Journal of Microscopy and Ultrastructure

: 2022  |  Volume : 10  |  Issue : 4  |  Page : 168--173

A comparison of pre- and postbreakfast tooth brushing in caries prevention through the estimation of Streptococcus mutans counts: A prospective clinical and microbiological study

Sujit Ranjan Sahoo1, DB Nandini2, Praveen S Basandi3, M Selvamani4, Mandana Donoghue5,  
1 Department of Dentistry, PRM Medical College and Hospital, Baripada, Mayurbhanj, Odisha, India
2 Department of Oral Pathology and Microbiology, Dental College, Regional Institute of Medical Sciences, Imphal, Manipur, India
3 Department of Oral Pathology and Microbiology, College of Dental Sciences, Davangere, India
4 Department of Oral Pathology and Microbiology, Mahe Institute of Dental Sciences and Hospital, Union Territory of Puducherry, India
5 Founder-Director Oral Pathology 360, Belgaum, Karnataka, India

Correspondence Address:
Dr. Sujit Ranjan Sahoo
Department of Dentistry, PRM Medical College and Hospital, Baripada, Mayurbhanj, Odisha


Objectives: The purpose of this study was to compare the efficacy of prebreakfast tooth brushing (PrBTB) and postbreakfast tooth brushing (PoBTB) with or without a prebrushing rinse in caries prevention through the estimation of Streptococcus mutans count. Materials and Methods: Sixty consenting dental students were divided into three age-matched groups of 20 each and instructed to follow prebreakfast, postbreakfast tooth brushing, and prebreakfast rinsing plus PoBTB using the Bass technique. Plaque samples were collected at the start and at the end of every quarter for a total of 6 quarters (18 months) for the evaluation of S. mutans colony-forming unit (CFU) counts. The decayed missing filled surface (DMFS) index of participants was also recorded and compared at the beginning and end of the study period. Results: The post-breakfast tooth brushing group with pre-breakfast rinse (RPoBTB) and without pre-breakfast rinse (PoBTB) showed a highly significant reduction in total S. mutans CFU counts per ml (38% and 29% respectively) at the end of the study. The changes in DMFS value were not significant and did not show any correlation with the S. mutans counts. Conclusion: Our study revealed that PoBTB with or without a prebreakfast rinse reduces the total counts of the cariogenic bacteria S. mutans more efficiently than PrBTB. Although further proof in the form of clinical trials is essential, this study provides the proof of concept for a minor change in the tooth brushing habit, which can significantly enhance caries prevention.

How to cite this article:
Sahoo SR, Nandini D B, Basandi PS, Selvamani M, Donoghue M. A comparison of pre- and postbreakfast tooth brushing in caries prevention through the estimation of Streptococcus mutans counts: A prospective clinical and microbiological study.J Microsc Ultrastruct 2022;10:168-173

How to cite this URL:
Sahoo SR, Nandini D B, Basandi PS, Selvamani M, Donoghue M. A comparison of pre- and postbreakfast tooth brushing in caries prevention through the estimation of Streptococcus mutans counts: A prospective clinical and microbiological study. J Microsc Ultrastruct [serial online] 2022 [cited 2023 Mar 20 ];10:168-173
Available from:

Full Text


Dental caries is a multifactorial ecological disease in which diet, host, and the microbial flora interact over a period of time in such a way as to initiate demineralization of the tooth enamel with resultant caries formation.[1] Microbial plaque is a critical factor in the multifactorial etiology of caries. Acquired pellicle is an indispensable component of dental plaque formed just before or concomitant with the bacterial colonization that may facilitate the plaque formation.[2] Streptococcus mutans (S. mutans) is the chief initiator of caries; a major habitant of supragingival plaque. S. mutans possess several abilities, such as the ability to adhere to the salivary pellicle on enamel, produce acidic intermediate metabolites, build glycogen reserves, and synthesize extracellular polysaccharides, which are essential in dental caries initiation and propagation.[3]

Plaque removal by daily tooth brushing is recognized as an indispensable means of caries prevention.[1],[2],[3] The most commonly advised tooth brushing protocol is a twice-daily brushing protocol once at night and once in the morning. Brushing at night is essential because the dinner's leftover food residues would provide the ideal substrate for S. mutans to produce demineralizing acids throughout the night.[4] The reduced salivary secretion during the night (0.1 ml/min at rest compared to 1.0 ml/min stimulated secretion)[2] enables both increased volume and strength of the acid overnight by a combination of reduced clearance and buffering, respectively. It is generally agreed that night brushing is the most effective when completed after dinner or just before sleep.

On the other hand, no consensus exists on the most beneficial timing for the morning tooth brushing. Some dentists advise their patients to brush upon waking up, and others recommend brushing after breakfast.[5] Water rinsing cleans the oral cavity by increasing the oral clearance,[6] which can be assumed to help reduce dental caries.

This study was designed to compare pre and postbreakfast tooth brushing (PoBTB) and an additional rinse along with postbrushing on caries prevention through variations in S. mutans counts.

 Materials and Methods

The study was conducted in 2009-2010. Declaration of Helsinki and Informed Consent was obtained. The study was conducted on 60 dental students. Equal numbers of age-matched male and female dental students were included in the study after obtaining informed consent. Clearance for the study was obtained from the institutional ethical board. Students with ages ranging from 19 to 28 years with a minimum of 20 natural fully erupted permanent teeth were included in the study. Students suffering from acute caries, periodontitis, fluorosis, or any major systemic illnesses and those undergoing orthodontic therapy or long-term antibiotic therapy were excluded from the study. The students were divided randomly into three equal groups of 20 each. The groups were as follows:

Prebreakfast tooth brushing group (PrBTB)PoBTB groupPrebreakfast rinsing followed by PoBTB group (RPoBTB).

Bass brushing technique was demonstrated to all participants at the beginning of the study. The participants were instructed to brush with the Bass technique using soft toothbrushes and fluoridated toothpaste. All participants were asked to brush at night just before bed, change toothbrushes every 3 months, or when the bristles were damaged, whichever was earlier. Oral prophylaxis was performed on every participant to ensure a uniform oral hygiene status at the start of the study.

Two weeks after oral prophylaxis, the baseline decayed missing filled surface (DMFS) score[7] was calculated, and the plaque samples for the baseline colony-forming unit (CFU) counts of S. mutans were collected.

Microbiological sampling and counts

One side mandibular first molar was chosen for every sample collection in each subject. The plaque samples were collected from the middle third of the lingual surface; the sample collection site was chosen because of ease of access with a sterile toothpick. After collection, the toothpick ends with the plaque samples were placed in test tubes containing 2 ml of brain heart infusion broth. The test tubes with the samples were incubated at 37°C for 24 h to propagate bacteria. 100 μL of the diluted sample (1:10 dilution)[8] was transferred to Mitis Salivarius Bacitracin (MSB) Agar media, a selective and differentiating media for S. mutans containing Mitis Salivarius Agar base, 20% sucrose, 1% potassium tellurite, and 0.2 units/ml of bacitracin. The MSB agar plates were placed in an anaerobic candle gas jar and then incubated at 37°C for 48 h. The S. mutans colonies were identified based on individual colonies being 0.5–1 mm in diameter, usually gray-translucent to white, circular or irregular, sometimes with a rough surface[1] urther confirmation of colonies was done by biochemical tests of positive mannitol fermentation and negative arginase test.[9] Total CFU of S. mutans was counted using a colony counter [Figure 1]a,[Figure 1]b,[Figure 1]c and [Figure 2]. The total CFU counts were expressed as CFU per ml.{Figure 1}{Figure 2}

Statistical analysis

The results were analyzed using one-way analysis of variants for multiple comparisons followed by post hoc Tukey's test for group-wise comparison. An intragroup comparison was made by paired t-test. Spearman's rank correlation coefficient was done to analyze the correlation between DMFS and CFUs per ml counts of S. mutans at baseline and the end of the study.


The present study aimed to compare the effectiveness of PrBTB, PoBTB and RPoBTB in caries prevention by comparison of total S. mutans colony-forming unit counts and DMFS values. A total of 60 dental students were divided into three groups, i.e., PrBTB, PoBTB, and RPoBTB, comprising 20 subjects.

Changes in Streptococcus mutans colony-forming unit counts per ml at different time intervals observed in three groups

The baseline S. mutans CFU counts per ml for RPoBTB, PoBTB, and PrBTB groups ranged from 102.9–107.9 [Table 1] and [Figure 3]. The quarterly CFU counts of the PrBTB group showed a seesaw variation through the quarters. The counts showed a gradual increase through the 1st two quarters, a decline through the 3rd quarter, and subsequent rise through the remaining three quarters. Overall, the changes were not significant from that of the baseline till the end of the study. The PoBTB group showed a gradual decrease in the CFU counts per ml till the last quarter, when it increased slightly. The decline was more pronounced at the end of the 3rd quarter and 4th quarters.{Figure 3}{Table 1}

In terms of intragroup comparison, the PrBTB group showed a statistically insignificant reduction of 7% in the CFU counts per ml at the end of the study. The PoBTB group showed a highly significant (P < 0.001) total S. mutans CFU count reduction amounting up to 29% by the end of the study. The RPoBTB group also showed a highly significant (P < 0.001) total CFUs decrease of 38%, which was the maximum in the study [Table 2] and [Figure 4].{Figure 4}{Table 2}

Intergroup comparison of colony-forming unit counts per ml

The intergroup comparison was made by post hoc Tukey's test. The difference in the baseline CFU counts per ml between all the groups was statistically insignificant [Table 2] and [Figure 4]. While all groups showed a decline compared to the baseline, there were highly significant differences in decline levels. The fall in numbers was the least in the PrBTB group at 7%, followed by 29% in PoBTB and 38% in the RPoBTB group [Figure 5]. The counts in the PoBTB group were significantly lower than the PrBTB group. There was no statistically significant difference between CFU counts per ml of the PoBTB and RPoBTB group.{Figure 5}

Variation in decayed missing filled surface value at the end of the study as compared to baseline

By the end of the study, there were insignificant changes in the DMFS scores of groups [Table 3] and [Figure 6]. One occlusal pit lesion in the PrBTB group, two new buccal pit lesions in the PoBTB group, and one extraction and new occlusal caries in the RPoBTB groups were the cause of the changes [Table 4]. Spearman's rank correlation coefficient, to evaluate any correlation between DMFS and CFUs per ml at the end of the study, gave statistically insignificant values [Table 5].{Figure 6}{Table 3}{Table 4}{Table 5}


A prospective microbiological study was carried out on 3 equally divided groups, i.e., PrBTB, PoBTB, and RPoBTB groups to determine the effect of time of tooth brushing in the morning on S. mutans counts and caries. Plaque samples from all subjects were collected quarterly (3 months) for one and a half years (18 months).

The study demonstrated a statistically significant difference in CFUs between the three groups, with the subjects of RPoBTB group showing the most significant reduction. However, the difference in the DMFS value between baseline and at the end of study for the three groups was statistically insignificant. The above results were indicative that RPoBTB in the morning was far superior to PrBTB in terms of reducing the overall S. mutans CFU counts over a period of time. PrBTB removes bulk of the overnight accumulated plaque[5],[10],[11] and S. mutans, just prior to the breakfast. Even the most effective brushing, will not be able to remove all the cariogenic bacteria and few cariogenic bacteria will be left on the tooth surface.[5] Breakfast provides sucrose to the remaining S. mutans helping them to both recolonize and start the lactic acid production using their intrinsic enzymatic machinery activity.[3] This recolonization will continue throughout the day till the next scheduled tooth brushing, i.e., at night and growth is supplemented by substrate provided by intervening meals (the lunch and dinner) and snacks. Hence, the time duration for which S. mutans is active in acid production is 13–17 h, i.e., time interval between breakfast and night tooth brushing. We believe this to be the explanation for the high S. mutans CFU counts per ml in the PrBTB group at the end of the study as compared to other two groups. However, in the PoBTB group, the time duration for S. mutans to act upon substrate is decreased to 8–10 h, as PoBTB removes bulk of S. mutans along with food remnants left over from breakfast. Hence, effectively the substrate supplies to the left over S. mutans is cut off till lunch or till next snack is consumed. As the samples were collected just prior to lunch, the total CFU counts per ml gave a highly significant reduced value compared to baseline value for PoBTB group. In the RPoBTB group, the further reduction can be explained on the basis that in this group, PoBTB was further supplemented by prior water rinsing. Prebreakfast water rinsing in the RPoBTB group facilitated the dilution and removal of some of the buildup bacteria and acids[12],[13] accumulated overnight in addition to the previously explained effect of PoBTB.[13],[14] This is a very likely explanation of the greater decrease in total CFU counts per ml in this group as compared to the PoBTB group. The difference in the total CFUs per ml between PoBTB and RPoBTB groups was not significant, suggesting that additional rinsing has no significant implication in reducing total CFUs per ml when coupled with PoBTB. Our findings could not be compared with other studies since similar studies on timing of tooth brushing and microbial estimation are not available in existing published literature.

The PrBTB group showed a slight increase in CFU counts per ml between the baseline to 6th month followed by a prominent decline from 6th to 9th month and then a near plateau phase till the end of study period. PoBTB and RPoBTB groups showed a decline in the CFU counts throughout with more prominent decrease between 6th and 9th month. The introduction of Bass technique[13] in all groups could be expected to cause some loss of brushing efficacy in the initial months, demonstrated by the rise in CFU counts per ml in PrBTB group between the baseline to the 6th month. The CFU counts per ml of PoBTB and RPoBTB group, however, showed a slight decline in the same period, which might have been possibly due to the additional effect of the postbreakfast brushing time negating the loss of brushing efficacy. The prominent reduction in all the groups between 6th and 9th month demonstrated increase in efficiency of Bass technique with practice. The near plateau in the counts achieved from 12th month onward may be attributed to the microbial adaptation to the changing oral environment due to the new technique and timing of brushing.

The difference in DMFS value at the baseline and at the end of the study did not give any significant result. This could be explained by the time duration required for caries development.[14] The average time for incipient caries to develop into clinical caries is 18 ± 6 months,[2] which can explain the insignificant difference in DMFS value at the end of the study. Further, it could be attributed to overall improved technique in maintaining the oral hygiene producing an effective inhibitory effect on microbes. The use of fluoridated tooth paste also probably added to not significant DMFS difference. No significant correlation was noted between DMFS and CFUs per ml value in all the three groups. In the PrBTB group, one subject showed a new lesion, in the PoBTB group, two subjects developed new lesions, and in the RPoBTB, one subject underwent extraction for a preexisting deep carious tooth and another one developed an occlusal caries at the end of study period. This could probably be attributed to the multifactorial etiology of caries which were not considered in the study such as dietary details, previous oral hygiene measures, and variation in caries susceptibility of individual subjects. To the best of our knowledge, similar study was not available in existing literature for comparison of findings warranting further research.

Limitation of the study and future scope

PoBTB and RPoBTB groups showed a very marginal rise in CFU counts per ml in the last month, thus pointing to a possible microbial adaptation. This demonstrates a need for a longer duration of the study to develop a clear picture of the S. mutans adaptation that might follow in the postbreakfast brushing groups. The participants were not asked about their existing oral hygiene habits, which reflected in the baseline CFU count. Few cases were outlier in result in terms of CFU counts per ml because of which average of one group was higher (in RPoBTB group there was a preexisting deep carious tooth). Probable reason for this microbial count could be attributed to diet or lack of following of instructions given to the study group. Another shortcoming of the study is that at the beginning of the study, treatment should have been given to each participant as existence of caries could act as a source of infection in oral cavity. In addition, setting a maximum DMFS score for the inclusion of subjects in the study will ensure that subjects are equally distributed in the groups and thus preventing any bias being introduced due to variation in caries susceptibility and activity.


Timing of morning brushing has a highly significant effect in the reduction of S. mutans counts and PoBTB is the most effective in this regard. Additional water rinsing can reduce the S. mutans counts further; however, the reduction is not significantly more than when brushing alone is used. There is a severe scarcity of available literature on the effects of brushing in relation to timing of tooth brushing and caries development. This necessitates further research work in this area of cariology.


We thank Mr. Sangam D.K. for statistical analysis and Mr. Mallikarjun and Mr. Kumar, laboratory technicians, College of Dental Sciences, Davangere, Karnataka, for their technical help.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.


1Badjatia S, Badjatia RG, Thanveer K, Krishnan AC. Effects of fluoride varnish on Streptococcus mutans count in saliva. Int J Clin Pediatr Dent 2017;10:62-6.
2McDonald RE, Avery RD, Stookey GK. Dental caries in children. In: McDonald RE, Avery RD, Dean AH, editors. Dentistry, Child and Adolescent. 8th ed. St. Louis, Missouri: Elsevier; 2005. p. 203-35.
3Forssten SD, Björklund M, Ouwehand AC. Streptococcus mutans, caries and simulation models. Nutrients 2010;2:290-8.
4Melo P, Fine C, Malone S, Frencken JE, Horn V. The effectiveness of the Brush Day and Night programme in improving children's toothbrushing knowledge and behaviour. Int Dent J 2018;68 Suppl 1:7-16.
5Machiulskiene V, Richards A, Nyvad B, Baelum V. Prospective study of the effect of post-brushing rinsing behaviour on dental caries. Caries Res 2002;36:301-7.
6Ravindran S, Chaudhary M, Gawande M. Enumeration of salivary streptococci and lactobacilli in children with differing caries experience in a rural Indian population. ISRN Plast Surg 2013;2013:1-6.
7Klein H, Palmer CE. Studies on dental caries: VII. Sex differences in dental caries experience of elementary school children. Public Health Rep 1938;53:1685-90.
8Loesche WJ. Role of Streptococcus mutans in human dental decay. Microbiol Rev 1986;50:353-80.
9Newman MG, Takei HH, Carranza FA. Carranza's Clinical Periodontology. 9th ed. Philadelphia, Pennsylvania: Saunders Publication; 2003. p. 651-74.
10Chesters RK, Huntington E, Burchell CK, Stephen KW. Effect of oral care habits on caries in adolescents. Caries Res 1992;26:299-304.
11Hegde RJ, Kamath S. Comparison of the Streptococcus mutans and Lactobacillus colony count changes in saliva following chlorhexidine (0.12%) mouth rinse, combination mouth rinse, and green tea extract (0.5%) mouth rinse in children. J Indian Soc Pedod Prev Dent 2017;35:150-5.
12Chestnutt IG, Schäfer F, Jacobson AP, Stephen KW. The influence of toothbrushing frequency and post-brushing rinsing on caries experience in a caries clinical trial. Community Dent Oral Epidemiol 1998;26:406-11.
13Ashley PF, Attrill DC, Ellwood RP, Worthington HV, Davies RM. Toothbrushing habits and caries experience. Caries Res 1999;33:401-2.
14Zahir S, Sarkar S. Study of trace elements in mixed saliva of caries free and caries active children. J Indian Soc Pedod Prev Dent 2006;24:27-9.