1. INTRODUCTION:

The human oral cavity hosts a complex microbiome exceeding 700 bacterial species, with Streptococcus mutans recognized as a key etiological agent in dental caries^1,2,3,4,5. secondary caries development around enamel and dentine margins of class V restoration, using an in-vitro^6,7,8 bacterial caries model. S. mutans is a Gram-positive⁹, facultatively anaerobic lactic acid bacterium capable of thriving in low-pH environments generated by carbohydrate metabolism¹⁰. Its virulence is closely linked to adherence and biofilm formation^11-15, facilitated by glucosyltransferase-mediated synthesis of extracellular glucans on tooth surfaces¹⁶.

Reliable laboratory cultivation of S. mutans is essential for studies in cariology, antimicrobial screening, microbial pathogenesis, and molecular biology^17,18. As a fastidious organism, its recovery and maintenance depend heavily on nutrient-rich media¹⁹. Brain Heart Infusion (BHI) broth is the recommended standard for reviving lyophilized strains from collections such as MTCC and ATCC; however, its high cost, limited availability, and shorter shelf life can restrict routine use, particularly in resource-limited laboratories.^20, Alternative general-purpose media such as Nutrient Broth (NB)²¹ and Tryptic Soy Broth (TSB)²² offer practical substitutes. NB provides a simple peptone–beef extract base suitable mainly for non-fastidious organisms, whereas TSB contains casein and soy digests with dextrose, offering a more balanced nutrient profile capable of supporting broader microbial growth²³. Despite their widespread use, comparative assessments of NB and TSB specifically for the revival of lyophilized S. mutans are limited. Successful reactivation of lyophilized cultures depends on medium composition²⁴, buffering capacity, and ability to support early metabolic recovery²⁵. Media also influence subsequent colony morphology and maintenance on agar slants, which is critical for long-term strain viability^26,27. Understanding how NB & TSB and their corresponding agar media affect growth characteristics can aid in identifying cost-effective alternatives to BHI.²⁸

This study systematically evaluates NB and TSB for the revival, colony development, and maintenance of S. mutans MTCC. Parameters assessed include revival time, turbidity, colony morphology, and slant growth stability.²⁹ The goal is to establish an economical, reproducible, and efficient medium suitable for routine propagation of S. mutans, thereby supporting microbiology laboratories with limited resources while maintaining scientific reliability.

2. MATERIALS AND METHODS:

2.1 Bacterial Strain and Source:

A lyophilized culture of Streptococcus mutans (MTCC 497) was procured from the Microbial Type Culture Collection (MTCC), Chandigarh, India. The culture was maintained as per MTCC standard instructions for lyophilized bacterial strain revival.³⁰ All experimental procedures were performed under aseptic conditions within a Class II laminar airflow cabinet to prevent contamination.^31,32

2.2 Rehydration and Primary Revival:

The lyophilized pellet was aseptically rehydrated using 0.5mL sterile physiological saline (0.85% NaCl). The rehydrated suspension was gently mixed by tapping to disperse the pellet without causing cellular disruption. The entire suspension was immediately inoculated into two sterile test tubes containing 5mL each of Nutrient Broth (NB) and Tryptic Soy Broth (TSB), respectively.³³

· Nutrient Broth (NB) composition per liter: peptone 5.0 g, beef extract 3.0 g, sodium chloride 5.0 g; pH adjusted to 7.2±0.2.³⁴

· Tryptic Soy Broth (TSB) composition per liter: pancreatic digest of casein 17.0g, papaic digest of soybean meal 3.0g, dextrose 2.5g, sodium chloride 5.0g, dipotassium phosphate 2.5g; pH adjusted to 7.3±0.2.

All media were sterilized by autoclaving at 121°C for 15 minutes.

The inoculated broths were incubated at 37°C for up to 48 hours under microaerophilic conditions (achieved using sealed jars with a candle jar setup to reduce oxygen). Tubes were visually examined for turbidity every 6hours, and aliquots were withdrawn at 24h and 48 h intervals for optical density (OD) measurement at 600nm using a UV–Visible spectrophotometer (Shimadzu UV-1800, Japan).

Isolation by Streak Plate Technique:

Upon visible turbidity, each revived broth culture was streaked on agar plates using the four-way quadrant streaking technique for isolation of discrete colonies. The following broth-to-agar combinations were tested:

1. NB → Nutrient Agar (NA)

2. NB → Tryptic Soy Agar (TSA) (denoted as NA*)

3. TSB → Tryptic Soy Agar (TSA)

4. TSB → Nutrient Agar (NA) (denoted as TSA*)

Figure 1: Four quadrants streaking on plates

· Nutrient Agar (NA) composition per liter: peptone 5.0g, beef extract 3.0g, sodium chloride 5.0g, agar 20.0g.

· Tryptic Soy Agar (TSA) composition per liter: pancreatic digest of casein 15.0g, papaic digest of soybean meal 5.0g, sodium chloride 5.0g, agar 15.0 g, dextrose 2.5g, dipotassium phosphate 2.5g.

The plates were incubated at 37°C for 24–48 h in sealed Petri dishes containing moist tissue paper to prevent drying and maintain humidity. Colony morphology (color, form, elevation, edge, and opacity) and density were recorded after incubation.

2.4 Subculture and Maintenance on Agar Slants:

From the isolated colonies on each plate, representative colonies were aseptically picked using sterile nichrome inoculating loops and transferred to corresponding agar slants to establish stable subcultures. Four sets of slants were prepared to match the source medium:

· NA → NA slant

· NA* → NA* slant

· TSA → TSA slant

· TSA* → TSA* slant

Slants were incubated at 37°C for 24–48h, and growth coverage, consistency, and colony morphology were visually evaluated. Fully grown slants were stored at 4 °C for subsequent viability testing. The stability of each culture was assessed every 2 days by visual inspection and loop inoculation onto fresh plates to confirm purity.

2.5 Quantitative Growth Assessment:

Growth intensity in broth was quantified spectrophotometrically at 600nm, and results were expressed as mean±standard deviation (SD) from triplicate readings. Additionally, colony-forming units (CFU/mL) were estimated using the spread plate method by serially diluting broth samples up to 10⁻⁶ dilution and spreading 100µL aliquots on TSA plates. CFU counts were obtained after 48h incubation and expressed as log₁₀ (CFU/mL).

2.6 Morphological and Microscopic Characterization

Isolated colonies were subjected to Gram staining to confirm purity and cellular morphology. Stained smears were observed under oil immersion (1000× magnification) using a compound microscope (Olympus CX23). S. mutans was identified by its Gram-positive cocci morphology arranged in chains. Colony characteristics were recorded following Bergey’s Manual of Systematic Bacteriology descriptors.

2.7 Statistical Analysis:

All experimental data were recorded in triplicate and analyzed using GraphPad Prism v9.0. Mean and standard deviation (SD) values were calculated. Comparative analysis between media (NB vs TSB) was conducted using Student’s t-test, with significance set at p < 0.05. Graphical representations were generated to compare OD600 values and CFU counts across media and time intervals.

2.8 Quality Control and Aseptic Measures

All culture media batches were quality-checked for sterility by incubating uninoculated control tubes and plates. Glassware and inoculation instruments were sterilized in a hot air oven at 160 °C for 2 hours before use. Aseptic transfers were performed in a laminar flow cabinet previously sterilized by UV irradiation for 30 minutes and swabbed with 70% ethanol.

3. RESULTS AND DISCUSSION:

3.1 Revival Efficiency and Growth Kinetics in Different Broths:

The lyophilized S. mutans MTCC culture showed variable revival efficiency in Nutrient Broth (NB) and Tryptic Soy Broth (TSB). Turbidity was observed within 12–14 hours in TSB, while NB required 36–40hours to reach visible cloudiness. This difference indicates that TSB provides a superior nutritional environment for the resuscitation of dormant S. mutans cells.

Quantitative measurement of cell density using OD₆₀₀ confirmed this trend (Table 1). At 24h, the OD₆₀₀ of TSB-grown culture (0.85±0.07) was nearly 2.6 times higher than that of NB (0.32±0.04). The pH of the medium dropped from 7.0 to 6.1 in TSB, reflecting metabolic acid production typical of carbohydrate fermentation by S. mutans.

The enhanced growth in TSB can be attributed to its richer composition containing enzymatic digests of casein and soybean meal that supply abundant amino acids, peptides, and carbohydrates. These nutrients likely facilitated recovery of enzymatic systems such as glucosyltransferases and lactate dehydrogenase, essential for energy metabolism and acidogenicity in S. mutans.

Conversely, NB’s limited nutrient profile (peptone and beef extract) provided insufficient substrates for rapid repair of membrane and protein damage caused by lyophilization, leading to a prolonged lag phase.

3.2 Colony Morphology and Growth on Solid Media

Upon streaking the revived cultures onto agar plates, distinct morphological variations were observed among the four combinations: NB→NA, NB→TSA (NA*), TSB→NA (TSA*), and TSB→TSA.

Table 1: Comparative growth performance of S. mutans in Nutrient Broth (NB) and Tryptic Soy Broth (TSB)

Incubation Time (hr)	OD₆₀₀ in NB (Mean ± SD)	OD₆₀₀ in TSB (Mean ± SD)	pH (NB)	pH (TSB)	Visual Observation
0	0.08 ± 0.01	0.09 ± 0.01	7.0	7.0	Clear
12	0.15 ± 0.02	0.55 ± 0.05	6.8	6.5	Early turbidity in TSB
24	0.32 ± 0.04	0.85 ± 0.07	6.6	6.1	Moderate in NB, dense in TSB
36	0.45 ± 0.05	0.95 ± 0.08	6.4	6.0	Stationary phase in TSB
48	0.45 ± 0.05	1.02 ± 0.09	6.4	5.9	Stationary phase

Table 3: Viability and growth characteristics of S. mutans subcultures on different agar slants

Source Plate	Slant Medium	Growth Coverage (24 h)	Retention (Days at 4 °C)	Surface Appearance	Observation
NA	NA slant	60%	3–4	Dull, dry patches	Poor maintenance
NA*	NA* slant	95%	10–12	Creamy, moist	High viability
TSA*	TSA* slant	70%	5–6	Slightly dry	Moderate
TSA	TSA slant	100%	10–12	Smooth, uniform	Excellent

The extended viability on TSA and NA* can be attributed to enhanced nutrient retention and surface moisture, providing a semi-dormant yet metabolically active state conducive to survival. This aligns with prior studies showing that tryptic soy–based media sustain Streptococcus spp. better than basal media during low-temperature storage.

3.4 Statistical Evaluation of Media Performance:

Statistical comparison of OD₆₀₀ and CFU counts (n = 3) revealed significant differences between media types (p < 0.05). TSB and TSA supported the highest average growth, confirming their nutritional adequacy. The results clearly establish a positive correlation between the complexity of media composition and revival success, where nutrient-rich components enhance both growth rate and culture stability.

3.5 Biological Interpretation and Correlation with Literature

The superior growth of S. mutans in TSB and TSA media can be mechanistically linked to the high amino acid and peptide content derived from casein and soy hydrolysates. These compounds not only serve as primary nitrogen sources but also provide essential cofactors and buffering capacity that protect cells from acid stress during early revival.

Moreover, S. mutans being a fastidious, acidogenic bacterium, thrives in environments rich in carbohydrates and peptides, both of which are abundant in TSB. The lower performance in NB may stem from inadequate concentrations of reducing sugars and vitamins, which limits glycolytic flux and consequently slows acid production and growth.

The enhanced EPS formation and colony cohesion on TSA plates suggest that tryptic soy formulations better mimic the in vivo oral environment where S. mutans forms biofilms on tooth surfaces. Hence, these findings have implications for researchers studying virulence traits, biofilm formation, or antimicrobial susceptibility of S. mutans.

3.6 Implications and Future Directions:

The findings indicate that Tryptic Soy Broth (TSB) and Tryptic Soy Agar (TSA) serve as reliable, cost-effective alternatives to Brain Heart Infusion (BHI) for routine laboratory revival and maintenance of S. mutans. Additionally, NA* (NB→TSA) formulations demonstrated intermediate performance, suggesting that enriching nutrient agar with tryptic soy components can yield economical yet effective maintenance media.

Future work should focus on:

· Quantitative analysis of metabolic enzyme activity (e.g., glucosyltransferases, lactate dehydrogenase).

· Biofilm quantification assays to assess the influence of medium composition on adhesion and acidogenicity.

· Comparative studies on long-term cryopreservation or lyophilization recovery rates using TSA-based formulations.

3.7 Summary of Key Findings:

· TSB supported rapid revival of lyophilized S. mutans within 12–14 h.

· TSA and NA* slants-maintained viability for 10–12 days at 4°C.

· Colony morphology and EPS production were directly influenced by nutrient richness.

· Statistical analysis showed significant (p < 0.05) differences in OD₆₀₀ and CFU counts across media.

· Tryptic soy formulations are superior substitutes for BHI in S. mutans cultivation and maintenance.

4. CONCLUSION:

The present study provides a comprehensive comparative evaluation of Nutrient and Tryptic Soy media for the revival, cultivation, and maintenance of lyophilized Streptococcus mutans MTCC strains. The findings clearly establish that the composition and nutrient complexity of the medium play a decisive role in determining revival kinetics, colony morphology, and long-term culture viability. Among the tested formulations, Tryptic Soy Broth (TSB) supported the fastest revival rate and highest cell density within 24 hours of incubation, while Tryptic Soy Agar (TSA) facilitated the formation of large, smooth, and glistening colonies with typical S. mutans morphology. Furthermore, TSA and NA* slants (NB→TSA combinations) maintained active, moist cultures for up to 10–12 days at 4 °C, demonstrating superior viability and stability compared to NA and TSA*. The superior performance of TSB and TSA can be attributed to their rich amino acid and peptide content, which enhances metabolic reactivation, supports extracellular polysaccharide synthesis, and maintains osmotic balance during recovery from lyophilization-induced stress. These characteristics closely mirror the physiological requirements of S. mutans, a fastidious, acidogenic organism that thrives in peptide- and carbohydrate-rich environments. From a practical standpoint, the study highlights that TSB and TSA can serve as cost-effective, reliable alternatives to more expensive and complex media such as Brain Heart Infusion (BHI), without compromising revival efficiency or morphological fidelity. For laboratories involved in oral microbiology, dental biofilm research, or microbial preservation, these results offer a viable pathway to optimize culture maintenance protocols. The findings also provide an empirical framework for media optimization aimed at improving recovery rates of other lyophilized or fastidious Gram-positive bacteria. Future investigations should explore:

· The biochemical and enzymatic responses of S. mutans in various nutrient formulations,

· Biofilm production dynamics under different media conditions, and

· The application of TSB/TSA-based systems in routine dental pathogen screening and antimicrobial assays.

In conclusion, this study establishes that Tryptic Soy-based media significantly enhance the revival and propagation efficiency of lyophilized S. mutans strains, ensuring better viability, morphological consistency, and experimental reproducibility a critical advancement for both research and industrial microbiological practices.

5. ACKNOWLEDGEMENT:

The authors are grateful to Mahatma Gandhi Institute for Rural Industrialization, Wardha, Maharashtra and Ministry of MSME, Government of India, New Delhi for constant support and providing research facilities.

6. CONFLICT OF INTEREST:

The authors declare that they have no conflicts of interest.

7. AUTHOR’S CONTRIBUTIONS:

S.G.K. is major contributor behind the basic concept, literature survey, review, practical procedures, activation of S. mutans, anti-microbial study, original draft writing, schematic representation, figures and tables, creation of manuscript content, referencing, examination, citations, correction and editing of the manuscript; J.A.C. contributed in examination and correction of the manuscript. All authors read and approved the final manuscript; P.S.K. contributed in practical examination of the research work and anti-microbial study.

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Received on 27.01.2026 Revised on 04.03.2026

Accepted on 06.04.2026 Published on 21.04.2026

Available online from April 24, 2026

Res. J. Pharma. Dosage Forms and Tech.2026; 18(2):108-114.

DOI: 10.52711/0975-4377.2026.00017

This work is licensed under a Creative Commons Attribution-Non Commercial-Share Alike 4.0 International License. Creative Commons License.

Inoculum Source	Plating Medium	CFU (×10⁸/mL) ± SD	Colony Diameter (mm)	Morphology	Growth Observation
NB	NA	0.7 ± 0.2	0.6 ± 0.1	Irregular, opaque	Poor, delayed growth
NB	TSA (NA*)	1.8 ± 0.3	0.9 ± 0.1	Circular, dull	Moderate growth
TSB	NA (TSA*)	3.2 ± 0.4	1.1 ± 0.2	Smooth, convex	Good growth
TSB	TSA	4.5 ± 0.5	1.4 ± 0.2	Creamy, glistening, convex	Excellent growth