JOURNAL OF CLINICAL MICROBIOLOGY, June 1977, p. 578-583 Copyright 0 1977 American Society for Microbiology Vol. 5, No. 6 Printed in U.S.A. Comparative Recovery of Streptococcus mutans on Ten Isolation Media W. A. LITTLE,* D. C. KORTS, L. A. THOMSON, AND W. H. BOWEN National Caries Program, National Institute of Dental Research, Bethesda, Maryland 20014 Received for publication 1 February 1977 The ability of Streptococcus mutans (Bratthall serotypes a through e) to grow on 10 isolation media was examined. The number and morphology of the colonies were observed to vary on different media. The use of blood-sucrose media consistently produced the highest recoveries. Mitis salivarius agar (MS) had higher recovery values than modified medium 10 (MM1OSB), Trypticase-yeast extract-cystine medium (TYC), or MS with 1% tellurite (MST). MST with 40% sucrose (MS40S), MST with 20% sucrose and 0.2 U of bacitracin per ml (MSB), and Carlsson medium with 1% sulfasoxazole (MC), media formulated for the selection of S. mutans, were the most inhibitory for all serotypes. The morphol- ogy of several S. mutans strains was atypical on MC and MS4OS, making positive identification difficult. Absence of growth of serotype a strains on MSB and serotype d strains on MC were the two major differences observed among the serotypes. Results are discussed in terms of the difficulties in making quantitative determinations from cultural data. Studies investigating the association of Streptococcus mutans with dental caries have directed attention to methods for the isolation of this organism from clinical specimens. Isola- tion is usually accomplished by culturing sam- ples on media containing sucrose, which results in the formation of colonies with a characteris- tic morphology. Results of several investiga- tions (5, 8, 14) indicate that the percentage of recovery for S. mutans varies, depending in part, at least, on the medium used. The results of human population studies (5, 9, 11, 12; J. D. de Stoppelaar, Ph.D. thesis, Drakkerij Flinkwijk, the Netherlands, 1971) demonstrate that serotype c is the most pre- valent of the Bratthall (1) serotypes. Evidence indicates that the apparent prevalence of sero- types may be influenced by the isolation medium used. Serotype d strains have been reported to grow poorly on Carlsson sulfona- mide media (5, 6, 11), and several stock strains of serotype a have failed to grow on MSB agar (5, 13). The purpose of the present study was to ex- amine the ability of S. mutans to grow on 10 isolation media and to determine whether any of the serotypes were selectively inhibited. MATERIALS AND METHODS The ability of S. mutans to grow on the following media was determined: (i) BA-1, Trypticase soy agar (Baltimore Biological Laboratories, Cockeysville, Md.), 5% defibrinated sheep blood, and 1% sucrose; (ii) BA-5, Trypticase soy agar, 5% defibrinated sheep blood, and 5% sucrose; (iii) MS, mitis salivar- ius agar (Difco Laboratories, Detroit, Mich.); (iv) MST, MS, 1% Chapman tellurite (Difco) (4); (v) MS40S, MS, 1% Chapman tellurite, and 40% sucrose (total concentration) (8); (vi) MSB, MS, 1% Chap- man tellurite, 20% sucrose (total concentration), and 0.2 U of bacitracin per ml (The Upjohn Co., Detroit, Mich.); (vii) TYC, Trypticase (Casitone [Difco]), yeast extract cystine (Baltimore Biological Laboratories) (J. D. de Stoppelaar, Ph.D. thesis); (viii) MM1OSB, modified medium 10, with 5% sucrose and 2% sheep blood (10), (ix) MC, Carlsson medium, 0.1% sulfasoxazole (Gantrisin, Roche Lab- oratories, Nutley, N.J.) (2); (x) MC(.005), Carlsson medium, 0.005% sulfasoxazole (3, L. A. Thomson, doctoral dissertation, University of Michigan, Ann Arbor, 1970). All media were prepared within the same week according to manufacturer's or developer's instruc- tions and stored at 4°C. Media were used for a period of up to 1 month, except for MSB, which was pre- pared fresh weekly. Plates were stored in plastic bags to minimize evaporation. MC media were used containing sulfonamide con- centrations of 0.1%, as originally described (2), and 0.005%, which the results of previous work (16) indi- cated was closer to the concentration permitting growth of most S. mutans strains. Because the sul- fonamide (sulfadimetine [Elkosin], CIBA-GEIGY Limited, Basel, Switzerland) used by Carlsson (2) is not readily available in the United States, sulfasox- azole was used in the preparation of the MC media. The 18 strains of S. mutans listed in Table 1 were used in this study. Tubes containing 10 ml of Todd- Hewitt broth were inoculated with 0.1 ml of a pure culture of S. mutans and incubated for 20 h at 37°C. 578 on May 25, 2021 by guest http://jcm.asm.org/ Downloaded from on May 25, 2021 by guest http://jcm.asm.org/ Downloaded from on May 25, 2021 by guest http://jcm.asm.org/ Downloaded from
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JOURNAL OF CLINICAL MICROBIOLOGY, June 1977, p. 578-583Copyright 0 1977 American Society for Microbiology
Vol. 5, No. 6Printed in U.S.A.
Comparative Recovery of Streptococcus mutans on TenIsolation Media
W. A. LITTLE,* D. C. KORTS, L. A. THOMSON, AND W. H. BOWEN
National Caries Program, National Institute ofDental Research, Bethesda, Maryland 20014
Received for publication 1 February 1977
The ability of Streptococcus mutans (Bratthall serotypes a through e) to growon 10 isolation media was examined. The number and morphology of the colonieswere observed to vary on different media. The use of blood-sucrose mediaconsistently produced the highest recoveries. Mitis salivarius agar (MS) hadhigher recovery values than modified medium 10 (MM1OSB), Trypticase-yeastextract-cystine medium (TYC), or MS with 1% tellurite (MST). MST with 40%sucrose (MS40S), MST with 20% sucrose and 0.2 U of bacitracin per ml (MSB),and Carlsson medium with 1% sulfasoxazole (MC), media formulated for theselection of S. mutans, were the most inhibitory for all serotypes. The morphol-ogy of several S. mutans strains was atypical on MC and MS4OS, makingpositive identification difficult. Absence of growth of serotype a strains on MSBand serotype d strains on MC were the two major differences observed among
the serotypes. Results are discussed in terms of the difficulties in makingquantitative determinations from cultural data.
Studies investigating the association ofStreptococcus mutans with dental caries havedirected attention to methods for the isolationof this organism from clinical specimens. Isola-tion is usually accomplished by culturing sam-ples on media containing sucrose, which resultsin the formation of colonies with a characteris-tic morphology. Results of several investiga-tions (5, 8, 14) indicate that the percentage ofrecovery for S. mutans varies, depending inpart, at least, on the medium used.The results of human population studies (5,
9, 11, 12; J. D. de Stoppelaar, Ph.D. thesis,Drakkerij Flinkwijk, the Netherlands, 1971)demonstrate that serotype c is the most pre-valent of the Bratthall (1) serotypes. Evidenceindicates that the apparent prevalence of sero-types may be influenced by the isolationmedium used. Serotype d strains have beenreported to grow poorly on Carlsson sulfona-mide media (5, 6, 11), and several stock strainsof serotype a have failed to grow on MSB agar(5, 13).The purpose of the present study was to ex-
amine the ability of S. mutans to grow on 10isolation media and to determine whether anyof the serotypes were selectively inhibited.
MATERIALS AND METHODSThe ability of S. mutans to grow on the following
media was determined: (i) BA-1, Trypticase soy agar(Baltimore Biological Laboratories, Cockeysville,Md.), 5% defibrinated sheep blood, and 1% sucrose;
(ii) BA-5, Trypticase soy agar, 5% defibrinatedsheep blood, and 5% sucrose; (iii) MS, mitis salivar-ius agar (Difco Laboratories, Detroit, Mich.); (iv)MST, MS, 1% Chapman tellurite (Difco) (4); (v)MS40S, MS, 1% Chapman tellurite, and 40% sucrose(total concentration) (8); (vi) MSB, MS, 1% Chap-man tellurite, 20% sucrose (total concentration),and 0.2 U of bacitracin per ml (The Upjohn Co.,Detroit, Mich.); (vii) TYC, Trypticase (Casitone[Difco]), yeast extract cystine (Baltimore BiologicalLaboratories) (J. D. de Stoppelaar, Ph.D. thesis);(viii) MM1OSB, modified medium 10, with 5%sucrose and 2% sheep blood (10), (ix) MC, Carlssonmedium, 0.1% sulfasoxazole (Gantrisin, Roche Lab-oratories, Nutley, N.J.) (2); (x) MC(.005), Carlssonmedium, 0.005% sulfasoxazole (3, L. A. Thomson,doctoral dissertation, University of Michigan, AnnArbor, 1970).
All media were prepared within the same weekaccording to manufacturer's or developer's instruc-tions and stored at 4°C. Media were used for a periodof up to 1 month, except for MSB, which was pre-pared fresh weekly. Plates were stored in plasticbags to minimize evaporation.MC media were used containing sulfonamide con-
centrations of 0.1%, as originally described (2), and0.005%, which the results of previous work (16) indi-cated was closer to the concentration permittinggrowth of most S. mutans strains. Because the sul-fonamide (sulfadimetine [Elkosin], CIBA-GEIGYLimited, Basel, Switzerland) used by Carlsson (2) isnot readily available in the United States, sulfasox-azole was used in the preparation of the MC media.The 18 strains ofS. mutans listed in Table 1 were
used in this study. Tubes containing 10 ml of Todd-Hewitt broth were inoculated with 0.1 ml of a pureculture of S. mutans and incubated for 20 h at 37°C.
A 3-ml portion of the broth suspension was sonicallydispersed for 15 s at 20 W with a Branson W185sonifier (Heat Systems-Ultrasonics, Inc., Plainview,N.Y.) and special microtip. Dilutions producing col-ony-forming units within the 30 to 300 range werecalculated from standard turbidity curves read at660 nm with a Beckman model 2S spectrophotome-ter. Portions, 0.1 ml, were inoculated onto five repli-cate plates of each of the 10 media investigated.Diluting and plating procedures were performed un-der aerobic conditions. Plates were then incubatedin Gas-Pak (Baltimore Biological Laboratories) jarsfor 72 h, after which colonies were counted and meanvalues were calculated for each medium.
Colonies were photographed with a Bausch andLomb Stereozoom 7 microscope equipped with a Po-laroid camera and Polaroid 105 film. All plates wereilluminated with reflected light only. Magnificationwas identical for all colonies photographed.To facilitate comparison of media and to deter-
mine differences of statistical significance, meancounts were standardized as a percentage of themaximum mean count. The resulting standardizedpercentages were compared by using the Duncanmultiple comparison test (P = 0.05). The countswere analyzed a second time after logarithms (logo0)were taken to improve the homogeneity of the vari-ances. Only those differences among media thatwere significant both in the untransformed analysisand in the log-transformed analysis were declaredsignificant. One strain representing each serotype
TABLE 1. List of strains according to serotype
Serotype
a b c d eAHT Fa-1 10449 SL1 LM7E49 BHT GS5 KlR V1003720 JC1 B13 B2OMZ61 JC2 6715 B14
was examined on two separate occasions to estimatethe amount of variation inherent in the experimen-tal methods.The ultimate goal in comparing the growth of
pure cultures on different media is to be able tointerpret the results as an indication of what occurswhen plaque is studied. The examination of plaquewas not practical, because samples containing rela-tively high numbers of the individual serotypeswere not readily available. An additional experi-ment was performed in which representative strainsfrom each serotype were introduced into plaquesamples that were free of S. mutans as determinedby cultural and fluorescent-antibody (FA) methods.These mixtures were then dispersed and plated withthe same methods as those used to study the growthof pure cultures.
RESULTS
The results of the pure culture experimentare presented in Table 2. The percent recoveryvalue beneath each medium abbreviation is amean value based on the individual strainswithin each serotype. A line underscoring anytwo media indicates that the differences fromone to another are not statistically significant(P = 0.05). When the results of the individualstrains are grouped according to serotype, therecovery patterns for the 10 media appearedquite similar. For all serotypes, the blood-su-crose media (BA-1, BA-5) produced the highestrecoveries, with little difference between thetwo levels of sucrose. MST, MM1OSB, and TYChad lower recoveries than MS. MS40S, MSB,and the two MC media, all of which are selec-tive for S. mutans, were also the mostinhibitory. There was little difference betweenthe two levels of sulfasoxazole tested. It can beobserved from Table 3 that some strains from
TABLE 2. Media results presented as the percent recovery of the maximum mean count
each serotype failed to grow on one or more ofthe media; these were usually MSB, MS40S,MC, or MC(.005). In two separate experiments,one serotype c strain (JC1) failed to grow on
TYC agar. For a particular medium, the varia-tion in recovery levels observed within a sero-
type was generally as great as that observedamong strains of different serotypes. The mostpronounced differences among the media were
the failure of serotype a strains to grow on MSBand the absence of serotype d on either of thesulfasoxazole media.
Colonies of a given strain ofS. mutans variedin both number and morphology on differentmedia. Figure 1 illustrates the range in colonymorphology within a single strain. The coloniesof most strains appeared very small on MC andMS40S, making identification difficult in mixedculture.When a strain of S. mutans, in pure culture
and in plaque suspension, was grown simulta-neously on the different media, results were inclose agreement. Comparison of recovery levelsfor all media produced correlation coefficientsequal to 0.87, 0.93, 0.98, 0.96, and 0.99 for thestrains representing serotypes a through e, re-
spectively. These correlation values were gen-erally comparable to those resulting from a
pure culture examined on two separate occa-
sions (Table 4).
DISCUSSIONThe present study demonstrates the effect of
different media on the enumeration of S. mu-
tans. When a strain ofS. mutans was grown on
several media, a considerable range was ob-served in the percent recovery values and incolonial morphology. For a particular medium,a wide range of results was observed not onlyfor strains of different serotypes, but also forstrains within a serotype, and occasionally forrepeated experiments of the same strain. Vari-ations in the ability of a single medium or
J. CLIN. MICROBIOL.
different media to support the growth of S.mutans are further evidenced in results re-ported by other investigators.The most notable differences in media per-
formance have related to the growth of S. mu-tans on MSB and MC media. The paper describ-ing MSB (7) reported no inhibition of several S.mutans strains, including AHT (serotype a).However, other investigators (5, 13) have beenunable to grow serotype a strains on MSB.Serotype a strains AHT, OMZ61, E49, and 3720did not grow on MSB in the present study. Inaddition, serotype b strain FA-1 was com-pletely inhibited by MSB in one of the replica-tion experiments.
Shklair and Keene (12) observed no inhibi-tion of strains SL1, 6715, K1R, B13, or OMZ176when they used a medium containing sulfadi-metine. Furthermore, they were able to isolateserotype d from 15 of 194 plaque samples withsimilar media. Four of seven serotype d plaqueisolates were reported by Perch et al. (11) to besensitive to sulfathiazole. Emilson et al. (6)noted that a higher proportion of S. mutansfrom plaque grew on MS agar than on MC.They concluded that it was probable that somed strains are sensitive to sulfadimetine. In alater study (5), Emilson and Bratthall reportedthat 13 of 22 fresh isolates and 6 culture collec-tion strains failed to grow on MC agar. In thepresent experiment, strains SL1, 6715, K1R,and B13 were inhibited by media containing 0.1or 0.005% sulfasoxazole. Furthermore, at leastone strain representing serotypes a, b, c, and efailed to grow on one of the sulfasoxazole me-dia. Because sulfonamide inhibition will be re-duced by incorporation into insufficientlycooled media or into media containing traceamounts of certain nutrients, it is not surpris-ing to see considerable variation with the re-sults from studies that use MC agar.From Table 2, it is evident that the addition
of potassium tellurite to MS agar results in areduction in the number of colonies. This reduc-tion ranged from 6% for the serotype d strainsto 25% for the b strains, which parallels theobservation of Syed and Loesche (14), who re-ported a three- to fourfold decrease in S. mutansplaque counts on MS medium containing tellu-rite. In contrast, Gold et al. (7) found that theaddition of tellurite to MS did not affect thegrowth of S. mutans. Furthermore, differenceswere not detected between S. mutans plaquecounts on MST and blood-sucrose agar (heartinfusion base). In the present study, blood-su-crose agar produced higher recoveries than MSor MST. When the data for each serotype aregrouped, the difference is statistically signifi-
FIG. 1. Strain JC2 (serotype c) incubated 48 h (GasPak) on (A) BA-5, (B) TYC, (C) MSB, (D) MS, (E)MC and (F) MS40S. Colony size ranges from 0.15 to 1.5 mm. (x 24).
cant, as illustrated in the bottom row of Table2.
It was observed by Syed and Loesche (14) thatrecoveries of S. mutans from plaque were simi-lar on MS and MM1OSB. This observation is incontrast to the results of the present study thatshow pure culture counts to be higher on MSthan MM1OSB. The differences were statisti-cally significant for the serotype a and d strainsand the grouped data representing all of thestrains tested.
The most variable results were observed onTYC agar (Table 3). Strain JC1 (serotype c)failed to grow, and strain B14 (serotype e) grewvery poorly, although colonial morphology var-ied little on this medium.The level of variability in cultural results
prevents the unqualified recommendation of asingle medium for isolating S. mutans. Al-though the blood-sucrose media gave the high-est recoveries in this study, other factors mustbe considered. Colony morphology can vary
considerably from medium to medium, and onblood-sucrose agar some strains of S. mutansproduce atypical colonies, especially when theplate is crowded. There is also the possibilitythat the presence of S. mutans will be maskedby the overgrowth of other organisms. Samplescontaining few S. mutans are likely to be foundnegative even on media giving the best recover-ies. The colony-forming units of S. mutanswould be lost in the dilution necessary to pro-duce an uncrowded plate. In this type of situa-tion, it may be advisable to use selective mediasuch as MSB, MC, or MS40S, provided theirdeficiencies are fully realized. If sample dilu-tion is minimal, the inhibitory effect of thesemedia may be reduced. This rationale is sup-ported by the study of Emilson et al. (6), whodetected S. mutans in all 36 plaque samplesexamined by FA methods. On MS agar, 8 of the36 samples were negative for S. mutans,whereas on MC agar, 7 of the negative sampleswere positive. The present study indicates thatthe sole use of either MSB or MC is limitedbecause of their failure to support the growth ofselected serotype a or d strains, respectively.Furthermore, the atypical morphology ob-served with several strains on MS4OS and MCcould result in the failure to recognize a posi-tive sample.
If the levels of recovery of S. mutans fromplaque parallel the results of pure culture ex-periments, it would be expected that the per-centage of S. mutans in the total cultivableflora would vary with the choice of a medium.The study ofEmilson and Bratthall (5) supportsthis premise, although the differences they ob-served in S. mutans plaque counts among sev-eral media were not as great as differencesobserved with pure cultures on the same media.
In the present study, the number of S. mutanscolonies on a particular medium was essen-tially the same whether pure cultures or plaquesuspensions were used as the inoculum.The results of this study demonstrate the
variation in recoveries of S. mutans on differ-ent media and the limitation this approachplaces on making quantitative determinationsfrom cultural results. A combination of FA andcultural methods is currently used in this labo-ratory for the detection and isolation of S. mu-tans from clinical specimens. The media em-ployed are the two that consistently gave thehighest recoveries in this study, i.e., BA-5 andMS. In situations where a specimen is negativeculturally but positive by FA, isolation is at-tempted by direct plating onto one or more ofthe selective media, depending on the sero-type(s) identified by the FA technique.
LITERATURE CITED
1. Bratthall, D. 1970. Demonstration of five serologicalgroups of streptococcal strains resembling Streptococ-cus mutans. Odontol. Revy 21:143-152.
2. Carlmon, J. 1967. A medium for isolation ofStreptococ-cus mutans. Arch. Oral Biol. 12:1657-1658.
3. Carlson, J. 1968. A numerical taxonomic study of hu-man oral streptococci. Odontol. Revy 19:137-160.
4. Chapman, G. H. 1946. The isolation and testing of fecalstreptococci. Am. J. Dig. Dis. 13:105-107.
5. Emilson, C. G., and D. Bratthall. 1976. Growth ofStreptococcus mutans on various selective media. J.Clin. Microbiol. 4:95-98.
6. Emilson, C. G., B. Kohler, and D. Bratthall. 1974.Immunofluorescent determination ofthe relative pro-portions of Streptococcus mutans. Arch. Oral Biol.19:1079-1081.
7. Gold, 0. G., H. V. Jordan, and J. van Houte. 1973. Aselective medium for Streptococcus mutans. Arch.Oral Biol. 18:1357-1364.
8. Ikeda, T., and H. J. Sandham. 1972. A high sucrosemedium for the identification of Streptococcus mu-tans. Arch. Oral Biol. 17:781-783.
9. Jablon, J. M., T. Ferrer, and D. D. Zinner. 1974. Quan-titation of Streptococcus mutans by the membranefilter fluorescent antibody technique. Arch. OralBiol. 19:929-934.
10. Loesche, W. J., R. N. Hocket, and S. A. Syed. 1972. Thepredominant cultivable flora of tooth surface plaqueremoved from institutionalized subjects. Arch. OralBiol. 17:1311-1325.
11. Perch, B., E. Kjems, and T. Ravn. 1974. Biochemicaland serological properties of Streptococcus mutansfrom various human and animal sources. Acta Pa-
thol. Microbiol. Scand. Sect. B 82:357-370.12. Shklair, I. L., and H. J. Keene. 1974. A biochemical
scheme for the separation of the five varieties ofStreptococcus mutan8. Arch. Oral Biol. 19:1079-1081.
13. Staat, R. H. 1976. Inhibition of Streptococcus mutansstrains by different mitis salivarius agar prepara-tions. J. Clin. Microbiol. 3:378-380.
14. Syed, S. A., and W. J. Loesche. 1973. Efficiency ofvarious growth media in recovering oral bacterialflora from human dental plaque. Appl. Microbiol.26:459-465.
Comparative Recovery of Streptococcus mutans on TenIsolation Media
W. A. LITTLE, D. C. KORTS, L. A. THOMSON, AND W. H. BOWENNational Caries Program, National Institute ofDental Research, Bethesda, Maryland 20014
Volume 5, no. 6, p. 578, column 2, line 16: "medium, 0.005% sulfasoxazole (3, L. A. Thomson.."should read "... sulfasoxazole (L. A. Thomson..."Page 578, column 2, line 27: "0.005%, which the results of previous work (16) .. ." should read
".... previous work (L. A. Thomson, PH.D. thesis, 1970)..."
Simple Disk-Plate Method for the Biochemical Confirmationof Pathogenic Neisseria
JOSEPH A. VALUMicrobiology Research Laboratories, Akro-Medic Engineering, Inc., Denville, New Jersey 07834
Volume 3, no. 2, p. 172, column 2, line 15: "the aid of approximately 16.0 ml of 1 N NaOH ..should read ".... 1.6 ml of 1 N NaOH ...".
