Modified TSBB Culture Media Enhance Faster Growth of Streptococci
Modified TSBB Culture Media Enhance Faster Growth of Streptococci
Modified TSBB Culture Media Enhance Faster Growth of Streptococci
Received on 08 April, 2016; received in revised form, 28 May, 2016; accepted, 13 June, 2016; published 01 September, 2016
INTRODUCTION: Streptococcus mutans has For this purpose several selective media have been
emerged as one of the most significant caries developed. Among all the media MSB( Mitis
causing organism 10, 3, 1, 18. Mostly researches are salivarius bacitracin) was the first widely accepted
related with Streptococcus mutans etiology, medium 5 but it has several disadvantages like
epidemiology, transmission of caries and many sometimes it is tough to differentiate visually
molecular biological studies which require its easy between S. mutans and non- S. mutans colonies and
and quick isolation. serotype a of Streptococcus mutans is failed to
grow on MSB 4, 15, 8.
QUICK RESPONSE CODE
DOI: Kimmel & Tinaoff 1991 7 and Llena et al 2007 9
10.13040/IJPSR.0975-8232.7(9).3689-94
modified MSB and gave the names MSKB and
MSBTPF respectively. MSKB was supplemented
Article can be accessed online on: Mitis salivarius agar with kanamycin sulfate,
www.ijpsr.com
sorbitol, potesium tellurite & bacitracin and
DOI link: http://dx.doi.org/10.13040/IJPSR.0975-8232.7 (9).3689-94 MSBTPF by adding 5% potassium tellurite and 64
µg/ml fluconazol, but result of this MSKSB is not Mutans sanguis agar (Hi Media) was used in this
satisfactory as selectivity of this media for is study as reference medium, which was autoclaved
Streptococcus mutans very less but MSBTPF‘s and dispersed in sterile petri plate. In an effort to
results were relatively better. There are many improve the selectivity and accuracy of the
media which were prepared to overcome the isolation of Streptococcus mutans we have
shortcomings of MSB medium. Such as tryptone- formulated an enriched and selective medium
yeast extract- cysteine with sucrose and bacitracin TSBB which contains - Thioglycolate agar which
(TYCSB) 19, 20, 21, 22 trypticase soy with sucrose and consists of Trypticase soy agar 4g, Yeast extracts
bacitracin (TSY 20B) [14], MM 10 4, Mannitol 1g, Thioglycolate 3g, Sucrose 20g, D/W 100ml.
containing broth 2, glucose-sucrose-tellurite-
bacitracin (GSTB) 17. After the medium was autoclave at 121°C, 15 lbs
pressure for 15 mins, cool it at 55°C. Add two disk
Wade et al 1986 21 proved best recovery of S. of bacitracin (each contains 10 U) for at least 15
mutans with a final sucrose concentration of 20% mins, so that antibiotic get suspends in the medium.
and bacitracin 2 units /ml. Sucrose is added before For maintaining temperature of medium at 55°C, it
autoclaving the media and bacitracin is added after was kept in water bath and then 2 ml of blood was
sterilization. added to it.
The aim of the present study was to compare the Dilution and inoculation of reference strains
growth ability of S. mutans on five media including ATCC 25175 and OMZ 61:
new modified medium named as Thioglycolate Colonies of reference strains were pelleted by
sucrose blood bacitarcin agar (TSBB) which was centrifugation (10,000 rpm for 20 minutes) and
isolated from clinical samples along with two then re-suspended in sterile PBS. These inocula
reference strains. were disaggregated by sonication bath for 3 min for
40 kHz. Bacteria were finally suspended in PBS to
MATERIAL AND METHODOLOGY: make a suspension equal to a 0.5 McFarland
Bacterial strain: standard (1.2 × 108 CFU/ml). It was then further
The standard strain of S. mutans which was used in diluted from 1:10 to 1:107. 0.1 ml portion of each
this study was ATCC 25175. It was stored in - dilution were plated on different media by spread
70°C, thawed and sub-cultured onto Thioglycolate plate method. Plates were incubated for 48 hours at
agar. As this is anaerobic medium there was no 37o C in candle jar.
need to give anaerobic condition. Gram staining
and biochemical tests were done to confirm the Dilutions and inoculation of Streptococcus
purity of the culture. Further bacterial strain was mutans isolated from clinical samples:
grown on same above mentioned medium The samples were homogenized by vortexing for 5
Thioglycolate agar in candle jar. minutes, 1 ml samples were diluted from 1:10 to
1:10 6. After dilution, 0.1 ml portion of appropriate
Clinical samples: dilution was plated by using a bent glass rod on
The dental plaque sample were collected from all different agar media (triplets of every medium).
surfaces of the teeth and tongue by sterilized cotton After incubation of 48 hr suspected colonies were
swabs. 10 samples were collected from 10 caries isolated and identified by using following tests –
active school going children. Each sample was Gram staining, Catalase test, Esculine hydrolysis
placed in test tubes containing fluid thioglycolate test, sugars (mannitol, sorbitol, melibiose and
medium and stores at 4oC till plated. inulin) fermentation test.
The colonies on plates were enumerated by colony It was observed that moderate growth of
counter. Streptococcus mitis, and Streptococcus salivarius
in the, TSY 20B, MM 10, GSTB media (RGI <
Statical analysis: 10%) and in TSBB (RGI < 5%) in relation to
Analysis of variance for multiple comparison control medium. Recovery of Streptococcus
(ANOVA) using stativa 6.0, statsoft (Tulsa USA) sobrinus in TYCSB was high (RGI > 35%). While
was performing in order to confirm statistical Staphylococcus aureus, Lactobacillus,
significance of differences among samples Actinomycetes were inhibited (RGI < .05) in
(P<0.05). Mean values were compared using the TSY20B, MM10 and GSTB media. RGI was less
Tukey test at P<0.05. than 0.001% in TYCSB and TSBB.
Streptococcus mutans as on this medium colonies 2. Ellen RP, Fillery ED, Banting DW: Comparison of
selective broth and plating methods for isolation of
appears like beads droplet and puddle shape which Streptococcus mutans from root surface dental plaques.
contain soluble extracellular polysaccharide with α Journal of Clinical Microbiology 1980; 11:205-8.
& γ haemolysis. 2. It was found that there was no 3. Emilson CG: Prevalence of Streptococcus mutans with
different colonial morphologies in human plaque and
need to incubate the cultures in anaerobic condition saliva. Scandinavian journal of dental research 1983;
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7. Kimmel N Tinanoff: A modified mitis salivarius medium
and favorable for the growth Streptococcus mutans for a caries diagnostic test. Oral Microbiology and
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Comparative recovery of Streptococcus mutans on ten
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and count of Streptococcus mutans. It helps to 578–583.
easily identify the colonies of Streptococcus 9. Llena MC, Pérez-Gracia MT, Férnández-Barredo
S, Galiana C: New culture media for the isolation of
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thioglycolate present in media contains sodium 11. Momeni SS, Patrick P, Wiener HW, Cutter GR, Ruby JD,
Cheon K, Whiddon J, Moser SA, Childers NK: mutans
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ACKNOWLEDGMENT: The authors are Journal of Dental Research 1986; 65:906-908.
14. Staat RH: Inhibition of Streptococcus mutans strains by
indebted of Department of Microbiology, different mitis-salivarius agar preparations. Journal of
Barkatullah University, Bhopal and People’s Clinical Microbiology 1976; 3:378-380.
College of Medical Science, Bhopal for providing 15. Takada K, Hirasawa M: A novel selective medium for
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16. Tanzer JM, Borjesson AC, Laskowski L, Kurasz AB,
CONFLICT OF INTERESTT: There are Testa M: Glucose-sucrose potassium tellurite-bacitracin
agar, an alternative to mitis salivarius-bacitracin agar for
nonfinancial competing interests (political, enumeration of Streptococcus mutans. Journal of Clinical
personal, religious, ideological, academic, Microbiology 1984; 20:653-659.
intellectual, commercial, or any other) to declare in 17. van Houte J: Role of micro-organisms in caries etiology.
Journal of Dental Research 1994; 73:672-681.
relation to this manuscript. 18. van Loveren C, Buijs JF, Bokhout B, Prahl-Andersen B,
Ten Cate JM: Incidence of mutans Streptococci and
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