Effect of Toothpaste on Oral Microbial Flora (Bacteria and Fungi)

Transcription

1 International Journal of Bioengineering & Biotechnology 2018; 3(3): Effect of Toothpaste on Oral Microbial Flora (Bacteria and Fungi) Obiazi Helen A. K. 1, *, Dic-Ijiewere Ebenezer Oseremen 2 1 Department of Microbiology, Ambrose Alli University, Ekpoma, Nigeria 2 Department of Chemical Pathology, Ambrose Alli University, Ekpoma, Nigeria address * Corresponding author To cite this article Obiazi Helen A. K., Dic-Ijiewere Ebenezer Oseremen. Effect of Toothpaste on Oral Microbial Flora (Bacteria and Fungi). International Journal of Bioengineering & Biotechnology. Vol. 3, No. 3, 2018, pp Received: April 21, 2018; Accepted: May 15, 2018; Published: July 2, 2018 Abstract This study was carried out to evaluate the effect of toothpaste on oral microbial flora (bacteria and fungi) isolated of students of Ambrose Alli University, Ekpoma. The sample size of the study consisted of 50 subjects from Ekpoma, randomly selected across Ekpoma, consisting of 20 males and 30 females. Sterile swab sticks were used to collect samples from the oral cavity (tongue, teeth and the inner cheek) aseptically from each subject using swab sticks. The organisms isolated were Bacteria: Streptococcus mutans (25), Staphylococcus aureus (10), Escherichia coli (2), Klebsiella specie (5), and Fungi: Candida albicans (4) and Rhizopus spp (2). The antibiotics sensitivity indicated Close-Up Deep action (Red hot) and Macleans (Fresh mint) were sensitive to all the organisms isolated, Pepsodent - Natural white (Gel), Oral B (Pro-Health), Close-Up Herbal and Colgate were sensitive to three (3) organisms, while the antibiotics sensitivity of the oral fungal flora indicated Close-Up Deep action (Red hot), Macleans (Fresh mint) and Oral B (Pro-Health) showed sensitivity to all the fungal isolates while Pepsodent - Natural white (Gel), Close-Up Herbal and Colgate were sensitive to one (1) of the fungal isolate. Analysis of the inhibition of bacteria by the toothpastes studied shows that they are effective for some bacterial species than others. However, the microbial quality of toothpaste formulations marketed in the open markets has met the drug standard which excludes the presence of the index, indicator or pathogenic organism in drugs to be consumed by the populace. Keywords Toothpaste, Teeth, Microbial Flora, Oral, Students 1. Introduction Teeth are small, calcified, whitish structures found in the jaws or mouths of many vertebrates that are used to break down food. Some animals particularly are embedded in the mandible bone or the maxillary bone and are covered by gums. Teeth are made up multiple tissues of varying diversity and hardness. Dental biofilm formation is a natural process that must be controlled by regular brushing to prevent the development of dental caries and periodontal disease [1-2]. Dental caries and related oral diseases like gingivitis and periodontitis are most common oral diseases throughout the world including both developed and developing countries, affecting people from all ages of life. The frequency of these oral diseases is continuously increasing with change in eating habit of among peoples of different age group and increased consumption of sugar. Standard toothpaste formulas generally contain a combination of fluoride and detergents, enhancing the efficacy of biofilm control [2-3]. The addition of different antimicrobial agents has been suggested as a potential method for reduction, control and prevention of the accumulation of cariogenic and periodontopathogenic microorganisms [4]. However, the antimicrobial ability of such agents in combination with fluoride-containing toothpastes has not yet been effectively tested or proven [5]. Toothpaste is a gel dentifrice used with a toothbrush as an accessory to clean and maintain the aesthetics and health of teeth. Toothpaste is used to promote oral hygiene. Triclosan, an antibacterial agent, is a common toothpaste ingredient in

2 25 Obiazi Helen A. K. and Dic-Ijiewere Ebenezer Oseremen: Effect of Toothpaste on Oral Microbial Flora (Bacteria and Fungi) the United Kingdom. Triclosan or zinc chloride prevents gingivitis and, according to the American Dental Association, helps reduce tartar and bad breath. Herbal toothpastes contain baking soda, aloe, eucalyptus oil, myrrh, plant extract, and essential oils. In developing countries like Nigeria, a very significant portion of dental problems results from microbial infections. Dental problems consists mainly of three types, they are dental plaques, dental caries and periodontal diseases [6]. Plaque (a layer that forms on the surface of a tooth, principally at its neck; composed of bacteria in an organic matrix) has been linked to gingivitis, periodontal disease, or dental caries. Previous studies have shown that dental plaque can be controlled by physical removal of plaque, use of antimicrobial toothpastes as well as mouthwash. Dental caries is one of the commonest infectious microbial diseases of the world since ages and has an increased incidence in recent past due to drastic changes in lifestyle habits. Streptococcus mutans, a bacterium seen in the mouth is known to be one of the major causes of dental plague and may also cause dental caries [7]. Mouth bacteria have been linked to plaque, tooth decay and toothache, though caries effectively avoidable by simple inexpensive and easy to practice personal hygiene habits with tooth brushing habit, the most effective of them. Among various factors of this practice, antibacterial efficacy of the tooth paste has a major role to play in the outcome. Anti-microbial agents have been used as chemotherapeutic agents to help improve the state of oral health as such it is imperative to ascertain the efficacy of the different brands of toothpaste on the oral micro-flora [8]. However, relatively few published articles in the literature have compared the effect of toothpastes based dentifrices on plaque associated microbial flora [9-12]. However, the oral biofilms associated with caries and periodontal diseases are generated by multi-species interactions. Dentifrices need to contain various antimicrobial agents in order to reduce, control and prevent different kinds of dental diseases. Many dentifrices claim to have antimicrobial properties but very little research has been conducted to investigate these claims. Considering that antimicrobial efficacy of chemical agents may be reduced or inactivated when associated to other ingredients of toothpastes. Hence, the present study was carried out to determine the effect of some selected toothpastes on oral microbial (bacteria and fungi) flora. 2. Methodology 2.1. Area of Study This study was carried out in Ekpoma, Esan West Local Government Area of Edo State. Edo state lies between longitude I E and I E and latitude I N and I N with a land mass of 17, 450 sq.km located in the South-South geopolitical zone of Nigeria with a population of 3.1 million people. Ekpoma is a semi- urban town with the major occupation of farming, trading, civil servants and students [13] Collection of Sample The sample size of the study consisted of 50 subjects from Ekpoma, randomly selected across Ekpoma, consisting of 20 males and 30 females. Sterile swab sticks were used to collect samples from the oral cavity (tongue, teeth and the inner cheek) aseptically from each subject using swab sticks. All samples used for this study were collected between the months of June and August Ethical Consideration and Informed Consent Ethical approval was obtained from the Ambrose Alli University Ethical Committee and informed consent was obtained from the subjects before collection of samples Research Design Six (6) toothpastes were selected for assessment of their in vitro antimicrobial activities. They were purchased from local provision shops in Ekpoma, Esan West Local Government Area, Edo State, Nigeria Culture Media Preparation The media prepared were nutrient agar, MacConkey agar, Chocolate agar, Blood agar and Sabroud dextrose agar; the media were all prepared following the manufacturer s instructions apart from blood agar, which had blood added to prepared nutrient agar. The selected dentifrices solutions were made by mixing the calculated amount of toothpastes (2.0 gm) in measured volume (2 ml) of sterile pyrogen-free distilled water to give 1:1 dilution; they were further diluted in sterile distilled water and four different dilutions of 1:2, 1:4, 1:8 and 1:16 were made. Nutrient agar and brain heart infusion agar plates were prepared to assess the antimicrobial activity of dentifrices against the pathogens. All other chemicals and reagents used were of analytical grade Isolation of Organisms The different test organisms were isolated from dental swabs of some selected individuals in Ekpoma, Edo state, Nigeria. The test organisms isolated from the study include; Bacteria (Staphylococcus aureus, Streptococcus mutans, Escherichia coli and Klebsiella spp) and fungi (Candida albicans and Rhizopus spp). This was done by careful observation of the growth colony morphology, gram staining as described by Monica Cheesbrough [14] and biochemical tests Biochemical Tests The following biochemical tests were carried out; catalase test, oxidase test and coagulase test as described by Monica Cheesbrough [14] as well as the temporary and permanent direct mount for the fungal identification. All isolates for this study were identified by their colonial morphology on the media which include Size, Shape,

3 International Journal of Bioengineering & Biotechnology 2018; 3(3): Elevation, Opacity, Edge, Colour, haemolysis and fermentation, Gram stain reaction, biochemical test characterization and sugar fermentation test were carried out. 3. Results The present study investigates the antimicrobial effects of some selected toothpaste on oral microbial flora (bacteria and fungi). The organisms isolated are Bacteria: Streptococcus mutans, Staphylococcus aureus, Escherichia coli, Klebsiella spp and Fungi: Candida albicans and Rhizopus spp. The toothpastes evaluated in this study are shown in Table 1, along with their ingredients as listed on the package and the manufacturer's name. Table 1. Toothpastes evaluated in the study and their respective ingredients. Toothpaste Close-Up Deep action (Red hot) Macleans (Fresh mint) Pepsodent Natural white (Gel) Oral B (Pro-Health) Close-Up Herbal Colgate Ingredients PEG-32, Aroma, Cellulose gum, Triclosan, Sodium saccharin, Sodium fluoride, Zinc sulphate, Mica, Sodium hydroxide, Glycerin, Eugenol, CL 12490, CL 16035, CL 17200, CL 77491, CL Sodium fluoride 0.306% w/w, Aqua, Hydrated silica, Sorbitol, Glycerin, PEG-6, Sodium lauryl sulphate, Flavour, Titanium dioxide, Xanthan, gum, Sodium saccharin, Triclosan. PEG-32, Aroma, Perlite, Cellulose gum, Sodium saccharin, Sodium fluoride, Calcium gluconate, Glycerin, Limonene, CL 74160, CL 74260, CL Aroma, Cellulose gum, Trisodium phosphate, Sodium phosphate, Sodium saccharin, Sodium fluoride, Carboner, Limonene, Eugenol, CL 19140, CL PEG-32, Aroma, Cellulose gum, Sylodent, Sodium saccharin, Sodium fluoride, Aloe babadensis leaf extract, Limonene, CL 73360, CL 74260, CL , CL 77492, CL Sorbitol, Aqua, Sylodent, Hydrated silica, Sodium lauryl sulphate, Aroma, Benzene sulphate, Sodium saccharin, Sodium fluoride, Cellulose gum, Limonene, CL 72360, CL organisms isolated are Bacteria: Streptococcus mutans (25), Staphylococcus aureus (10), Escherichia coli (2), Klebsiella spp (5) and Fungi: Candida albicans (4) and Rhizopus spp (2). Table 2. Microorganisms isolated from the mouth of subjects studied. Microorganisms Organisms Isolated No. of Isolates Bacteria Streptococcus mutans 25 Staphylococcus aureus 10 Escherichia coli 2 Klebsiella spp 5 Fungi Candida albicans 4 Rhizopus spp. 2 TOTAL 48 Organisms isolated according to gender are shown in table 3. Male subjects are 20 (40%) while the female subjects are 30 (60%). The organisms isolated from the male subjects are; Streptococcus mutans, Staphylococcus aureus, Klebsiella spp, Candidaalbicans and Rhizopus spp. while from the female subjects, Streptococcus mutans, Staphylococcus aureus, Escherichia coli and Candida albicans was isolated. Table 3. Organisms isolated according to gender. Gender No Examined (%) Organisms Isolated Male 20 (40) Streptococcus mutans, Staphylococcus aureus, Klebsiellaspp, Candida albicans Rhizopus spp. Female 30 (60) Streptococcus mutans, Staphylococcus aureus, Escherichia coli Candida albicans TOTAL 50 The antibiotics sensitivity of the oral bacterial flora of the subjects sampled is shown in table 4. Close-Up Deep action (Red hot) and Macleans (Fresh mint) were sensitive to all the organisms isolated, Pepsodent - Natural white (Gel), Oral B (Pro-Health), Close-Up Herbal and Colgate were sensitive to three (3) organisms. Table 2. Shows the organisms isolated from the study. The Table 4. Antibiotics Sensitivity of the Oral Bacterial Flora of the subjects sampled. Toothpaste Zone of Inhibition (mm) Staphylococcus aureus Streptococcus mutans Escherichia coli Klebsiellaspp Close-Up Deep action (Red hot) Macleans (Fresh mint) Pepsodent - Natural white (Gel) Oral B (Pro-Health) Close-Up Herbal Colgate Control Gentamycin Streptomycin Table 5 shows the antibiotics sensitivity of the oral fungal flora of the subjects sampled. Close-Up Deep action (Red hot), Macleans (Fresh mint) and Oral B (Pro-Health) showed sensitivity to all the fungal isolates while Pepsodent - Natural white (Gel), Close-Up Herbal and Colgate were sensitive to one (1) of the fungal isolate in difference.

4 27 Obiazi Helen A. K. and Dic-Ijiewere Ebenezer Oseremen: Effect of Toothpaste on Oral Microbial Flora (Bacteria and Fungi) Table 5. Antibiotics Sensitivity of the Oral Fungal Flora of the subjects sampled. Toothpaste Zone of Inhibition (mm) Candida albicans Rhizopusspp Close-Up Deep action (Red hot) 8 4 Macleans (Fresh mint) 10 6 Pepsodent - Natural white (Gel) 5 0 Oral B (Pro-Health) 4 2 Close-Up Herbal 0 3 Colgate 2 0 Control Fluconazole Ketoconazole 8 5 Table 6 and Table 7 shows the Cultural Characteristics and Biochemical analysis of bacterial and fungal isolates. Table 6. Cultural Characteristics and Biochemical Analysis of Bacterial Isolates. Isolates Cultural characteristics Biochemical analysis Shape Elevation Consistency Colour Opacity Gram Catalase Coagulase Indole 1 Cocci in Cluster Spherical Moist Pale Translucent _ 2 Rod Convex Mucoid Rose Pink Opaque _ + _ + 3 Rod Raised Mucoid Light Pink Opaque 4 Cocci in Chains Raised Moist Creamy Translucent + + Table 6. Continue. Isolates Biochemical analysis Sugar Fermentation Organism Motility Oxidase Citrate Urease Glucose Maltose Sucrose Lactose _ Staphylococcus aureus 2 + _ + + _ + Escherichia coli _ + + Klebsiellaspp 4 _ + + _ + + Streptococcus mutans KEY + = Positive - = Negative Table 7. Morphological and Cultural Characteristics of Fungi Isolates. Fungal Isolates Macroscopy Microscopy Grows quickly and cover agar surfacewith white fluffy that later turns grey, Hyphae practically non-septate, sporangiophores Candida albicans reverse side is white. are long, often branched Rhizopusspp Grows quickly and cover agar surfacewith white fluffy, reverse side is white. Sporangiospore, non-septate, positive to mould 4. Discussion The toothpastes tested in this study contained several different constituents with anti-fungal and anti-bacterial properties, which theoretically could be useful in controlling the oral microbial population. It has been established that Streptococcus mutans plays a major role in tooth decay by metabolizing sucrose to lactic acid. Candida albicans, yeast like fungus and a relatively common inhabitant of the oral cavity, is the chief causative agent of candidiasis an opportunistic infection. Studies have shown the growth of E. coli in sub gingival flora of aggressive and chronic periodontitis [15]. Thus it would be beneficial if oral formulations have established antimicrobial properties against common oral micro-organisms. Maintenance of a good and proper oral hygiene is important for the prevention of various dental diseases [16]. Activities of the oral micro flora are the causes for most oral diseases and mouth odour. Microorganisms can also enter the mouth through food, water and air [17]. The addition of antibacterial agents in the production of toothpaste aids in keeping these oral organisms to a level consistent with oral health [18]. Halitosis is due to the activities of oral micro flora, hence antimicrobial agents are necessary additions to toothpaste and mouth rinses. When these substances are added to oral products, they kill microorganisms by disrupting their cell walls and inhibiting their enzymatic activity. They also prevent bacterial aggregation, slow multiplication and release endotoxins [19]. Data from the present study is in support of this assertion as all the investigated dental care products exhibited wide variations in their effectiveness against the microorganisms isolated from oral cavity of the subjects sampled. The results showed that six organisms were isolated from the oral cavity of the subjects, they include: Bacteria: Streptococcus mutans (25), Staphylococcus aureus (10), Escherichia coli (2), Klebsiella spp (5) and Fungi: Candida albicans (4) and Rhizopus spp (2). This is in accordance with Marsh [3], S. aureus was the most frequently isolated (28.7%) while Candida albicans (10%) was the least frequently isolated organism. S. epidermidis (15.3%) Streptococcus

5 International Journal of Bioengineering & Biotechnology 2018; 3(3): pyogenes (19.3%) Streptococcus mutans (16.7%). Among all the investigated toothpastes, Macleans and Close-Up Deep action (Red hot) emerged as the most effective, based on the mean diameter of the zone of microbial inhibition produced by the toothpastes in disc diffusion method, against the microorganisms isolated from the study. Macleans and Close-Up Deep action (Red hot) contains triclosan and zinc sulfate in it. Our study results were in agreement with other studies [4]. Next to Macleans and Close-Up Deep action (Red hot), Close-Up Herbal and Colgate with sodium fluoride and Sylodent as main ingredients showed maximum zone of inhibition against Streptococcus mutansande. coli. Sylodent acts as a polishing agent which safely cleans and helps in eliminating stains to whiten teeth. Fluorides are abundantly used in many oral health products including toothpastes and mouth rinses as they help in caries prevention [20]. Fluoride products such as toothpaste have shown to reduce caries between 30 and 70% compared with no fluoride therapy. The effectiveness of fluoride toothpastes are concentration dependent [21]. Excess of fluoride concentration can lead to dental fluorosis during developing stage of tooth. When formulated correctly and used as directed, fluoride toothpaste will help to prevent tooth decay more effectively. It is well documented that fluoride can inhibit or even reverse the initiation and progression of dental caries. However, if the bacterial challenge is too high, it is not possible for fluoride to overcome the challenge completely [4]. It is followed by Close-Up herbal and Colgate containing hydrated silica and sodium fluoride as ingredients against streptococcus mutans and E coli. Least efficacy was shown by Oral B containing Amine fluoride as a main ingredient against Candida compared to other test formulations this may be due to the ingredients present. C. albicans was isolated on Sabouraud dextrose agar; white, fluffy colonies were observed. Candida albicans is the most common cause of candidiasis, which could be acute, sub-acute or a chronic infection that may involve any part of the body. This organism is found as part of the normal flora in the mouth, skin, vaginal tract and gastrointestinal tract [22]. Healthy mouth means a balance exists between oral immunity and microorganisms and other factors like maintaining hygiene, intrinsic and extrinsic factors of saliva, tooth position and composition etc. If this harmony breaks then proliferation of microorganisms takes place initiating the disease process. These toothpastes removed teeth bacteria more than 50% and so it is not in agreement with the work done by Okpalugo et al. [23] which said that No brand of toothpaste removed bacteria (teeth) more than 50%. The addition of antimicrobial agents to conventional toothpastes aims to increase effectiveness in the control or elimination of microorganisms involved in a wide variety of microbial infections in the human mouth and body, such as Streptococcus mutans and Streptococcus sobrinus, the primary etiological agents of dental caries [24]. Further studies are needed to evaluate their clinical advantages in the treatment or prevention of biofilm-mediated diseases. 5. Conclusion Analysis of the inhibition of bacteria by the toothpaste studied shows that they were effective for some bacterial species than others. However, the microbial quality of toothpaste formulations marketed in the open markets has met the drug standard which excludes the presence of the index, indicator or pathogenic organism in drugs to be consumed by the populace. Further studies are needed to know the proper concentration of ingredients. In vivo studies required to know the role of saliva, plaque and mechanism of action of these active ingredients. Nevertheless, the in vitro method is a well-established technique that is commonly used in screening the antimicrobial efficacy of chemicals before in vivo testing. Hence further studies have to be conducted in vivo to know the efficacy of oral care products. We recommend the combine use of toothpastes with different Microbial coverage for better oral health. Acknowledgements The authors wish to express their gratitude to Management and Staff of the Diagnostic and Research Laboratory of Ambrose Alli University, Ekpoma, Nigeria for the use of their facility. References [1] Arweiler, N. B., Auschill, T. M., Reich, E. and Netuschil, L. (2002). Substantivity of toothpaste slurries and their effect on reestablishment of the dental biofilm. J ClinPeriodontol. 29: [2] Davies, R. M. (2008). Toothpaste in the control of plaque/gingivitis and periodontitis. Periodontol. 48: [3] Marsh, P. D. (2010). Controlling the oral biofilm with antimicrobials. J Dent. 38 Suppl 1: S11-S15. [4] Prasanth, M. (2011). Antimicrobial efficacy of different toothpastes and mouth rinses: an in vitro study. Dent Res J Isfahan. 8: [5] Maltz, M. and Beighton, D. (2012). Multidisciplinary research agenda for novel antimicrobial agents for caries prevention and treatment. Adv Dent Res. 24: [6] Manupati, P. (2011). Antimicrobial Efficacy of Different Toothpastes and Mouth rinses: An in vitro Study. Dent, Res. J. (Isfahan). 8 (2): [7] William, M. I. and Cummuns, D. M. (2003). The Technology behind close-up total advanced fresh, comprehensive continue education on dentals, Brit. J. of Dent. Surg. 24 (5): 4-9. [8] Mohan, K. K. P., Priya, N. K. and Madhushankari, G. S. (2013). Anti-Cariogenic Efficacy of Herbal and Conventional Toothpastes - A Comparative In-Vitro Study, J. of Int. Oral Health. 5 (2): [9] Lee, S. S., Zhang, W. and Li, Y. (2004). The antimicrobial potential of 14 natural herbal dentifrices: results of an in vitro diffusion method study. J Am Dent Assoc. 135:

6 29 Obiazi Helen A. K. and Dic-Ijiewere Ebenezer Oseremen: Effect of Toothpaste on Oral Microbial Flora (Bacteria and Fungi) [10] Netuschil, L., Brexc, M., Heumann, C. and Hoffman, T. (2005). Clinically controlled 6- month study of the influence of toothpastes with anti-inflammatory ingredients on plaque and gingivitis. Quintessenz. 56: [11] Ozaki, F., Pannuti, C. M., Imbronito, A. V., Pessotti, W., Saraiva, L. and Maria de Freitas, N. (2006). Efficacy of a herbal toothpaste on patients with established gingivitis - a randomized controlled trial. Braz Oral Res. 20: [12] Peck, M. T., Africa, C. W. J., Stephen, L. X. G., Marnewick, J. M. and Abdul, M. (2011). An in-vitro analysis of the antimicrobial efficacy of herbal toothpastes on selected primary plaque colonizers. Int Journal of Clinical Dental Science. 2 (3): [13] World Gazetteer (2007): Population of Cities, news, divisions. htt://world gazetteer.com/ng.php. [14] Monica Cheesbrough. (2005). Discrete Laboratory Practice in Tropical Countries Part 1, Cambridge Second Editions. Published by Press Syndicate of the University of Cambridge. [15] Asmara, W., Astuti, I., Tandelilin, R. T. C. and Jonarta, A. L. (2010). Systemic IL-1β and TNF-α production of E. coli Lipopolysaccharide-Induced Periodontitis model on rats. The Indonesian Journal of Dentistry. 1 (1): [16] Gamboa, F., Estupinan, M. and Galindo, A. (2004). Presence of Streptococcus mutans in saliva and its relationship with dental caries: Antimicrobial susceptibility of the isolates. Universitas Scientiarum. 9 (2): [17] Hoikhian, C. S. O. and Okoror, L. O. (2012). Resistance of oral bacterial species to varied toothpaste effects, Int. J. of Eng. Res. and Sci. and Tech. 1 (1): [18] Nwankwo, I. U. and Ihesiulo, S. C. (2014). Comparative Analysis of the Antibacterial Potentials of Some Brands of Toothpaste Commonly Used In Umuahia, Abia State. J. of Pharm. and Bio. Sci. (IOSR-JPBS). 9 (3): [19] Bou-Chacra, N. A., Gobi, S. S., Ohara, M. T. and Pinto, T. J. A. (2005). Antimicrobial activity of four different dental gel formulas on cariogenic bacteria evaluated using the liner regression method. Revista Brasileira De Ciencias Farmaceuticas. 41 (3): [20] Jenkins, S., Addy, M. and Newcombe, R. G. (1994). Dose response of chlorhexidine against plaque and comparison with triclosan. J Clin Periodontol. 21 (4): [21] Harper, P. R., Milsom, S., Wade, W., Addy, M., Moran, J. and Newcombe, R. G. (1995). An approach to efficacy screening of mouthrinses: studies on a group of French products (II). Inhibition of salivary bacteria and plaque in vivo. J Clin Periodontol. 22 (9): [22] Larone, D. H. (2002). Identification of fungi in culture, In: A guide to identification of medically important fungi fourth Edition, ASM Press, Washington DC; Pp [23] Okpalugo, J., Ibrahim, K. and Inyang, U. S. (2009). Toothpaste formulation efficacy in reducing oral flora, Trop. J. of Pharm. Res. 8 (1): [24] Saravia, M. E., Nelson-Filho, P., Silva, R. A., De Rossi, A., Faria, G. and Silva, L. A. (2013). Recovery of mutans streptococci on MSB, SB-20 and SB-20M agar media. Arch Oral Biol. 58: