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Recovery of mutans streptococci on MSB, SB-20 and SB-20M
agar media
Marta Estela Saravia
a,b
, Paulo Nelson-Filho
b,
*, Raquel Assed Bezerra Silva
b
,
Andiara De Rossi
b
, Gisele Faria
c
, Le a Assed Bezerra Silva
b
, Claes-Go ran Emilson
d
a
Department of Preventive Dentistry, School of Dentistry, National University of Tucuma n, T4000, San Miguel de Tucuma n, Argentina
b
Department of Pediatric Clinic, Preventive and Community Dentistry, School of Dentistry of Ribeira o Preto, University of Sa o Paulo, Av. do
Cafe , s/n Monte Alegre, 14040-904 Ribeira o Preto, SP, Brazil
c
Department of Restorative Dentistry, Araraquara Dental School, UNESP University Estadual Paulista, Rua Humaita , 1680, 14801-903
Araraquara, SP, Brazil
d
Department of Cariology, Institute of Odontology, The Sahlgrenska Academy at Gothenburg University, Box 450, SE-405 30 Gothenburg,
Sweden
a r c hi v e s of o r a l b i ol o gy 5 8 ( 2 0 1 3 ) 3 1 1 3 1 6
a r t i c l e i n f o
Article history:
Accepted 1 October 2012
Keywords:
Selective culture media
Streptococcus mutans
Streptococcus sobrinus
a b s t r a c t
Objective: The recovery of mutans streptococci in saliva and dental biolm samples
depends, in part, on the culture medium used. In this study, we compared (i) the culture
media Sucrose-Bacitracin agar (SB-20), Modied SB-20 (SB-20M) and Mitis Salivarius Baci-
tracin agar (MSB) in the count of colony forming units (cfu) of mutans streptococci and (ii) in
the morphological and biochemical differentiation between Streptococcus mutans and Strep-
tococcus sobrinus.
Design: Samples of non-stimulated saliva from 20 children were plated on SB-20, SB-20M
and MSB, and incubated in microaerophilia at 37 8C for 72 h. Identication of microorgan-
isms was basedonanalysis of colony morphology under stereomicroscopy. The biochemical
identication of colonies was done by biochemical tests using sugar fermentation, resis-
tance to bacitracin and hydrogen peroxide production.
Results: There was no signicant difference ( p > 0.05) in the number of cfu of mutans
streptococci recovered on SB-20 and SB-20M agar. Comparing the media, SB-20 and SB-
20M yielded a larger number of mutans streptococci colonies ( p < 0.05) and were more
effective thanMSB inthe identicationof S. sobrinus ( p < 0.05), but not of S. mutans ( p > 0.05).
Conclusion: There was no signicant difference between SB-20 and SB-20Mculture media in
the count of mutans streptococci, demonstrating that the replacement of sucrose by coarse
granular cane sugar did not alter the efcacy of the medium. Compared with MSB, SB-20 and
SB-20M allowed counting a larger number of mutans streptococci colonies and a more
effective morphological identication of S. sobrinus.
# 2012 Elsevier Ltd. All rights reserved.
* Corresponding author. Tel.: +55 16 3602 4099; fax: +55 16 3633 0999.
E-mail address: nelson@forp.usp.br (P. Nelson-Filho).
Available online at www.sciencedirect.com
journal homepage: http://www.elsevier.com/locate/aob
00039969/$ see front matter # 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.archoralbio.2012.10.010
Author's personal copy
1. Introduction
Considering the important role of mutans streptococci in the
aetiology of dental caries,
14
their quantication and identi-
cation are relevant for epidemiological and early intervention
studies.
5
Detection and identication of mutans streptococci
have been performed by different methods, namely microbial
culture techniques,
6,7
biochemical identication,
8,9
bacterio-
cin typing
10,11
and molecular techniques.
12
Mutans streptococci are the main etiologic agents of dental
caries in humans, and are classied into seven species. Of
these, Streptococcus mutans and Streptococcus sobrinus are the
most frequently isolated from the humans oral cavity.
Moreover research indicates that the coexistence of S. sobrinus
and S. mutans is an important factor in the development of
dental caries.
1,7
When microbial culture techniques are used, the recovery
of mutans streptococci in saliva and dental biolm samples
depends, in part, on the culture medium used.
5,13,14
However,
some of these media are not considered fully adequate
because they affect the growth, differentiation and count of
S. mutans and S. sobrinus. Infection with S. sobrinus represents a
relevant additional risk factor for dental caries, which means
that identication and count of this microorganism in a simple
and reliable manner are essential for the determination of
caries risk and activity.
7
Counting and identication of mutans streptococci are
traditionally undertaken based on the morphology of colo-
nies in selective culture media, such as Mitis Salivarius
Bacitracin agar (MSB),
5,1517
Sucrose-Bacitracin agar (SB-
20)
10,18
and Modied Sucrose-Bacitracin agar (SB-20M).
7,9,19
The general characteristics for differentiating species within
the mutans streptococci are S. mutans (serotype c, e, f, and k in
humans
20
), Streptococcus ferus (serotype c in wild rats),
Streptococcus cricetus (serotype a in hamster, humans and
wild rats), S. sobrinus (serotype d, g in humans), Streptococcus
rattus (serotypeb, in rats and humans) and Serotype h (serotype
h in monkeys). On the two SB-20 and SB-20M agar media,
colonies of S. mutans have a granular surface, similar to
ground glass, with or without a scintillant polysaccharide
drop on the surface. Colonies of S. sobrinus are circular,
sometimes star-shaped, and opaque milky white and are
surrounded by a milky white halo, frequently exhibiting
polysaccharide drops.
7
The general biochemical character-
istics for the S. mutans colonies on agar are production of acid
from mannitol, sorbitol, rafnose, lactose, inulin, salicin,
mannose andtrehalose, resistance to bacitracin, and absence
of hydrogen peroxide release. S. sobrinus colonies on agar, on
the other hand, produce acid from mannitol, inulin and
lactose, present positive or negative ability to ferment
sorbitol according to specic strains, do not ferment
melibiose and rafnose, and produce H
2
O
2
.
Common ingredients in these growth media are bacitracin
and high concentrations of sucrose, which facilitate the
identication of mutans streptococci on the basis of colony
morphology. Bacitracin is an antibiotic that inhibits the
growth of oral streptococci, except for mutans group strepto-
cocci, which makes it a coadjutant in the selective character-
istic of bacitracin-containing media. However, some strains
and serotypes are inhibited in some selective culture
media,
21,22
tending to underestimate the bacterial counts.
13,23
Although SB-20 and SB-20M media have been employed in
research studies, their efcacy in the count of mutans
streptococci has not yet been compared to nd out whether
the replacement of sucrose by coarse granular cane sugar in
SB-20M affects the efcacy of this culture medium.
The use of selective culture media that are easy to prepare
and allow the differentiation between S. mutans and S.
sobrinus is of clinical importance. However, there are no
published studies comparing SB-20, SB-20M and MSB agar
media in the count of S. mutans and S. sobrinus and the
differentiation between these species. The aim of the
present study was therefore to (i) compare the culture
media SB-20, SB-20M and MSB in the count of salivary
mutans streptococci and (ii) to study the morphological and
biochemical differentiation between S. mutans and S.
sobrinus on SB-20, SB-20M and MSB.
2. Materials and methods
2.1. Selection of patients
Twenty 412-year-old children of both sexes (10 boys and 10
girls) in good general health who were not under dental
treatment or using antibiotics or antiseptic solutions for a
minimal period of 3 months prior to the study, were recruited
from a public daycare centre and the Paediatric Dentistry
Clinic of the School of Dentistry of Ribeira o Preto, University
of Sa o Paulo, Brazil. The study was approved by the
Institutional Ethics Committee (protocol #0002.0.138.000-10)
and written informed consents were obtained from the
parents/guardians.
2.2. Microbial sampling and analysis
Approximately 2 h after toothbrushing, 2.03.0 mL of non-
stimulated salivary samples were collected into bottles
containing glass beads. Saliva was homogenised in an agitator
(Mixtron-Toptronix; Sa o Paulo, SP, Brazil) during 1 min and
subjected to serial dilution in 4.5 mL of phosphate buffer saline
(PBS, pH 7.0). Aliquots of 0.5 mL of pure and diluted saliva
suspensions were deposited on agar plates of SB-20,
10
SB-20M
7
and MSB.
15
The composition of the media per litre was:
- SB-20: bacto-casitone (15.0 g); yeast extract (5.0 g); L-cysteine
(0.2 g); sodium sulphite (0.1 g); sodium acetate (20.0 g);
sucrose (200.0 g); agar (15.0 g); distilled water (qsp); and
bacitracin added to a nal concentration of 0.2 U/mL agar.
- SB-20M: bacto-casitone (15.0 g); yeast extract (5.0 g); L-
cysteine (0.2 g); sodium sulphite (0.1 g); sodium acetate
(20.0 g); coarse granular cane sugar (200.0 g); agar (15.0 g);
distilled water (qsp); and bacitracin added to a nal
concentration of 0.2 U/mL agar.
- MSB: Difco
TM
Mitis Salivarius agar (pancreatic digest of
casein, 6.0 g; proteose peptone no.3, 9.0 g; proteose peptone,
5.0 g; dextrose, 1.0 g; saccharose, 50.0 g; dipotassium
a r c hi v e s o f o r a l b i o l og y 5 8 ( 2 0 1 3 ) 3 1 1 3 1 6 312
Author's personal copy
phosphate, 4.0 g; trypan blue, 75.0 mg; crystal violet, 0.8 mg;
agar 15.0 g); saccharose 150.0 g; distilled water (qsp);
potassium tellurite (1%) and bacitracin added to a nal
concentration of 0.2 U/mL agar.
Seeding was done with a micropipette (duplicate plates),
according to the method described by Westergren and
Krasse.
24
The plates were incubated for 72 h at 37 8C in
microaerophilia using the candle jar technique.
Enumeration of mutans streptococci cfu from 20 saliva
samples was performed by a single calibrated examiner
(Kappa > 0.8) under a dissecting microscope with a 20
magnication. Enumeration on SB-20 and SB-20M (clear
media) was performed with the plates against a dark
background in order to highlight the characteristics of the
colonies: colonies of S. mutans showed a granular surface,
similar to ground glass, with or without a scintillant
polysaccharide drop on the surface; colonies of S. sobrinus
were circular and opaque milky white and were surrounded by
a milky white halo, frequently exhibiting polysaccharide
drops. These colonies were sometimes star-shaped and
appeared penetrating the surface of the agar.
Colonies grown on the SB-20, SB-20M and MSB with the
typical morphology of S. mutans and S. sobrinus were randomly
selected and transferred to tubes containing thioglycollate
broth without glucose or indicator. The tubes were incubated
overnight at 37 8C in candle jars for biochemical identication
(biotyping). The broth cultures were then used as the
inoculum to test for the ability of each isolate to ferment
mannitol (with and without bacitracin), sorbitol, rafnose,
melibiose and bacitracin resistance (2UI)
8
and to produce
hydrogen peroxide.
25
2.3. Statistical analysis
Differences in cell counts of mutans streptococci were analysed
statistically by the Wilcoxon test and Fishers exact test, using
the GraphPad Prism
1
software version 5.0 (GraphPad Software
Inc., San Diego, CA, USA). The signicance level was set at 5%.
3. Results
3.1. Recovery of mutans streptococci
The median numbers of cfu of mutans streptococci on SB-20,
SB-20M and MSB culture media were 1.1 10
6
, 1.0 10
6
and
0.6 10
6
, respectively (Table 1). There was no statistically
signicant difference in the recovery between SB-20 and SB-
20M agar ( p > 0.05) whereas the yield on MSB agar was lower
compared with the other two culture media ( p < 0.05).
3.2. Efcacy of SB-20, SB20-M and MSB culture media in
the morphological and biochemical differentiation between S.
sobrinus and S. mutans
Fifty-four colonies with typical morphology of S. sobrinus
(n = 28) and S. mutans (n = 26) on each culture medium, as
illustrated in Fig. 1, were subjected to biochemical tests to
conrm the microbial identity. It was found that 25 of the 28
and 26 of the 28 colonies with typical morphology of S. sobrinus
in SB-20 and SB-20M, respectively, were also positive in the
biochemical tests, conrming biotyping in 89% of the cases for
SB-20 and 93% for SB-20M. On the MSB medium, only 6 out of
28 colonies with positive morphology for S. sobrinus were
positive in the biochemical tests, i.e. conrmation of the
microbial identity occurred in only 21% of the cases. Of the 26
Table 1 Number of mutans streptococci (T10
6
) on SB-20,
SB-20M and MSB culture media in saliva samples
obtained from 10 boys and 10 girls aged 412 years-old.
Culture medium Median Q1Q3 P
*
SB-20 1.1 0.601.93 a
SB-20M 1.0 0.752.00 a
MSB 0.6 0.131.60 b
*
Wilcoxon test. Values with the same letters are not statistically
different from each other. Q1 = first quartile and Q3 = third
quartile.
Fig. 1 (A) Morphological aspect of S. mutans and S. sobrinus isolated in MSB, revealing the difficulty in the morphological
differentiation between these two species in this culture medium (20T magnification). (B): Morphological aspect of S.
mutans and S. sobrinus (arrow) representative of colonies isolated in SB-20 and SB-20M, with typical whitish halo of S.
sobrinus. It can be seen the presence of larger colonies with a more accentuated morphological appearance, which
facilitates the differentiation between S. mutans and S. sobrinus (20T magnification).
a r c hi v e s o f or a l b i ol o gy 5 8 ( 2 0 1 3 ) 3 1 1 3 1 6 313
Author's personal copy
S. mutans colonies with typical morphology on the SB-20
medium, 23 were positive in the biochemical tests, while out of
26 colonies with typical morphology on the SB-20M medium,
22 were positive in the biochemical tests. Out of 26 colonies
with typical morphology on the MSB medium, 18 were positive
in the biochemical tests. Thus, biotyping conrmed that 88%
of S. mutans colonies plated on SB-20, 85% on SB-20M and 69%
on MSB agar were characterised correctly.
Therewas a statistically signicant difference betweenthe
culture media in the identication of S. sobrinus ( p < 0.005),
but not in the identication of S. mutans ( p > 0.05) (Figs. 2
and 3).
4. Discussion
In the present study there was no difference in the number of
cfu of mutans streptococci in the SB-20 and SB-20M culture
media. This result shows that replacing the laboratory
sucrose used in SB-20
10
by the coarse granular cane sugar
used in SB-20M
7
did not affect the efcacy of the medium for
the count of mutans streptococci. The use of coarse granular
cane sugar instead of laboratory sucrose is advantageous
because the bacterial cells can use the same type of sugar
substrate to grow on the SB-20M agar they would use in the
mouth after intake of sugar-containing foods. This maybe the
reason for the presence of larger and more exuberant colonies
with a more characteristic morphology on the SB-20M
compared with SB-20, a nding which facilitated the
differentiation between S. mutans and S. sobrinus from the
background ora.
7
The SB-20 and SB-20M media yielded a larger number
of mutans streptococci colonies and showed greater
efcacy in the identication of S. sobrinus than did the
MSB agar.
Although the majority of selective media enhance the
detection of the target microorganism, there are some
limitations in the differential identication and quantica-
tion of S. mutans and S. sobrinus strains. The MSB agar
15
has
been used in numerous studies over the years and can be
regarded as the reference medium when examining the
growth ability on other media. It medium detects all
serotypes of mutans streptococci, except for serotype a.
23
However, in the clinical patient situation this is not a
problem as serotype a is extremely rare or even non-existent
in humans. When comparing the two common strains of
mutans streptococci in humans, MSB causes greater selec-
tive suppressionof S. sobrinus comparedwith S. mutans.
5,15,26
One of the reasons for this observation might be that MSB
agar contains trypanblue, whichimpairs the visualisation of
the whitish halo characteristic of S. sobrinus. Both SB-20 and
SB-20M are transparent and colourless media, which
facilitates the identication of S. sobrinus colonies based
on their morphological characteristics showing a milky
white halo.
7
The SB-20 medium, and particularly the SB-20M medium, is
easily and inexpensively formulated compared with other
selective media. In addition, both media may be stored at room
temperature for up to 3 months without notable change of
selectivity, as long as bacitracin and coarse granular cane
sugar are added only shortly before use.
The results of the present study support the ndings of
Dasanayake et al.
14
and Hildebrandt and Bretz
5
that the
growth and recovery standard of salivary mutans streptococ-
ci vary qualitatively and quantitatively according to the
culture medium. Therefore, comparisons between studies
that used different culture mediafor quantication of mutans
streptococci should be done with caution and selective media
that are more effective in microbial quantication should be
preferred.
Fig. 2 Comparison of the percentage of positive and
negative biotyping of S. sobrinus isolated in SB-20, SB-20M
and MSB culture media (Fishers exact test).
Fig. 3 Comparison of the percentage of positive and
negative biotyping of S. mutans isolated in SB-20, SB-20M
and MSB culture media (Fishers exact test).
a r c hi v e s o f o r a l b i o l og y 5 8 ( 2 0 1 3 ) 3 1 1 3 1 6 314
Author's personal copy
5. Conclusions
Based on the obtained results, we can reach the following
conclusions:
1. There was no signicant difference between SB-20 and SB-
20M culture media in the count of mutans streptococci,
demonstrating that the replacement of sucrose by coarse
granular cane sugar did not alter the efcacy of the
medium.
2. SB-20 and SB-20M permitted the count of a larger number of
colonies of mutans streptococci and a more effective
morphological identication of S. sobrinus, compared with
MSB.
Funding
This study was supported in part by a fellowship from the
Brazilian Ministry of Educations Federal Agency for Support
and Evaluation of Graduate Education (CAPES) to the rst
author, Marta Estela Saravia.
Conict of interest
The authors deny any conicts of interest. We afrm that
we have no nancial afliation (e.g., employment, direct
payment, stock holdings, retainers, consultantships, patent
licensing arrangements or honoraria), or involvement with
any commercial organisation with direct nancial interest
in the subject or materials discussed in this manuscript, nor
have any such arrangements existed in the past three
years.
Ethical approval
The study was approved by the Ethics Committee of the School
of Dentistry of Ribeira o Preto, University of Sa o Paulo, Brazil,
with Certicate of Presentation to Ethics Appreciation regis-
tered under number 0002.0.138.000-10.
Acknowledgements
The authors are indebted to Dr. Izabel Yoko Ito (in memorian) of
Department of Clinical Analysis, Toxicology and Bromatology,
School of Pharmaceutical Sciences of Ribeira o Preto, Univer-
sity of Sa o Paulo, for helpful assistance during microbiological
processing and analysis.
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