Indian Journal of Dental ResearchIndian Journal of Dental ResearchIndian Journal of Dental Research
Indian Journal of Dental Research   Login   |  Users online: 1312

Home Bookmark this page Print this page Email this page Small font sizeDefault font size Increase font size         


ORIGINAL RESEARCH Table of Contents   
Year : 2009  |  Volume : 20  |  Issue : 3  |  Page : 304-307
The effect of combined bleaching techniques on oral microbiota

1 Department of Physiological Sciences, Piracicaba Dentistry School, UNICAMP, Brazil
2 Physiological Sciences, São Leopoldo Mandic Dental School, Brazil
3 Restorative Dentistry, Guarulhos University, Brazil
4 Restorative Dentistry, Piracicaba School of Dentistry, UNICAMP, Brazil

Click here for correspondence address and email

Date of Submission26-Mar-2008
Date of Decision20-Jul-2008
Date of Acceptance06-Oct-2008
Date of Web Publication30-Oct-2009


Aims : To evaluate the antimicrobial activity of 10% and 37% carbamide peroxide during dental bleaching in three different modes.
Materials and Methods : This five-week double-blind randomized controlled trial included 32 volunteers assigned to four groups (n = 8). Each group received bleaching agents or placebo as an in-office and at-home treatment. The dental bleaching techniques were: In-office bleaching (37% carbamide peroxide: CP37); at-home bleaching (10% carbamide peroxide: CP10) and the association of both (CP37 and CP10). Saliva samples were collected right before (baseline), right after, 12 hours after, and seven days after the treatment. Counts of total microorganisms, Streptococci, and Mutans streptococci were carried out. Friedman test (α = 0.05) was used to compare the microorganism counts.
Results : The number of the all oral microorganisms remained stable during all experiment.
Conclusions : No bleaching agent (CP37, CP10 or the combination of both) was able to reduce the oral microorganisms tested.

Keywords: Antibacterial activity, carbamide peroxide, dental bleaching, oral microorganisms, randomized clinical trial, Streptococcus mutans

How to cite this article:
Franz-Montan M, Ramacciato JC, Rodrigues JA, Marchi GM, Rosalen PL, Groppo FC. The effect of combined bleaching techniques on oral microbiota. Indian J Dent Res 2009;20:304-7

How to cite this URL:
Franz-Montan M, Ramacciato JC, Rodrigues JA, Marchi GM, Rosalen PL, Groppo FC. The effect of combined bleaching techniques on oral microbiota. Indian J Dent Res [serial online] 2009 [cited 2019 Sep 22];20:304-7. Available from:
Dental bleaching, performed at home or in an office, is a simple and conservative procedure for aesthetic restoration of vital and nonvital discoloured or stained teeth. [1],[2]

First described in 1989 by Haywood and Heymann, [3] the at- home dental bleaching, proven safe and effective, [4] has been associated with the in-office dental bleaching to achieve tooth whitening in a shorter period of time, especially in the cases of more severe stains. [5]

Although previous studies have shown some antimicrobial effect of bleaching systems containing carbamide peroxide on oral microorganisms, [6],[7],[8] there are still controversies concerning the in vivo antimicrobial activity of the bleaching agents.

The present study was conducted to evaluate the effect of the association of two different dental bleaching techniques, on oral microorganisms, from human saliva.

   Materials and Methods Top

The Ethical Committee of Piracicaba Dental School, State University of Campinas, approved this research. All volunteers signed a written informed consent.

This double blind study evaluated 32 healthy volunteers between the age range of 18 to 31 years. They were in good health, had no history of allergy to the dental bleaching agents used, and had all teeth (no fixed or removable dentures). The ones wearing orthodontic appliances, pregnant, nursing, smokers, having dentin sensitivity, ingesting medicines that would decrease salivary flow, or taking antimicrobial agents/mouthrinses at least three months prior to the study were also excluded.

Dental bleaching

The volunteers were randomly distributed into four different groups (n = 8) and received the combination of the in-office (IO) and the at-home (AH) dental bleaching, according to each group [Table 1]. The IO dental bleaching comprised a 37% carbamide peroxide gel (CP-37 - Whiteness Super- FGM, Joinville, Brazil), and the AH dental bleaching was 10% carbamide peroxide gel (CP-10 - Whiteness Perfect- FGM, Joinville, Brazil). A placebo gel (PG - Carbopol 934P gel, FGM, Joinville, Brazil) was also used in combination with the IO or the AH treatment.

Two weeks before the treatment began, the volunteers received toothbrushes (Oral B 40, Gillete do Brasil Ltd., Manaus, Brazil), and dentifrices without fluoride (52.5% of calcium carbonate, 25% of glycerin, 18% of natrosol, 2% of sodium lauril sulphate, and distilled water - Proderma Drugstore, Piracicaba, Brazil). They were instructed to use both, toothbrush and dentifrice, during all the experiment.

Impressions were taken of the upper and lower arches in order to manufacture trays with reservoirs for the AH dental bleaching. [8] The trays were trimmed and polished up to the cervical margin of the teeth.

The IO dental bleaching was performed under rubber dam isolation in three different sections lasting one hour, considering seven days interval. The treatment was then applied on the surface of all teeth (CP 37% or PG, according to each group) during one hour. After this time, the gel was carefully removed with air/water spray.

The AH dental bleaching treatment was kept for three weeks and the volunteers were instructed to use the trays all night long for at least six hours. Depending on the group, the treatment was either CP 10% or PG.

Saliva samples collection and microbiological methods

Saliva samples were collected after stimulation (paraffin wax chewing), as previously described by Dasanayake et al., [9] during five weeks according to the following periods of time: Right before the treatment starts (baseline); right after the IO application (zero hour), and 12 hours after the treatment (12 hours). During the fourth and the fifth weeks, when the treatments stopped, just the baseline collections were performed. The subjects deposited the saliva samples in sterilized eppendorfs tubes by using sterilized straws in order to avoid skin contamination. [10]

Immediately after the saliva collection, the samples were submitted to dispersion of cell aggregates in an ultrasonic cell disrupter (VibraCell 400 W, Sonics and Materials Inc. - at 5% amplitude, 9.9 seconds cycle, 6 pulses), followed by 10-fold dilutions in 0.9% NaCl solution (1:10, 1:100, 1:1000, 1:10000).

Aliquots of each dilution (5 μL) were spread on three different culture media in  Petri dish More Detailses (5 ΄ 2 cm 2 ), in duplicates. The media were blood agar (Difco Co - agar base + 5% sheep blood), MSA (Difco Co - Mitis Salivarius Agar, supplemented with 1% potassium tellurite and 15% sucrose), and MSB (Difco Co - Mitis Salivarius Bacitracin agar, supplemented with 200 IU of bacitracin/mL, 1% potassium tellurite, and 15% sucrose), in order to isolate the total microorganisms, streptococci, and mutans streptococci, respectively. [11] The plates were placed in an incubator (Jovan IG 150) with 10% of CO 2 at 37 o C, for 48 hours. Blood agar dishes were also incubated in an aerobic incubator at 37 o C, for 24 hours.

The results were compared by the Friedman test and Wilcoxon sign-ranked test for pair-wise comparison as the post hoc test (Bioestat 4.0, Mamirauα Institute, Belιm, PA, Brazil). The significance level was set at 5%.

   Results Top

The numbers of all microorganisms were not affected (P > 0.05) during the period of time by any of the bleaching agents. There were also no significant differences (P > 0.05) among the groups concerning any of the isolated microorganisms.

[Table 2],[Table 3],[Table 4] shows the bacterial growth (ufc/μL) on blood agar, MSA, and MSB, respectively.

   Discussion Top

Hydrogen peroxide was first used in 1913 to improve healing after gingival surgery as well as to inhibit plaque or periodontal microorganisms. [12] Hydrogen peroxide, with more than 100 years of use, is the active bleaching agent in carbamide peroxide. [13],[14] It is a potent and biologically active substance, reacting with lipids, proteins, nucleic acids, and other structures causing oxidative cell damage. [15] Therefore, the present study evaluated the antimicrobial activity of a bleaching agent against oral microorganisms. [6],[7],[8],[16]

Anaerobic bacteria showed high susceptibility against carbamide peroxide solutions at different concentrations. [16],[17] However, the susceptibility of aerobic and facultative oral- microorganisms varies according to the species. Although Streptococcus mutans, Streptococcus mitis, Streptococcus sanguis, Lactobacillus casei, and  Lactobacillus acidophilus Scientific Name Search e inhibited in vitro by three commercial 10% carbamide peroxide bleaching agents; a 10% carbamide peroxide solution failed to reduce salivary levels of mutans streptococci in a 6-week AH dental bleaching treatment. [6] In previous studies, no antimicrobial activity against S. mutans was observed for 10% carbamide peroxide or 7.5% hydrogen peroxide applied on both dental arches one hour a day for three weeks. [8] These results were similar to the ones observed in the present study focusing on carbamide peroxide at different concentrations (10 and 37%).

The present study aimed at the combination of both, IO and AH dental bleaching techniques according to a previous study, showing better bleaching results for such combination. A complete elimination of Grade 1 and Grade 2 tetracycline stains was observed for this combination used in a two-month bleaching treatment. [5] Despite the well known better bleaching results, little information on the antimicrobial activity concerning this combination was previously reported in the literature.

In the present study, carbamide peroxide was tested for three weeks as an AH (10%), and IO (37%) dental bleaching treatment, different from other studies focusing on AH dental bleaching only. However, no reduction in the oral microorganisms tested was observed for this association.

Such lack of antimicrobial activity observed in the present study could be accounted for the well known ability of the microorganisms tested, especially viridans streptococci, to survive in hydrogen peroxide environments; furthermore, some strains can produce hydrogen peroxide.

Hydrogen peroxide produced by S. sanguis I, S. sanguis II, and S. mitior strains can be metabolized by  Neisseria More Details sicca, Hemophilus segnis, H. parainfluenzae, Actinomyces viscosus, and  Staphylococcus epidermidis Scientific Name Search ins. [18] Also, some oral microorganisms were observed to degrade hydrogen peroxide.

Results observed in the present study showing no antimicrobial activity could be explained by the ability of some oral microorganisms, such as cariogenic streptococci, to adapt to different stress conditions. [19] Both supragingival and subgingival plaque have been reported to be resistant to oxidative stress. [20]

Salivary lactoperoxidase, an oral enzyme, catalyzes the reaction between thiocyanate and hydrogen peroxide, producing hypothiocyanite, which is much less toxic than hydrogen peroxide for bacteria, protecting the Streptococci against hydrogen peroxide. [21],[22]

In an in vitro study, Bentley et al., [6] reported that carbamide peroxide was bacteriostatic against S. mutans and lactobacilli, testing three peroxide carbamide based products (Nitewhite, Opalescence, and Proxigel). However, in an in vivo model, these authors observed no antimicrobial activity for a 10% carbamide peroxide solution (Proxigel) against salivary S. mutans; these results were similar to those observed in the present study.

Further studies are needed to verify the antimicrobial activity of carbamide peroxide in association with other agents, such as chlorexidine, which was reported to have better antimicrobial properties when associated with hydrogen peroxide. [23]

In conclusion, the carbamide peroxide when used at 37%, 10%, or in combination, did not affect human salivary microorganisms tested in the present study.

   Acknowledgments Top

This work was supported both by the Coordenaηao de Aperfeiηoamento de Pessoal de Nνvel Superior (Capes) and Conselho Nacional de Desenvolvimento Cientνfico e Tecnolσgico (CNPq).

   References Top

1.Sulieman M. An overview of bleaching techniques: 2, Night guard vital bleaching and nonvital bleaching. SADJ 2006;61:352,354,356.  Back to cited text no. 1      
2.Kihn PW. Vital tooth whitening. Dent Clin North Am 2007;51:319- 31, viii.  Back to cited text no. 2      
3.Haywood VB, Heymann HO. Nightguard vital bleaching. Quintessence Int 1989;20:173-6.  Back to cited text no. 3      
4.Leonard RH Jr, Bentley C, Eagle JC, Garland GE, Knight MC, Phillips C. Nightguard vital bleaching: A long-term study on efficacy, shade retention side effects, and patients' perceptions. J Esthet Restor Dent 2001;13:357-69.  Back to cited text no. 4      
5.Fiedler RS, Reichl RB. Combined professional and home care nightguard bleaching of tetracycline-stained teeth. Gen Dent 2000;48:257-61.  Back to cited text no. 5      
6.Gurgan S, Bolay S, Alaηam R. Antibacterial activity of 10% carbamide peroxide bleaching agents. J Endod 1996;22:356-7.  Back to cited text no. 6      
7.Bentley CD, Leonard RH, Crawford JJ. Effect of whitening agents containing carbamide peroxide on cariogenic bacteria. J Esthet Dent 2000;12:33-7.  Back to cited text no. 7      
8.Alkmin YT, Sartorelli R, Flσrio FM, Basting RT. Comparative study of the effects of two bleaching agents on oral microbiota. Oper Dent 2005;30:417-23.  Back to cited text no. 8      
9.Dasanayake AP, Caufield PW, Cutter GR, Roseman JM, Kφhler B. Differences in the detection and enumeration of mutans streptococci due to differences in methods. Arch Oral Biol 1995;40:345-51.  Back to cited text no. 9      
10.Groppo FC, Ramacciato JC, Simυes RP, Flσrio FM, Sartoratto A.Antimicrobial activity of garlic, tea tree oil, and chlorhexidine against oral microorganisms. Int Dent J. 2002;52:433-7.  Back to cited text no. 10      
11.Gold OG, Jordan HV, Van Houte J. A selective medium for Streptococcus mutans. Arch Oral Biol 1973;18:1357-64.  Back to cited text no. 11      
12.Marshall MV, Cancro LP, Fischman SL. Hydrogen peroxide: A review of its use in dentistry. J Periodontol 1995;66:786-96.   Back to cited text no. 12      
13.Li Y. Toxicological considerations of tooth bleaching using peroxide- containing agents. J Am Dent Assoc 1997;128:31S-6S.   Back to cited text no. 13      
14.Tredwin CJ, Naik S, Lewis NJ, Scully C. Hydrogen peroxide tooth- whitening (bleaching) products: Review of adverse effects and safety issues. Br Dent J 2006;200:371-6.  Back to cited text no. 14      
15.Imlay JA. How oxygen damages microbes: Oxygen tolerance and obligate anaerobiosis. Adv Microb Physiol 2002;46:111-53.  Back to cited text no. 15      
16.Scherer W, Boylan R, Bhatt S. Vital bleaching agents and oral antiseptic: Effect on anaerobic bacteria. J Esthet Dent 1992;4:84-5.  Back to cited text no. 16      
17.Gautier G, Noguer M, Costa N, Canela J, Viρas M. Mouthrinses: A comparative microbiological study. Bull Group Int Rech Sci Stomatol Odontol 2000;42:23-9.  Back to cited text no. 17      
18.Ryan CS, Kleinberg I. Bacteria in human mouths involved in the production and utilization of hydrogen peroxide. Arch Oral Biol 1995;40:753-63.  Back to cited text no. 18      
19.Lemos JA, Abranches J, Burne RA. Responses of cariogenic streptococci to environmental stresses. Curr Issues Mol Biol 2005;7:95-107.   Back to cited text no. 19      
20.Marquis RE. Oxygen metabolism, oxidative stress and acid-base physiology of dental plaque biofilms. J Ind Microbiol 1995;15:198-207.   Back to cited text no. 20      
21.Carlsson J. Salivary peroxidase: An important part of our defense against oxygen toxicity. J Oral Pathol 1987;16:412-6.   Back to cited text no. 21      
22.Thomas EL, Milligan TW, Joyner RE, Jefferson MM. Antibacterial activity of hydrogen peroxide and the lactoperoxidase-hydrogen peroxide-thiocyanate system against oral streptococci. Infect Immun 1994;62:529-35.  Back to cited text no. 22      
23.Steinberg D, Heling I, Daniel I, Ginsburg I. Antibacterial synergistic effect of chlorhexidine and hydrogen peroxide against Streptococcus sobrinus, Streptococcus faecalis and Staphylococcus aureus. J Oral Rehabil 1999;26:151-6.  Back to cited text no. 23      

Correspondence Address:
Francisco C Groppo
Department of Physiological Sciences, Piracicaba Dentistry School, UNICAMP
Login to access the Email id

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0970-9290.57367

Rights and Permissions


  [Table 1], [Table 2], [Table 3], [Table 4]


    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Email Alert *
    Add to My List *
* Registration required (free)  

    Materials and Me...
    Article Tables

 Article Access Statistics
    PDF Downloaded289    
    Comments [Add]    

Recommend this journal