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. Author manuscript; available in PMC: 2011 Jul 29.
Published in final edited form as: J Appl Microbiol. 2006 Jun;100(6):1339–1347. doi: 10.1111/j.1365-2672.2006.02877.x

Comparison of culture media and chairside assays for enumerating mutans streptococci

GH Hildebrandt 1, WA Bretz 2,3
PMCID: PMC3146258  NIHMSID: NIHMS313204  PMID: 16696682

Abstract

Aim

This study compared several traditional culture-based media and chairside cultural assays for ability to recover mutans streptococci (MS) from pure cultures and from saliva samples.

Methods and Results

When pure cultures were used with traditional culture-based media, mitis-salivarius bacitracin (MSB) agar demonstrated less support for bacterial recovery than trypticase-yeast extract-cysteine sucrose-bacitracin (TYCSB) agar and the modified medium of Ritz (HLR-S). One species of MS, Streptococcus ferus (c), was not recovered on MSB medium. Chairside cultural tests displayed considerable disparity between tests in recovering bacteria from pure cultures. On the glass adherence assay (Mucount®), S. ferus was not detected and Streptococcus criceti was not detected on the dipslide assay (Carie-screen SM®) or on the plastic adherence assay (Dentocult SM Strip mutans®). The frequency of isolation of pure strains of bacteria other than MS was common. From saliva samples, the frequency of isolation of MS on HLR-S and TYCSB media and the glass adherence assay was 91–97%. The frequency of isolation on MSB medium and on the dip-slide and plastic adherence assays was significantly decreased (37, 47 and 69%, respectively). Recovery scores varied considerably among the culture methods studied and tended to be highest on the HLR-S medium and on the glass adherence assay.

Conclusions

Growth and recovery profiles of pure bacterial cultures and of saliva samples for the MS varied according to different media.

Significance and Impact of the Study

Caution should be exercised in comparing results between studies that employ different cultural methods for MS enumeration.

Keywords: culture media, mutans streptococci, pure strains, rapid assays, saliva

Introduction

The pattern of dental caries prevalence is changing. Today, a small segment of the population is experiencing the majority of the burden of this infectious disease (Glass 1982; White et al. 1995). This has resulted in a great interest in caries risk assessment (Anderson 2002). By identifying risk factors associated dental caries, individuals most likely to develop caries lesions can be identified and targeted for preventive and therapeutic treatments (Pitts 1998).

The mutans streptococci (MS) have long been implicated as a significant contributor to the development of human dental caries on both coronal and root surfaces (Loesche 1986; van Houte et al. 1990). The risk of developing caries lesions is related to the levels of MS on the dentition, which is often estimated by culturing stimulated saliva (Keene 1986; Mundorff et al. 1990). Isolation of MS from dental plaque or saliva samples involves culturing samples on selective media or chairside cultural assays. Traditional selective culture-based media are generally used in research, e.g., epidemiological surveys and clinical trials. Traditional culture-based media techniques generally involve the plating of sonicated and serially diluted samples onto selective agar media, incubation under anaerobic conditions, and enumeration with the aid of a stereomicroscope.

Chairside cultural tests (rapid assays) for the enumeration of MS have been introduced which lower the cost, reduce the amount of equipment needed, and simplify the enumeration process for the purpose of making MS screening available to the practicing dentist. Screening for MS in clinical practice is seen by some as important for assessing caries risk and monitoring the success of caries control efforts (Bretz et al. 1992; Krasse 1988). Chairside cultural tests are often used in epidemiological and caries intervention studies in place of conventional microbiological techniques. As these methods become more popular and find their way into new applications, the limitations of these tests and their comparability to traditional culture-based media needs to be documented.

There is concern that the various traditional culture-based media methods and chairside cultural tests differ in terms of recovery of MS, both qualitatively (which strains are recovered) and quantitatively (comparing relative recovery rates) (Dasanayake et al. 1995). Accordingly, the purpose of this study was to compare several traditional culture-based media methods and chairside assays in terms of recovery of MS.

Materials And Methods

Rationale for selecting study strains

The MS are cariogenic streptococci often found in plaque biofilms and in saliva which exhibit distinct biochemical profiles, produce extracellular polysaccharides from sucrose, and are associaged with dental caries in animal models (Coykendall 1974). Eight serotypes can be recognized on the basis of carbohydrate antigens (Perch et al. 1974) whereas DNA hybridization studies demonstrate the existence of four genetic groups (Coykendall 1974). In fact, the genotypic heterogeneity of MS human isolates has become apparent since two or three genotypes within serotypes (c-g) have been ascertained (Saarela et al. 1993).

Streptococcus mutans strains possess the c, e or f antigens, where the c serotype accounts for about 70–100% of the human isolates of MS. Streptococcus mutans therefore has been individually and collectively named as the MS group. Other human isolates of MS possess d, g and h carbohydrate antigens such as Streptococcus sobrinus. S. rattus (serotype b) and S. cricetus (serotype a) are essentially laboratory-bred (Hamada and Slade 1980) and recently have been renamed to Streptococcus criceti and Streptococcus ratti (Truper and De Clari 1997). A serotype c MS has been isolated from wild rats and because of its genetic unrelatedness to S. mutans and the other MS was named as the new species Streptococcus ferus (Whatmore and Whiley 2002). In addition, certain serotype c strains from monkeys have shown to be significantly different from S. mutans and have been named a new species, Streptococcus macacae (Beighton et al. 1984).

We elected to include in the MS group strains that are isolated from humans and animals. We additionally included a non-MS group as control species often found in plaque and saliva such as Streptococcus sanguinis and Streptococcus salivarius. Veillonella dispar thrives on S. mutans by-products and therefore was also selected. These non-MS organisms were chosen because they are commonly found among the nonspecific growth from plaque and saliva samples.

Laboratory phase

Strains

The study was carried out in two phases: a laboratory and a clinical phase. For the laboratory phase, pure cultures of the following strains of MS were obtained from the American Type Culture Collection: S. mutans (c/e/f, ATCC 25175), S. ratti (b, ATCC 19645), S. criceti (a, ATCC 19642), S. sobrinus (d/g, ATCC 33478), and S. ferus (c, ATCC 33477). Pure cultures of the following additional species were also obtained: S. sanguinis (ATCC 10556), S. salivarius (ATCC 13419), S. macacae (ATCC 35911), and V. dispar (ATCC 17748).

Cultural procedures

Cultures were revived, tested for purity, and their identity confirmed by using the biochemical scheme of Shklair and Keene (1974). The fermentation of raffinose, melibiose and manitol for the most part can distinguish MS serotypes. Tubes containing 10 ml sterile trypticase soy broth were inoculated with 100 μl of one or two different pure cultures in late log phase and incubated overnight at 35 °C in an anaerobic chamber (Coy Laboratory Inc., Ann Arbor, MI, USA; 85% N2, 10% H2 and 5% CO2) yielding 108–109 CFU ml−1. The broth suspensions were agitated for 5 s with a vortex mixer (Vortex Genie, Model K-550-G, Scientific Industries Inc., Springfield, MA, USA) and cell aggregates dispersed for 10 s with a sonifier cell disrupter (Model W185D, Heat-Systems-Ultrasonics, Inc., Plainview, L.I., NY, USA). Serial dilutions were prepared utilizing reduced transport fluid (RTF) (Syed and Loesche 1972) and 50 μl of each prepared dilution was plated with an automatic spiral plater (Model D, Spiral Systems Inc., Cincinnati, OH, USA) on the following agar media: mitis-salivarius bacitracin (MSB), trypticase-yeast extract-cysteine sucrose-bacitracin (TYCSB), and the modified medium of Ritz (HLR-S) (Table 1).

Table 1.

Constituents of culture media

Media constituents
Media Base Sucrose (%) Bacitracin
(U ml−1)		 Other anti-microbial
agents (μgml−1)		 References
MSB Mitis salivarius agar 20 0·2 None Gold et al. 1973
TYCSB Trypticase-yeast-cystine agar 20 0·1 None van Palenstein Helderman et al. 1983
HLR-S Trypticase soy agar 20 0·3 Polymyxin B sulfate (1·75),
 crystal violet (0·5)		 Ritz 1967; Lang et al. 1987
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MSB, mitis-salivarius bacitracin; TYCSB, trypticase-yeast extract-cysteine sucrose-bacitracin; HLR-S, modified medium of Ritz.

Plates were incubated in an anaerobic chamber at 35 °C for 3–5 days and colony forming units (CFU) enumerated under a stereomicroscope by the sector-count method (Loesche and Straffon 1979). Counting was performed by two calibrated investigators. In case of doubt, colony identity was confirmed by mannitol (broth test) fermentation.

Chairside assays

Sonicated pure cultures were applied to the following chairside cultural tests: Cariescreen SM® (John O. Butler Co., Chicago, IL, USA)-20 μl into 10 ml diluent; Mucount® (Showa Yakuhin Kako Co., Tokyo, Japan)-4 μl into 2 ml liquid medium; and Dentocult SM Strip mutans® (Orion Diagnostica, Helsinki, Finland)-10 μl onto plastic test strip then immersed in 5 ml liquid medium. Instructions provided by the manufacturer were used to determine the parameters of incubation (temperature, duration and orientation). A detailed description of chairside cultural procedures can be seen in Table 2. It should be noted however that when using pure cultures an adaptation of sampling procedures was necessary to verify growth of pure cultures on the various assays. In this manner, the number of CFU was determined by utilizing a compromise scale that included the scores of the assays provided by the manufacturers (Table 3). Two calibrated examiners made this measurement with consensus on disagreements. Representative colonies of MS were tested for mannitol fermentation (broth test). Counting of both media plates and commercial kits were performed without knowledge of sample source or counts on other media.

Table 2.

Synopsis of manufacturer instructions for use of chairside cultural tests

Cariescreen SM (John O. Butler Co.) (Jordan et al. 1987)
 (1) Add bacitracin tablet (750 IU) to diluent vial (10 ml diluent containing 85 mg sodium chloride, 39 mg monobasic potassium phosphate,
   and 107 mg dibasic potassium phosphate)
 (2) Subject chews paraffin and expectorates directly into diluent vial (approx. 1 ml) (Note: in this study for consistency, saliva volume
   was measured)
 (3) Gently mix 5 s
 (4) Immerse dipslide in diluent vial, remove and blot off excess fluid at edge of slide, insert slide into dipslide vial containing activated carbon
   dioxide tablet
 (5) Incubate vertically 48 h at 37°C
 (6) Mutans streptococci levels estimated by comparison against a gradient density scale
Mucount (Showa Yakuhin Kako Co.) (Matsukubo et al. 1981)
 (1) Prepare reagent solution by adding paper discs impregnated with bacitracin, potassium tellurite and trypan blue to 1 ml of dissolving
   buffer and mixing gently.
 (2) Add one drop (approx. 40 μl) reagent solution to glass vial containing the liquid medium (2 ml)
 (3) Add 0·1 ml saliva to the medium, cap and mix gently
 (5) Incubate angled-horizontally 24 h at 37°C
 (6) Mutans streptococci levels estimated by comparison against a gradient density scale
Dentocult SM strip mutans (Orion Diagnostica) (Jensen and Bratthall 1989)
 (1) Add bacitracin impregnated wafer to culture broth vial
 (2) Subject chews paraffin and then introduces roughened end of test strip into mouth, rotates in over tongue about ten times, and then
   removes it through lightly closed lips
 (3) Inoculated test strip is then immersed in culture broth vial
 (4) Incubate vertically 48 h at 37°C
 (5) Mutans streptococci levels estimated by comparison against a gradient density scale
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Table 3.

Compromise study scale for chairside cultural tests used in this study

Study scale
 Cariescreen SM® scale
 Mucount scale
 Dentocult SM strip mutans
scale
Score Density of growth† Score Density of growth† Score Density of growth† Score Density of growth†
0 ND 0 ND − ND 0 ND
1 104 1 104 + <3 × 104 1 <105
2 5 × 104
2 105 3 105 ++ 3 × 104–105 2 105–106
4 2·5 × 105
5 5 × 105
3 106 6 106 +++ >105 3 >106
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†

Colony forming units ml−1.

ND, not detectable.

Clinical phase

Saliva collection and secretion rate

Saliva samples were employed in order to compare the ability of the various selective agar and chairside cultural tests to recover MS from saliva samples. The following study protocol was reviewed and approved by the Human Subjects Committee of the University of Michigan, USA. Informed consent was obtained from each subject. Three-minute paraffin-stimulated saliva samples were collected from 35 subjects (20 males and 15 females, 12–49-years old, mean 27) selected from patients at the clinics of the University of Michigan School of Dentistry, USA based on the diagnosis of active dental caries. Active caries is defined by an area of whitish/chalky enamel namely white spot lesion or any other cavitation with softened tissue. Three-minute salivary flow rates yielded on average 2·8 ml (SD 0·8).

Microbiological procedures

Bacteriological procedures followed the same format as for the pure cultures in the laboratory phase for traditional culture-based media except that saliva replaced the bacterial broth suspensions. Samples were sonified, serially diluted in RTF and introduced onto traditional culture-based media plates. Chairside cultural assays were processed according to manufacturer’s instructions and are briefly described (see Table 2): (i) The Cariescreen SM test requires 1 ml of stimulated saliva to be added to a diluent vial containing buffering solution; (ii) Mucount requires 0·1 ml of saliva to be added to a 2 ml dissolving buffer; (iii) The Dentocult SM Strip mutans was processed at the time the patient provided the salivary sample by rotating a spatula on the tongue about ten times and inserting it into a vial containing 5 ml of culture medium. Samples were subsequently incubated, and enumerated in the same manner as previously described (Table 3).

Statistical analysis

For the laboratory phase, relative recovery rates are descriptive since no statistical analysis was possible due to the small number of samples. Categorical data for the chairside cultural tests were not easily compared due to different scales assigned by the manufacturers. A compromise scale was employed for comparison purposes (Table 3).

In the clinical phase, comparison of MS recovery between traditional culture-based media and chairside cultural tests was performed by evaluating isolation frequency and by converting counts according to the four-point study scale (Table 3). These comparisons were carried out with Friedman overall analysis and Wilcoxon pairwise tests. Counts from traditional culture-based media were log10 transformed to control for variance in order to apply parametric statistical tests to compare MS recovery between the various selective agar media. These comparisons were carried out with anova and paired t-tests. The detection thresholds for MS on MSB, TYCSB and HLR-S media were 2·3 log10 CFU ml−1 of saliva relative to media with undetectable MS colonies.

Results

In the laboratory phase of the study, the abilities of traditional culture-based media and chairside cultural assays to support the growth of pure cultures of different strains of MS and non-MS organisms are presented in Tables 4 and 5. All five strains of MS were recovered from both plates of all three selective agar media, except S. ferus on MSB medium. Streptococci other than MS were also recovered on the three selective agar media. However, MSB medium did not support S. salivarius and supported S. sanguinis and S. macacae at lower levels. On the chairside cultural assays, all five strains of MS produced positive test results except S. criceti (on dipslide and plastic adherence assays) and S. ferus (on glass adherence assay). Nonspecific growth was also noticed on all three chairside cultural tests. The plastic adherence assay supported growth of the non-MS as well as of V. dispar.

Table 4.

Relative recovery rates of bacteria from pure cultures on various selective agar media (1 : 1000 dilution)

MSB TYCSB HLR-S
Control 0 0 0
Mutans streptococci
 Streptococcus mutans (c/e/f) 2 3 3
 S. ratti (b) 1 3 3
 S. criceti (a) 1 2 3
 S. sobrinus (d/g) 1 3 3
 S. ferus (c) 0 3 3
 S. macaccae (c) 1 3 3
Other bacteria
 S. sanguinis 1 3 3
 S. salivarius 0 1 1
 V. dispar 0 0 0
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0, no growth; 1, intermittent colonies, 2, discontinuous lawn, 3, continuous lawn.

MSB, mitis-salivarius bacitracin; TYCSB, trypticase-yeast extract-cysteine sucrose-bacitracin; HLR-S, modified medium of Ritz.

Table 5.

Recovery rates of bacteria from pure cultures on various chairside cultural tests

Dipslide assay
(cariescreen
SM)		 Glass
adherence
assay
(Mucount)		 Plastic
adherence
assay
(Dentocult SM)
Control 0 0 0
Mutans streptococci
 S. mutans (c/e/f) 2 3 3
 S. ratti (b) 3 1 2
 S. criceti (a) 0 3 0
 S. sobrinus (d/g) 1 3 3
 S. ferus (c) 1 0 3
 S. macaccae (c) 0 2 3
Other bacteria
 S. sanguinis 1 0 3
 S. salivarius 1 0 1
 V. dispar 0 0 1
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0, no growth; 1 = 104; 2 = 105; 3 = 106.

The isolation frequency of MS saliva samples for the various assays were as follow (data not shown): HLR-S (97%), glass adherence assay (97%), TYCSB medium (91%), plastic adherence assay (71%), dipslide assay (47%), and MSB medium (40%). The mean levels of MS recovered from the saliva samples on the traditional culture-based media were for MSB, TYCBS and HLR-S, respectively, 2·6± 0 ·7, 5·1 ± 1·1, 6·3 ± 1·0 log10 CFU ml−1 of saliva (data not shown). Mitis-salivarius bacitracin recovered significantly lower levels of the MS than did HLRS and TYCBS (P < 0·001).

Comparison of MS recovery from clinical samples between traditional culture-based media and chairside cultural tests is presented in Table 6. There are considerable differences between methods. Scores 2 and 3 (corresponding to MS levels ≥105 CFU ml−1) were most frequently recorded for HLR-S medium (33/35) and the glass adherence assay (32/35), with significantly lower frequencies for TYCSB medium (23/35), the plastic adherence assay (16/35), the dipslide assay (2/34), and MSB medium (1/35).

Table 6.

Number, mean and median values of various scores using the study scale for mutans streptococci recovered from 35 saliva samples*

Study
scale		 MSB TYCSB HLR-S Dipslide assay
(Cariescreen SM)		 Glass adherence
Assay (Mucount)		 Plastic adherence
assay (Dentocult SM
strip mutans)
0 22 3 1 18 1 11
1 12 9 1 14 2 8
2 1 15 5 2 8 11
3 0 8 28 0 24 5
Mean (SD) 0·4 (0·5) 1·8 (0·9) 2·7 (0·7) 0·5 (0·6) 2·6 (0·7) 1·3 (1·1)
Median 0 2 3 0 3 1
Pairwise
P < 0·01† 		TYCSB,
HLR-S,
 glass adherence
 assay, plastic
 adherence assay		 HLR-S dipslide
 assay, glass
 adherence assay		 Dipslide assay,
 plastic adherence
 assay		 Glass adherence
 assay, plastic
 adherence assay		 Plastic adherence
 assay
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*

Overall significance for mean comparisons among cultured media and assays, P < 0·001 by Friedman’s two-way analysis.

†

By Wilcoxon signed rank test with Bonferroni’s correction.

MSB, mitis-salivarius bacitracin; TYCSB, trypticase-yeast extract-cysteine sucrose-bacitracin; HLR-S, modified medium of Ritz.

Discussion

Isolation of MS from plaque biofilms or saliva samples for epidemiological and caries intervention studies have involved culturing samples on certain agar-based media with traditional anaerobic microbiological techniques. Because identifying and enumerating MS colonies amidst a heavy growth of other bacterial types can be labour intensive, several isolation media selective for MS have been developed to facilitate identification and enumeration in clinical samples. These selective media were formulated by the addition of bacitracin and high concentrations of sucrose to various plating media. Sucrose and bacitracin act to inhibit the growth of most commonly recovered oral bacteria other than MS. Sucrose fosters the growth of MS colonies with a characteristic morphology because of synthesis of extracellular polysaccharides which can facilitate identification.

On these selective media, MS demonstrate characteristic colonial morphology with a ‘wet sugar’ appearance, being firmly affixed to the underlying medium, and often studded with droplets or surrounded by a puddle. Occasionally, the colonies are surrounded by an opaque halo on TYCSB and HLR-S agar. Although some nonspecific growth is observed, MS colonies can readily be distinguished by morphology on these media. These characteristic colonial morphologies are not so readily identifiable in the rapid chairside tests, which may render comparisons among assays difficult.

Media selective for MS may failed to recover certain strains and serotypes (Liljemark et al. 1976; Staat 1976) and tended to quantitatively underestimate MS levels, although they are present in relatively high proportions in clinical samples (Emilson and Brathall 1976; Little et al. 1977). Percent recovery of MS depends to some degree on the isolation medium employed (Emilson and Brathall 1976; Little et al. 1977; van Palenstein Helderman et al. 1983). The exclusive use of one selective medium for the quantification of MS could conceivably lead to an inaccurate picture of MS levels in clinical samples (Loesche and Syed 1973; Tanzer and Clive 1986).

This study looked at relative recovery rates (differences in recovery between methods) for various bacterial strains and not absolute recovery rates (differences in recovery between bacterial strains). We acknowledge that it would not be appropriate to analyse differences in numbers of colonies between strains from pure cultures because no effort was taken to start with inocula of identical concentration nor did we use a nonselective medium as the gold standard count. The relative recovery rates measured in this study demonstrate that in terms of the five strains of MS chosen (representing each of the five species), MSB medium is less effective at recovery than TYCSB and HLR-S media (Table 4).

In the laboratory phase of the study, all strains of MS were recovered from both plates of all three selective agar media, except S. ferus on MSB media (Table 4). The lack of support for S. criceti (serotype a) by MSB media has been reported previously (Liljemark et al. 1976; Staat 1976; Little et al. 1977). Since these species are not common to man, this difference is of little clinical importance, except in epidemiological studies aimed at surveying species distribution. Because not all species of MS are supported by MSB, using this medium to study distributions of MS serotypes or species could conceivably lead to inaccurate conclusions. The two species that are the most clinically relevant (S. mutans and S. sobrinus) were recovered at lower levels on MSB medium compared to the other two selective agar media.

On the chairside cultural tests, all five strains of MS produced positive test results except S. criceti (on dipslide and plastic adherence assays) and S. ferus (on glass adherence assay) (Table 5). This is not surprizing for the dipslide method since the agar utilized in this test is similar to MSB medium. For the clinically relevant MS species (S. mutans and S. sobrinus) the dipslide assay recorded MS at slightly lower levels than the adherence assays. The implication is that the dipslide assay may have a lower sensitivity than the adherence assays tested. Nonspecific growth was also noticed on all three chairside cultural tests. The dipslide assay supported all three streptococci tested as well as V. dispar. Support of these non-MS bacteria suggest a less than ideal specificity. This corroborates the findings of other authors that the dipslide and plastic adherence assays have low sensitivity and moderate specificity (Newbrun et al. 1984; Stecksen-Blicks 1985; Alaluusua et al. 1990). The glass adherence assay being less supportive of nonspecific growth is corroborated in the literature (Matsukubo et al. 1981). These findings (Tables 4 and 5) should be put into perspective as the clinical use of cultural and chairside tests with saliva samples begins to show its value not with pure cultures, but in the presence of high density, competing bacteria. In the clinical phase of the study, there was considerable disparity between methods. Isolation frequencies of MS were higher for TYCSB and HLR-S media and the glass adherence assay than for the other methods. Recovery levels of MS were highest for the same three methods (Table 6).

Estimating mean MS load for the group of 35 subjects with traditional culture-based media resulted in values that were 2–3 log10 units lower (100–1000 times lower) when derived from growth on MSB medium rather than on TYCSB or HLR-S media. This agrees with the laboratory phase finding where the species of MS most commonly found on the human dentitions (S. mutans and S. sobrinus) were not as well supported by MSB medium compared to TYCSB and HLR-S media (Table 4). The finding that MSB medium is less supportive of MS than other selective agar is corroborated in the literature (van Palenstein Helderman et al. 1983; Tanzer et al. 1984; Schaeken et al. 1986; Wan et al. 2000).

Using the compromise study scale to compare MS recovery on the three traditional culture-based media and three chairside cultural tests, MSB medium and the plastic adherence assay more frequently displayed low-end scores and HLR-S medium and the glass adherence assay more frequently displayed high-end scores (Table 6). These high-end-scores could potentially reflect nonspecific growth on these assays albeit difficult to ascertain. The clinical relevance of these differences depends on the study design. In clinical intervention studies, these differences are not particularly significant since changes in MS load over time are monitored on the same type of selective medium. In epidemiological studies, too, the differences in MS load between groups (cross-sectional) or over time (longitudinal) are assessed using the same type of selective medium throughout.

Studies, which enroll subjects based on MS load are usually biasing the study in favour of disease to evaluate a caries intervention strategy. If MSB medium is used for this screening, the subjects enrolled will likely harbour higher actual MS loads than if any of the other traditional culture-based media or chairside cultural tests are used. This may become significant when comparing effectiveness of various intervention strategies by comparing MS reductions between studies that used different cultural tests.

Studies involving the estimation of risk for developing caries lesions which base this risk (in whole or in part) on MS load will be affected by the choice of the cultural test employed. In clinical practice, caries risk assessment is used to target a segment of the practice population for caries control efforts. Using a threshold of ≥105 CFU ml−1 for risk assessment as an example, in this study, subjects were more frequently identified as at-risk with HLR-S medium (33/35) and the glass adherence assay (32/35) than with any of the other methods. If less sensitive tests are employed (e.g., MSB medium or the plastic adherence assay), a more select group of individuals will tend to be identified as at-risk. The effect of this on caries control efforts for the patient population of a practice is uncertain – but it is possible that some patients at-risk will fail to be identified and that caries control efforts for these individuals will not be instituted.

In conclusion, MSB medium is a less supportive medium compared to TYCSB and HLR-S media, recovering less S. mutans and S. sobrinus, as well as less nonspecific growth. MSB medium also will not support well S. ferus, although this shortcoming is of limited clinical significance. The chairside cultural tests are supportive of MS but tend to grow other nonmutans organisms. The dipslide assay was the least supportive of MS from clinical samples, the plastic adherence assay was intermediate, and the glass adherence assay was the most supportive. There was considerable disparity in MS recovery from clinical samples between the traditional culture-based media and chairside cultural tests studied. Therefore, caution should be used in comparing results from different studies where different media were employed and in designing studies it is advisable to maintain one cultural method throughout. Finally, these findings have to be put into perspective. Current advances in molecular microbial genetic analysis have provided a battery of assays including reverse capture checkerboard hybridization (Becker et al. 2002), DNA microarrays (Smoot et al. 2005), pulsed-field gel electrophoresis (Mineyama et al. 2004), real-time PCR (Yano et al. 2002), among others, that can enumerate and classify the MS in a significantly more precise and diverse manner. The applicability of these advances for epidemiological studies of the MS however, remains to be determined.

Acknowledgements

We would like to acknowledge the assistance of ‘Bonnie’ Harris and Janice Stoll in media preparation and the assistance of Robert Lucero and Dena Fisher in sample processing. This research was supported by grants from NCRR 2-S07-RR05321-30 and from NIDCR DE13534.

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