/home/opencode/cpanel/stomaeduj_hacked/uploads/2018/04/Article_5_4_2.pdf ORAL MICROBIOLOGY EFFECT OF POWDERED GREEN TEA MATCHA ON BIOFILM FORMATION Original Article BY MUTANS STREPTOCOCCI Mochamad Fahlevi Rizal1,2a , Noboru Kaneko1a* , Hiroshi Ogawa1b 1 Division of Preventive Dentistry, Department of Oral Health Science, Graduate School of Medical and Dental Sciences, Niigata University, Japan 2 Department of Pediatric Dentistry, Faculty of Dentistry, Universitas Indonesia, Indonesia DDS, PhD, Assistant Professor a DDS, MDSc, PhD, Professor and Head b ABSTRACT DOI: 10.25241/stomaeduj.2018.5(4).art.2 Introduction: Antibacterial ingredients with high safety and mild taste that can be OPEN ACCESS This is an Open used for mouthwash for children are required. Matcha is one of the natural beverages Access article under the CC BY-NC 4.0 license. that is made from the leaves of Camellia sinensis and widely consumed by Japanese and Peer-Reviewed Article Asian people. This study aimed to evaluate the effectiveness of matcha in suppressing Citation: Rizal MF, Kaneko N, Ogawa H. biofilm formation by mutans streptococci, typical cariogenic bacteria. Effect of powdered green tea matcha on Methodology: Five laboratory strains of Streptococcus mutans (serotype c, e and f ) biofilm formation by mutans Streptococci. Stoma Edu J. 2018;5(4):220-225. and Streptococcus sobrinus (serotype d and g) were used to evaluate the antibacterial Academic Editor: Mircea Ioan Popa effect of matcha. Matcha extract was added to bacterial cells in Heart Infusion broth MD, PhD, Professor, Head Microbiology Depart- supplemented with 1% sucrose (HIS). After incubation for 24 hours, the formed biofilm ment II, “Carol Davila”University of Medicine and Pharmacy, Bucharest, Romania was dyed by Crystal Violet, and optical density at 490 nm was determined as the Received: November 30, 2018 amount of biofilm formation. The effect of 0.02% chlorhexidine in HIS and MilliQ in HIS Revised: December 07, 2018 were also measured as well. Accepted: December 17, 2018 Published: December 18, 2018 Results: The amounts of biofilm formation by all serotypes of mutans streptococci in *Corresponding author: matcha + HIS were significantly lower than those in MilliQ + HIS as were chlorhexidine Assistant Professor Noboru Kaneko, DDS, in HIS, except S. sobrinus serotype g. The differences of amounts biofilm formation by PhD, Division of Preventive Dentistry, Department of Oral Health Science, Gradu- all S. mutans (serotype c, e and f ) and S. sobrinus serotype d in matcha + HIS and in ate School of Medical and Dental Sciences, Niigata University, 2-5274, Gakkocho-dori, chlorhexidine + HIS were not significant statistically. Chuo-ku, Niigata City, Japan Conclusion: Matcha extract has an equivalent effectiveness of 0.02% chlorhexidine Tel: +81 25 227 2857, Fax: +81 25 227 0807, e-mail: nkaneko@dent.niigata-u. against most serotypes of mutans streptococci. ac.jp Keywords: Streptococcus mutans, Streptococcus sobrinus, chlorhexidine, green tea, Copyright: © 2018 the Editorial Council mouthwash. for the Stomatology Edu Journal. 1. Introduction in the market in three forms: Loose leaf, bagged and Efforts to obtain antibacterial ingredients that meet powdered (matcha) [6]. Matcha is generally consumed the criteria of comfort, especially taste, are needed to in Japan as a beverage that has high anti-oxidants such improve the quality of the oral cavity. Oral health is as flavonoids [7]. Many natural foods and beverages inseparable from the normal flora balance of the oral that are commonly consumed by people in a region, cavity in order to prevent the occurrence of caries such as honey in the tropical regions, olive oil, virgin and gingivitis. The use of natural ingredients that coconut oil and others have been partially known to are commonly consumed by the wider community have antibacterial effects especially in the oral cavity in a place needs to be expanded in the functions of [8]. With many natural sources like ginger and garlic maintaining public health, especially oral health [1]. that also have flavonoid and are commonly used in The ease of getting it and the consumption habits Asia one needs to observe the antibacterial effect on of the community makes it easy for materials like oral bacteria [9]. As a comparison, chlorhexidine has this to be accepted by the community if proven to been used for many years as a strong antibacterial have good effects on oral health. Because caries standard and is also used in dentistry for preventive and gingivitis are also a problem in children in many measures and treatment but the taste becomes an countries, especially with developmental disabilities, issue especially for children [10]. natural ingredients as an antibacterial for the oral The streptococci that are commonly related to the cavity are urgently needed to be easily accepted by human caries process are Streptococcus mutans and the child community, both in terms of safety and ease Streptococcus sobrinus. These cariogenic bacteria are of use [2-4]. Each community has unique ingredients divided into 5 serotypes (c, e, f, d and g) [11]. There developed in its area, but has not been utilized has been no study on the antibacterial effects of optimally in addition to regular consumption of food matcha against each serotype of mutans streptococci. and beverages [5]. It is expected that the results of research related Green tea (Camellia sinensis) is commercially available to antibacterial from natural sources can be used 220 Stoma Edu J. 2018;5(4): 220-225. http://www.stomaeduj.com EFFECT OF POWDERED GREEN TEA MATCHA ON BIOFILM FORMATION BY MUTANS STREPTOCOCCI Original Article (a) (b) (c) (d) Figure 1. Biofilm formation in NC (MilliQ alone), MQ (MilliQ + bacterial cells in HIS) and PC (bacterial cells in HIS) by each strain of mutans streptococci: (a) S. mutans ATCC25175 serotype c, (b) S. mutans LM7 serotype e, (c) S. mutans OMZ175 serotype f, (d) S. sobrinus ATCC33478 serotype d, (e) S. sobrinus 6715 serotype g. (e) to prevent infection in order to minimize chemical ˚C overnight and then also sub-cultured in BHI broth. substances [12]. This study aimed to evaluate the The bacterial solution was centrifuged and dissolved effectiveness of matcha in suppressing biofilm in Heart Infusion broth (Becton Dickinson Maryland, formation by each serotype of mutans streptococci. USA) supplemented with 1% sucrose (HIS) until it reached 1 x 106 CFU/ml which was then used for the biofilm assay as strain culture. 2. Material and Methods 2.1. Preparation of antibacterial solutions 2.3. Biofilm assay As a matcha solution, 5 gram of powdered green The biofilm formation of each strain was evaluated tea matcha (Itoen, Tokyo, Japan) was added to 100 using a 96 well plate. Each plate was divided into 5 mL of water at 100˚C and boiled for 15 minutes to areas of 12 wells and labeled as MT for wells that were extract. Prior to use in the laboratory, the solution filled by matcha solution and strain culture, CHX for was filtered using 0.20 µm Minisart filter (Sartorius, wells that were filled by 0.02% chlorhexidine and Göttingen, Germany). The chlorhexidine solution strain culture, MQ for wells that were filled by MilliQ used in this study was 0.02% and was prepared from and strain culture, NC for wells that were filled by 20% chlorhexidine gluconate (Wako Pure Chemical, milliQ alone as negative control and PC for wells that Osaka, Japan). were filled by HIS and strain culture as positive control (Table 2). 2.2. Culture of mutans streptococci After 24 hours of incubation, the culture supernatant As mutans streptococci, we used 3 laboratory strains was discarded, and each well was washed twice with of S. mutans (serotype c, e, f) and 2 laboratory strain 200 µL Phosphate Buffered Saline (PBS). A 200 µL of S. sobrinus (serotype d, g) (Table 1). Each strain volume of 0.5% crystal violet was then added and from -80 ˚C stock was cultured in Brain Heart Infusion incubated at 37 ˚C for 15 minutes. (BHI) broth (Becton Dickinson Maryland, USA) at 37 The remaining crystal violet solution was discarded, the Stomatology Edu Journal 221 EFFECT OF POWDERED GREEN TEA MATCHA ON BIOFILM FORMATION BY MUTANS STREPTOCOCCI Original Article Table 1. Strains of mutans streptococci used in this study. Table 2. Composition of cultures for biofilm assay. Strain Serotype Antibacterial Bacterial HIS MilliQ Total Streptococcus mutans solution cells in HIS ATCC25175 c (type strain) PC 0 µL 100 µL 100 µL 0 µL 200 µL LM7 e MT 100 µL 100 µL 0 µL 0 µL 200 µL OMZ175 f CHX 100 µL 100 µL 0 µL 0 µL 200 µL Streptococcus sobrinus ATCC33478 d (type strain) MQ 0 µL 100 µL 0 µL 100 µL 200 µL 6715 g NC 0 µL 0 µL 0 µL 200 µL 200 µL PC: positive control; MT: matcha solution; CHX: 0.02% chlorhexi- dine gluconate; MQ: milliQ solution; NC: negative control biofilm was washed once with 200 mL PBS, and then 200 strains of mutans streptcocci. µL of 96% ethanol was added to each well. The optical This in-vitro research provides an overview of the density of the biofilm formation was then measured at antibacterial effects of matcha on various strains 490 nm (OD490) using a microplate reader [13]. of mutans streptococci. Mutans streptococci are considered as typical cariogenic bacteria [14,15]. 2.4. Statistical analysis Mutans streptococci can be classified into five The OD490 values evaluated as biofilm formation were serotypes, with dominant serotypes being slightly compared between NC, MQ and PC to confirm the different locally. The effect of matcha against S. validity of the measurement. Then OD490 values of MT, sobrinus serotype g was not significant among all CHX, NC and MQ were compared to determine the mutans streptococci strains of serotype examined differences in their effects on biofilm formation. In in this study. However, in oral cavity of humans order to compare the differences, t-test and Bonferoni worldwide, S. sobrinus serotype g is not a type that correction were used. found dominantly [16-19]. In other study S. sobrinus All statistical analyses were performed using SPSS serotype g more resistance to chlorhexidine than S. statistics 19 (IBM, USA); p values of 0.05 or less were mutans and S. sobrinus serotype d [20]. Therefore, considered significant. matcha is thought to have the effect of inhibiting biofilm formation for most of mutans streptococci in oral cavity. One of the cariogenic properties 3. Results of these bacteria owes the high performance of Fig. 1 shows OD490 values which mean amount of glucan synthesis to sucrose, which contributes to biofilm formation of NC (MilliQ alone), MQ (MilliQ + the biofilm formation [21,22]. So, the effectiveness bacterial cells in HIS) and PC (bacterial cells in HIS) of matcha in suppressing the biofilm formation by by S. mutans (serotype c, e and f) and S. sobrinus mutans streptococci colonization was important to (serotype d and g), respectively. The OD490 of PC be evaluated in this study. The better antibacterial will showed significantly higher values than MQ, and suppress more types of strains, meanwhile caries are those of MQ showed significantly higher values than not only caused by a single bacterial but related to the NC in all strains (serotype c, e, f, d and g). biofilm activity involving many bacteria [23]. Fig. 2 shows OD490 values which mean amount of Chlorhexidine is a common ingredient as an biofilm formation of MT (matcha solution + bacterial antibacterial agent and is widely used for cells in HIS), CHX (CHX solution + bacterial cells in mouthwashes [24-28]. However, chlorhexidine has a HIS), NC (MilliQ alone) and MQ (MilliQ + bacterial taste issue, and it has problems such as side effects on cells in HIS), respectively. The OD490 of MT, CHX and the mucosa and tooth coloring, so it is not suitable for NC showed significantly lower values than MQ and mouthwashes in children [29,30]. The concentration no significant difference was found between MT, of chlorhexidine used clinically is 0.12% and 0.2%, CHX and NC in all S. mutans (serotype c, e and f) and while in this study using a concentration of 0.02%. It S. sobrinus (serotype d). In S. sobrinus serotype g, the was proved that the concentration of 0.03% was as OD490 of MT showed lower value than MQ though effective as 0.12% or 0.2% concentration clinically the difference did not reach statistical significance. [31-33]. The result of this study revealed that the low The OD490 of CHX and NC showed significantly lower concentration of 0.02% chlorhexidine can inhibit values than MQ and MT in S. sobrinus serotype g. biofilm formation of mutans streptococci. Matcha has long been used for drinking and the safety has been established. The requirements for 4. Discussion mouthwash that can be used by children are related The OD490 of PC showed significantly higher values to safety issue [34,35]. It is also considered to have less than MQ and those of MQ showed significantly higher problems with taste. Therefore, it is considered that values than NC in all strains, which indicated that the matcha is suitable as an ingredient for mouthwash biofilm assay used in this study could evaluate the for children. The bacteria investigated this time are amount of biofilm formation adequately. The OD490 mutans streptococci and are strongly related to dental of MT and CHX showed significantly lower values caries. However, there are many others bacteria in than MQ and the differences between MT, CHX and the oral cavity related to dental caries and other oral NC were not significant in all strains except S. sobrinus disease such as periodontitis. In this study, we did serotype g. This means that the antibacterial effect not consider the influence on other such cariogenic of matcha solution resemble CHX solution in most bacteria and periodontal disease related bacteria. 222 Stoma Edu J. 2018;5(4): 220-225. http://www.stomaeduj.com EFFECT OF POWDERED GREEN TEA MATCHA ON BIOFILM FORMATION BY MUTANS STREPTOCOCCI Original Article (a) (b) (c) (d) Figure 2. Effect of matcha extract and chlorhexidine on biofilm formation by each strain of mutans streptococci: (a) S. mutans ATCC25175 serotype c, (b) S. mutans LM7 serotype e, (c) S. mutans OMZ175 serotype f, ( (d) S. sobrinus ATCC33478 serotype d, (e) S. sobrinus 6715 serotype g. (e) Hereafter, it seemed necessary to investigate the Department of Oral Health Science, Graduate School antibacterial effect on those bacteria. of Medical and Dental Sciences, Niigata University. The authors declare that the research was conducted in the absence of any commercial or financial 5. Conclusion relationship that could be construed as a potential Matcha has the equivalent effectiveness of 0.02% conflict of interest. chlorhexidine against most serotypes of mutans streptococci, namely S. mutans serotype c, e and f and S. sobrinus serotype d. It is suggested that matcha References is a promising ingredient as a safe and effective for 1. Jacob, PS. Editorial: Natural products in oral health. The Open Dent J. 2016;10(Suppl-1, M1): 158-159. mouthwashes in children. [Full text links] [Free PMC Article] [PubMed] Google Scholar (0) Scopus (0) 2. Norwood KW, Slayton, RL. Oral health care for children with developmental disabilities. Pediatrics. 2013;131(3): 614-619. Author Contributions [Full text links] [PubMed] Google Scholar (53) Scopus (22) MFR: Idea, experimental design, data analysis, 3. Lachenmeier DW, Monokhova YB, Markova M, et al. What happens if people start drinking mouthwash as surrogate wrote the manuscript. NK: Performed biofilm and alcohol? A quantitative risk assessment. Food Chem Toxicol. spectrophotometry assay, substantial contributed 2013; 51: 173-178. to writing manuscript. HO: Performed substantial [Full text links] [PubMed] Google Scholar (13) Scopus (8) 4. Werner CWdeA, Seymour RA. Are alcohol containing contributed to writing manuscript. mouthwashes safe? Br Dent J. 2009;207(10): 488-489. [Full text links] [PubMed] Google Scholar (37) Scopus (16) 5. Palombo EA. Traditional medicinal plant extracts and natural products with activity against oral bacteria: Acknowledgement potential application in the prevention and treatment The authors have no conflict of interest. This of oral diseases. Evid Based Complement Alternat Med. 2011;2011: 680354. research funding by Division of Preventive Dentistry, [Full text links] [Free PMC Article] [PubMed] Google Scholar Stomatology Edu Journal 223 EFFECT OF POWDERED GREEN TEA MATCHA ON BIOFILM FORMATION BY MUTANS STREPTOCOCCI (381) Scopus (196) 21. Hahnel S, Mühlbauer G, Hoffmann J, et al. Streptococcus Original Article 6. Farooq S, Sehgal A. Antioxidant activity of different forms mutans and Streptococcus sobrinus biofilm formation and of green tea: loose leaf, bagged and matcha. Curr Res Nutr metabolic activity on dental materials. Acta Odontol Scand. Food Sci. 2018;6(1): 35-40. 2012;70(2): 114-121. [Full text links] Google Scholar (1) Scopus (0) [Full text links] [PubMed] Google Scholar (13) Scopus (9) 7. Dietz C, Dekker M, Fiszman BP. An intervention study on 22. Merritt J, Qi F. The mutacins of Streptococcus mutans: the effect of matcha tea, in drink and snack bar formats, on regulation and ecology. Mol Oral Microbiol. 2012;27(2): 57- mood and cognitive performance. Food Res Int. 2017; 99(Pt 69. 1): 72-83. [Full text links] [Free PMC Article] [PubMed] Google Scholar [Full text links] [PubMed] Google Scholar (5) Scopus (3) (81) Scopus (59) 8. Lavine P, Fauziah E, Rizal MF, Budiardjo SB. Antibacterial 23. Sanz M, Beighton D, Curtis MA, et al. Role of microbial effect of virgin coconut oil on (Actinomyces Sp.) that biofilms in the maintenance of oral health and in the causes dental black stain in children. Asian J Pharm Clin Res. development of dental caries and periodontal diseases. 2018;11(2): 333-335. Consensus report of group 1 of the Joint EFP/ORCA [Full text links] Google Scholar (0) Scopus (0) workshop on the boundaries between caries and 9. Ali AMA, El-Nour MEM, Yagi SM. Total phenolic and periodontal disease. J Clin Periodontol. 2017 Mar;44 Suppl flavonoid contents and antioxidant activity of ginger 18: S5-S11. (Zingiber officinale Rosc.) rhizome, callus and callus treated [Full text links] Google Scholar (45) Scopus (27) with some elicitors. J Genet Eng Biotechnol. 2018. 24. Kocak MM, Ozcan S, Kocak S, et al. Comparison of the [Full text links] Google Scholar (2) Scopus (1) efficacy of three different mouthrinse solutions in 10. Calogiuri GF, Di Leo E, Trautmann A, et al. Chlorhexidine decreasing the level of streptococcus mutans in saliva. Eur J hypersensitivity: a critical and updated review. J Allergy Dent. 2009;3(1): 57-61. Ther. 2013;4(4): 1-7. [Full text links] [Free PMC Article] [PubMed] Google Scholar [Full text links] Google Scholar (35) Scopus (0) (51) Scopus (0) 11. Coykendall AL, Bratthall D, O’Connor K, Dvarskas RA. 25. Simratvir M, Singh N, Chopra S, Thomas AM. Efficacy of 10% Serological and genetic examination of some nontypical povidone iodine in children affected with early childhood Streptococcus mutans strains. Infect Immun. 1976;14(3): caries: an in vivo study. J Clin Pediatr Dent. 2010;34(3): 233- 667-670. 238. [Full text links] [Free PMC Article] [PubMed] Google Scholar [Full text links] [PubMed] Google Scholar (30) Scopus (17) (48) Scopus (17) 26. Cieplik F, Kara E, Muehler D, et al. Antimicrobial efficacy of 12. Kiany F, Niknahad H, Niknahad M. Assessing the effect alternative compounds for use in oral care toward biofilms of pomegranate fruit seed extract mouthwash on dental from caries-associated bacteria in vitro. Microbiology Open. plaque and gingival inflammation. J Dent Res Rev. 2016;3(4): 2018: e00695. 117-123. [Full text links] [PubMed] Google Scholar (0) Scopus (0) [Full text links] Google Scholar (1) Scopus (0) 27. Vinod KS, Sunil KS, Sethi P, et al. A novel herbal formulation 13. Linggriani A, Rizal MF, Fauziah E, Suharsini M. Differences in versus chlorhexidine mouthwash in efficacy against oral the effect of 0.05% and 0.1% propolis flavonoids on in vitro microflora. J Int Soc Prev Community Dent. 2018;8(2): 184- biofilm formation by Streptococcus mutans from children’s 190. dental plaque. Asian J Pharm Clin Res. 2018;11(2): 215-218. [Full text links] [Free PMC Article] [PubMed] Google Scholar [Full text links] Google Scholar (0) Scopus (0) (0) Scopus (0) 14. Sánchez-Acedo M, Montiel-Company JM, Dasí-Fernández F, 28. James P, Worthington HV, Parnell C, et al. Chlorhexidine Almerich-Silla JM. Streptococcus mutans and Streptococcus mouthrinse as an adjunctive treatment for gingival health. sobrinus detection by Polymerase Chain Reaction and Cochrane Database Syst Rev. 2017;3: CD008676. their relation to dental caries in 12 and 15 year-old [Full text links] [PubMed] Google Scholar (3) Scopus (23) schoolchildren in Valencia (Spain). Med Oral Patol Oral Cir 29. Gunasekaran G, Lakshmanan R. Effect of chlorhexidine Bucal. 2013;18(6): e839-e845. mouthwash on taste alteration. Asian J Pharm Clin Res. [Full text links] [Free PMC Article] [PubMed] Google Scholar 2016;9(13): 102-104. (12) Scopus (12) [Full text links] Google Scholar (2) Scopus (0) 15. Okada M, Kawamura M, Oda Y, et al. Caries prevalence 30. Bagis B, Baltacioglu E, Özcan M, Ustaomer S. Evaluation associated with Streptococcus mutans and Streptococcus of chlorhexidine gluconate mouthrinse-induced staining sobrinus in Japanese schoolchildren. Int J Paediatr Dent. using a digital colorimeter: an in vivo study. Quintessence 2012;22(5): 342-348. Int. 2011;42(3): 213-223. [Full text links] [PubMed] Google Scholar (31) Scopus (13) [Full text links] [PubMed] Google Scholar (36) Scopus (18) 16. Nakano K, Nemoto H, Nomura R, et al. Serotype distribution 31. Bonesvoll P. Oral pharmacology of chlorhexidine. J Clin of Streptococcus mutans a pathogen of dental caries in Periodontol. 1977;4(5): 49-65. cardiovascular specimens from Japanese patients. J Med [Free text links] [PubMed] Google Scholar (77) Scopus (46) Microbiol. 2007;56(Pt4): 551-556. 32. Dogan S, Günay H, Leyhausen G, Geurtsen W. Effects of low- [Full text links] [PubMed] Google Scholar (59) Scopus (38) concentrated chlorhexidine on growth of Streptococcus 17. Holbrook WP, Beighton D. Streptococcus mutans levels sobrinus and primary human gingival fibroblasts. Clin Oral in saliva and distribution of serotypes among 9-year-old Investig. 2003;7(4): 212-216. Icelandic children. Scand J Dent Res. 1987;95(1): 37-42. [Free text links] [PubMed] Google Scholar (10) Scopus (8) [Full text links] [PubMed] Google Scholar (46) Scopus (0) 33. Sylvie S, Catherine F, Jean-Philippe G, Christine R. In vitro 18. Fauziah E, Sutadi H, Bachtiar EW. Prediction of baby bottle bactericidal assay under simulated practical conditions for tooth decay syndrome using the salivary mucin MG2. Int J comparison of chlorhexidine mouthrinses: chlorhexidine Clin Prev Dent. 2013;9(4): 211-214. concentration isonly one of the in vitro activity criteria. Oral [Full text links] Google Scholar (1) Scopus (0) Health Dentl Manag. 2016;15(6): 1-6. 19. Kozai K, Nakayama R, Tedjosasongko U, et al. Intrafamilial [Full text links] Google Scholar (0) Scopus (0) distribution of mutans streptococci in Japanese families 34. Massey CC, Shulman JD. Acute ethanol toxicity from and possibility of father-to-child transmission. Microbiol ingesting mouthwash in children younger than age 6, Immunol. 1999;43(2): 99-106. 1989-2003. Pediatr Dent. 2006;28(5): 405-409. [Full text links] [PubMed] Google Scholar (131) Scopus (57) [Full text links] [PubMed] Google Scholar (12) Scopus (0) 20. Grönroos L, Mättö J, Saarela M, et al. Chlorhexidine 35. Sharma R, Hebbal M, Ankola AV, et al. Effect of two herbal susceptibilities of mutans streptococcal serotype and mouthwashes on gingival health of school children. J Tradit ribotypes. Antimicrob Agents Chemother. 1995;39(4): 894- Complement Med. 2014;4(4): 272-278. 898. [Full text links] [Free PMC Article] [PubMed] Google Scholar [Full text links] [Free PMC Article] [PubMed] Google Scholar (12) Scopus (6) (51) Scopus (33) 224 Stoma Edu J. 2018;5(4): 220-225. http://www.stomaeduj.com EFFECT OF POWDERED GREEN TEA MATCHA ON BIOFILM FORMATION BY MUTANS STREPTOCOCCI Original Article Mochamad Fahlevi RIZAL DDS, PhD, Assistant Professor Department of Oral Health Science Division of Preventive Dentistry Graduate School of Medical and Dental Sciences Niigata University, Niigata, Japan CV Mochamad Fahlevi Rizal is a Pediatric Dentist in Indonesia. He has been a lecturer in the Department of Pediatric Dentistry, Faculty of Dentistry, Universitas Indonesia since 1998. In 2018 he became visiting lecturer in the Niigata University and made collaborative research with the Division of Preventive Dentistry, Department of Oral Health Science, Graduate School of Medical and Dental Sciences, Niigata University. Questions 1. Which was the matcha used in this study? qa. 5 g in 100 mL water; qb. 0.5 g in 100 mL water; qc. 0.05 g in 100 mL water; qd. 50 g in 100 mL water. 2. What species of mutans streptococci were used in this study? qa. Streptococcus ferus and Streptococcus pneumonia; qb. Streptococcus mutans and Streptococcus sobrinus; qc. Streptococcus macacae and Streptococcus ratti; qd. Streptococcus criceti and Streptococcus downei. 3. What was the antibacterial used to compare the effectiveness of matcha in this study? qa. Ethil alcohol 70%; qb. Povidone iodine; qc. Chlorhexidine; qd. Gentian violet. 4. What was the result of this study? qa. All strain of mutans streptococci were suppressed significantly by matcha; qb. Only serotype c was suppressed significantly by matcha; qc. Only serotype c, e, and d were suppressed significantly by matcha; qd. Only serotype c, e, f, and d were suppressed. http://www.efcd.eu/conseuro-meetings/ Stomatology Edu Journal 225