious dentine in deciduous and permanent human teeth the aseptic rubber dam technique was given preference to remove successive samples of dentine from the cavity floors. The application of the rubber dam made it possible to establish and maintain a surgically clean field of operation and were an effective safeguard against contamination through seepage of saliva.

[The distribution of mutans streptococci and lactobacilli depending on the caries prevalence in 11- to 12-year-old children]. / Die Verbreitung von Mutans-Streptokokken und Laktobazillen in Abhängigkeit vom Kariesbefall bei 11- bis 12jährigen Kindern.

20 Korean children, aged 11 to 12 years, participated in this study with the aim to compare the microbial composition of plaque samples in order to estimate, which genera (lactobacilli, streptococci) or species are associated with coronal caries. Significant differences in the microbial composition of plaque samples between children with healthy and carious dentitions were found. S. mutans (c, e, f) was dominating under Mutans-streptococci.

The effects of non-fluoridated and fluoridated milk on experimental caries in rats.

The aim of the present investigation was to determine the caries-protective potential of non-fluoridated and fluoridated milk and to compare the efficacy of different compounds of fluoride as additives to milk. OM rats were maintained in three experiments in a frequency-controlled feeding machine of König et al. (1968) or, during one study, in metabolic cages with diet MIT 200 for three weeks. They received (1) milk with Ca-Fluoride [solubilized by KA1-Sulfate], (2) NaF, (3) NaMFP, and (4) Na-Silicofluoride. Controls were supplied with water or NaF solution of the same concentration of 10 or 15 ppm F. In addition, raw milk was provided ad libitum for the rats in a preliminary test. At the beginning and the end of the experiments, the pH of milk, its fluoride concentration, the body weight gain, the caries score, the fluoride concentration of the outermost enamel surface, the percentage of the interproximal bacteria, and the fluoride ingestion and excretion were determined. The raw milk significantly reduced the animal caries score by around 40%. This effect was lower but reproducible under programmed feeding with milk of a reduced fatty content (1.5%). The addition of Ca-Fluoride, which was not totally ionized (6.5 ppm F), reduced the caries score again by around 40%. Increasing concentrations of NaF (5, 10, 15 ppm F), Na-Silicofluoride, or NaMFP showed similar caries-inhibiting effects without remarkable influence of the fluoride dosage used. The percentage of streptococci ranged from 30 to 60 in the fluoridated milk and control groups as well. The increasing fluoride deposition in the enamel reflected the various fluoride dosages offered. The rats receiving non-fluoridated milk or distilled water had a significantly higher incidence of dental caries than those receiving fluoridated milk. The permanent availability of fluoride during the animal tests caused a higher caries-inhibiting effect than in clinical human studies reported.