Oral microbiology/ rotary endodontic courses by indian dental academy


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Indian Dental Academy: will be one of the most relevant and exciting training center with best faculty and flexible training programs for dental professionals who wish to advance in their dental practice,Offers certified courses in Dental implants,Orthodontics,Endodontics,Cosmetic Dentistry, Prosthetic Dentistry, Periodontics and General Dentistry.

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Oral microbiology/ rotary endodontic courses by indian dental academy

  2. 2. CONCLUSION INTRODUCTION: You get up in the morning and brush your teeth with the best available tooth paste and think you are clean. You rinse, you gargle a millions times, you floss religiously, use mouth washes and get professional prophylaxis done and you think you are safe……but, unfortunately, you are not. You can run and you can hide but you can never escape from these tiny, yet amazing, inhabitants of your mouth…….. the oral micro flora. A multitude of organisms are present in the mouth because of a wide range of sites with different environmental characteristics. Some organisms are almost always present in nos: > 1% and constitute the normal, constant, indigenous flora. These organisms do not comprise the host’s survival Eg: S. Sanguis, Neisseria Some organisms are present in low numbers (<1%) and are called supplemental flora. These become indigenous (i.e increase no:>1%) if the environment changes. A classic example is that of lactobaccilus sp, which becomes indigenous only when plaque pH drops and becomes acidic in a carious lesion. Thus, an environmental change has led to a supplemental flora to become indigenous. Certain microbes stay for a short time and comprise organisms that are just “passing through” a host. These constitute the transient flora and may be those organisms present in food/drink that temporarily get established in the mouth. ACQUISITION OF NORMAL FLORA: An infant’s mouth at birth is microbiologically sterile. Within a few hours of birth, streptococci (especially S. Salivarius) establish themselves in the mouth. These come from the mother or the environment. The streptococci alter the oral environment in such a way that it makes it more hospitable for other microorganisms.
  3. 3. During infancy and early childhood other species of streptococci, staphylococci, neisseria and veillonella occupy their habitats. Lactobacilli, actinomyces and fusobacteriumalso colonize in most children. With the eruption of teeth and the availability of a solid surface, the microbial flora undergoes dramatic changes. S.mutans, S.sanguis and A.viscosus establish themselves on dental hard tissues. Simultaneously, significant increase in the number of obligate anaerobes (strict O2 avoiding organisms) takes place. Due to the eruption of permanent teeth, gingival sulcus depth also increases. With adolescence spirochaetes and bacteroides tend to appear in the mouth. Adulthood brings in complex and varied organisms, which vary among different people, at different times and at different sites. The loss of normal teeth brings about another drastic change in microbial flora, which reverts back to the pre-eruptive days. Interestingly, use of artificial dentures yet again invites those bacteria, which are fond of hard dental tissue. The number of fungi can also increase. ORAL FLORA: Bacteria are by for the most predominant type of microorganisms present in the human oral cavity. These bacteria can be aerobic/ anaerobic depending on O2 requirement bacteria ,Gram-positive / Gram negative according to the gram stain and bacilli / cocci / treponemes / mycoplasmas according to shape. Bacteria: Gram +ve bacteria Cocci: Of all the bacteria present in the oral cavity Streptococci constitute the single largest group. A large number of its species are encountered in the mouth including S. faecalis S, Sanguis, S, mitis, S.mutans, S.milleri and S.salivarius. The other cocci include peptostreptococcus, micrococcus and staphylococcus. The last one is a transient member of the oral flora. S.mutans has been shown to be the most important organism in the initiation of caries. Bacilli: Lactobacilli are widely present in the human body and frequently found in the oral cavity. 27 recognized species of lactobacilli are known with important ones being
  4. 4. L. acidophilus, L.salivarius and L.casei They are known to be involved in the progression of caries. Other G + ve bacilli include actinomyces, Arachnia, Eubacterium, clostridium etc. Gram -ve bacteria: Cocci: Veillonella are the most numerous G -ve bacteria accounting for 10% of cultivable salivary and tongue flora. Neisseria and moraxella are also seen. Bacilli: Bacteroides, Fusobacterium, leptotrichia are the significant gram -ve bacilli Bacteroid species and F. nucleatum are most commonly occurring bacilli in gingival sulcus area. Treponemes: Spirochaetes such as T.denticola, T.orale, T.vincenti are common inhabitants of gingival crevices and are often associated with periodontitis Mycoplasma: These pleiomorphic organisms are regularly seen in plaque, calculus, periodontal pockets etc. M. salivarium is the most predominant species while others like M. pneumonione, M.orale, M. hominess have also been isolated. Fungi: Candida albicans is the most common fungus isolated from the oral cavity & is detected in high numbers in gingival flora, periodontal abscess, infected root canals etc. Other fungi isolated are Pencillium, Aspergillus, Hemispora. Protozoa: Are present in periodontal diseases Eg: Entamoeba Virus: EBV, mumps virus, measles and influenza virus can be observed during the active stage of the disease. MICROBIAL ECOSYSTEMS IN THE ORAL CAVITY
  5. 5. The mouth provides a congenial environment for the organisms to grow and survive. Variation in the different ecological niches causes differences in the number (quantitative) and in the type (qualitative) of the organisms. These variations are due to the complex interactions between the microbe and the host. The establishment, survival and persistence of the flora in the oral cavity is determined by: Microbial Factors Host Factors - Adherence Anatomical - Production of anti- microbial agents Saliva - Sensitivity to anti microbial agents Crevicular fluid - Metabolic capability Diet - Nutritional requirement Oral hygiene Oral and systemic disease Oral immune defenses Microbial Factors: Adherence: is a very important property for a bacteria to become a part of the oral flora. If it cannot be retained and subsequently multiply, it will be eliminated from the oral cavity by the washing action of the saliva. Adherence occurs reversibly initially via weak, non-specific electrostatic attraction forces and later becomes permanent involving specific bridging between host and normal flora. These 2 phases occur continuously. The factors influencing adherence are Microbial factors - Pilli and fimbriae - Cell wall components - Extra cellular polymers Host factors  Acquired Pellicle  Salivary factors like IgA lectins  Minerals  Anatomic factors
  6. 6.  Crevicular fluid Once adherence occurs via pilli or fimbriae, aggregation of homotypic and heterotypic organisms occur via extra cellular polymers, IgA, lectins etc. Antimicrobial Agents:Certain chemicals and biologically active substances are produced by one microorganisms against another. For eg: Bacteriocins are produced by certain organisms against other species. Certain metabolic end products like H2O2 can be antagonistic to unrelated species. Thus, the vulnerability of an organism to sucuumb to these antimicrobial factors can determine its survival in the oral cavity. Metabolic Capability: Organisms, which have the metabolic machinery to utilize the available nutrients and neutralize the deleterious chemicals produced in the oral cavity, have the capacity to survive and propagate Nutritional Requirements: Organisms use microbial sources such as intra cellular storage granules or extra cellular metabolic end products and host sources such as directory sucrose, salivary proteins and minerals and crevicular proteins for their growth. HOST FACTORS Aquired Pellicle: Salivary glycoproteins form a layer on the tooth surface within minutes of cleaning the tooth. The organism attach first to this pellicle and not directly to the enamel. Salivary Factors: saliva with its composition of 99% water and 1% solids influences the oral ecology in a number of ways:  Mechanical washing: prevents the overgrowth of microbes. Swallowing of saliva causes ingestion of aggregates of microbes.  Pellicle formation: salivary Glyco proteins serve as nutrition and also promotes / inhibits bacterial adhesion.  Inhibitory Action: - Lysozyme, lactoferrin, lactoperoxididase kills exogeneous bacteria  Buffering capacity: to maintain pH at 6.7 prevents overgrowth of microbes which need a high or a low pH for growth.
  7. 7.  IgA usually inhibits adherence and also alters microbial metabolism. Anatomical Factors: Untreated malocclusion, irregular teeth and failing or poorly contoured restorations can serve as stagnation sites for microbes to proliferate. Pits and fissures serve as natural stagnation areas. Crevicular Fluid: Is similar to serum in composition. It contains albumin, immunoglobulins (IgG,IgA,IgM) and transferin which aids is the host defense. Apart from electrolytes, it also contains enzymes such as proteinase, collagenase etc which helps to combat microbes. MISCELLANEOUS FACTORS: There are many other factors which significantly influence the ecology the oral cavity ⇒ A low redox potential supports the growth of anaerobic bacteria in the depth of the dental plaque. ⇒ Administration of antibiotics/ local antiseptics depresses the number of some species while allowing some other species to proliferate. ⇒ Smoking, tobacco chewing, poor oral hygiene, carbohydrate rich diet, systemic diseases conditions can influence the oral micro flora in an unpredictable manner. ANAEROBIOSIS: The transit of air through the mouth seems to preclude the possibility of any anaerobic organisms thriving in it. However, culturing procedures revealed that 50-70% of organisms cultured from the mouth were anaerobes. The factors determining the oral environment include: a) Oxygen tension: is the measure of the amount of oxygen in a gas. For air it is 21%. On the tongue it is 12-14%, in the buccal mucosa it is 1% and in a periodontal pocket it is 1- 2%. This indicates that the dento-gingival surface is mainly anaerobic especially where sub-gingival plaques form. The supra-gingival plaque on the labial, lingual and occlusal surface exhibited an O2 tension of 1-20%, thus producing an ideal niche for facultative anaerobes and microaerophillic species.
  8. 8. b) Oxidation- reduction potential (Eh) = is the tendency for a medium or compound to oxidize or reduce a molecule by the removal or addition of electrons. Microorganisms that need a +ve Eh are termed aerobes and those needing a –ve Eh are anaerobes. A low Eh would be expected in a microbial community which performs a fermentative metabolism as fermentation results in formation of reduced end products. Thus, Eh eventually becomes negative in a site where microbes accumulate. Portions of plaque keeps changing from aerobic to anaerobic environment with corresponding drop in Eh. This change coincides with a shift in the flora from a facultative microaerophillic to anaerobic. c) Super-oxide radical and presence of super-oxide dismutase : The super oxide radical is the most lethal form of O2 in biologic system as it causes both alteration is cell membrane and inactivation of enzymes. Aerobes contain the enzyme super-oxide dismutase which can destroy this super-oxide radical while anaerobes lack it. Hence, aerobes predominate on tongue and supra-gingival plaque while anaerobes are in sulcus areas. ORAL HABITATS: The oral habitat can either be a non-tooth habitat or a tooth habitat The non-tooth habitat includes:  Oral Mucosa (lip, cheeks, palate)  Dorsum of the tongue  Saliva and tonsillar areas While the tooth habitats include:  Root Surface  Sub- gingival areas  Pit and fissure areas  Smooth surfaces – gingival to proximal contact and gingival 1 /3 of facial and lingual surface of clinical crown. The oral mucosa: harbors organisms that can overcome abrasive forces of food, tongue and teeth for retention. Due to the washing effects of saliva, these organisms should be able to reproduce in great numbers to ensure survival by reattachment.
  9. 9. The dorsum of the tongue: with its surface papilla provides additional shelter to organisms. Streptococcus salivarius and Micrococcus mucilaginous are commonly found here and rarely on teeth. Saliva: has a wide variety of microbes as most of the microbes which get detached from the tissue / tooth surface will appear in saliva. All species of streptococcus especially S. oralis and S. salivarius are found in the saliva. The tooth surface: is unique as it is not protected by surface shedding mechanisms, which occur in other tissues. It is stable and gets covered by pellicle, which is ideal for streptococcal attachment. Tooth habitats favorable for harboring pathogenic plaque include: Pits and Fissures: provides excellent shelters for organisms especially S.Sanguis and other streptococci. S. mutans can also be isolated at these sites even in the absence of caries. Obligate anaerobes and gram –ve species are infrequently isolated or are absent. Smooth surface: The proximal area immediately gingival to the contact area is protected physically and is relatively free from the effects of mastication, tongue movements or salivary flushing. The composition of the microflora varies and is complex but is predominantly actinomyces and streptococci. Tooth topography (such as a rough surface due to defective/ poor restoration), the size and shape of gingival papilla (apically migrated papilla) and oral hygiene can predispose the tooth to caries or periodontal disease. Root surface: The proximal root surface near the CEJ is usually unaffected by flossing due to roughness / fluting. This favors formation of mature, isolated cariogenic plaque. Mainly, gram -ve obligate anaerobes and actinomyces are present here. Sub-gingival areas: The initial occupants of the sub- gingival area are an extension of the community from the adjacent tooth surface. Metabolites released from the plaque induce a strong inflammatory response in the sulcus leading to vascular changes and release of Ig, PMNL etc. This leads to a variation in the local environment by removal of some
  10. 10. species and introduction of newer ones. Thus, progressive changes from the cocci in the supra-gingival plaque to filamentous bacteria and spirochetes in the sub-gingival plaque is seen. Pathogenic Bacteroid melaninogenicus can exploit this habitat and cause destruction of the gingival epithelium. ECOLOGICAL DETERMINANTS: Factors determining the presence of different organisms in different anatomic sites: Nutrient Availability: Micro organisms like B melaninogenicus, B. gingivalis and capnocytophaga need hemin, certain bacteroids need vit. K, estradiol and progesterone while T. denticola need spermin as nutrients. As these nutrients are present in serum and not is saliva / food; these organisms take refuge in the gingival crevice where the crevicular fluid sustains them. Thus, an organism’s nutritional requirement decides on which niche they will occupy. Inhibitory factors: Specific antibodies, lyzozymes, lactoperoxidase and lactoferrin, which are present in saliva will inhibit bacteria from growing. Microbial acid production which reduces pH, their reduced end products with reduces Eh, production of H2O2 which oxidizes enzymes and presence of bacteriocins can prevent the growth of certain species while promoting the growth for another. Thus, interplay of these ecological determinants will decide which organism will reside where. BENEFITS OF THE ORAL MICROBIOTA Production of vitamins and co-factors =oral bacteria like the intestinal flora produce certain vitamins- the vit K, biotin and riboflavin- as well as co-factors required for our normal systemic functioning. Production of digestive enzymes= Small quantities of amylase, lipase and protease are produced by the oral microflora.
  11. 11. Prevention of colonization of exogenous organisms to establish themselves and produce disease= This may cause sensitivity to antimicrobial substances produced by the oral structures. DENTAL PLAQUE: It can be defined as a tenacious microbial deposit which forms on hard surfaces within the mouth and consists of microbial cells and their products along with host compounds mainly derived from saliva or crevicular exudates. Composition of Plaque: It mainly consists of proliferating micro organisms along with a scattering of epithelial cells, leukocytes and macrophages in an adherent intercellular matrix. Bacteria may be up 70-80% of this material. 1mm3 of plaque weighing 1mg contains more than 108 bacteria. 200-400 different species of micro organisms can be present in an extremely complex arrangement at one site alone. Other than bacteria, mycoplasma, protozoa and fungi can also be present. The material among the bacteria is termed “inter microbial matrix” and accounts for 25% of the plaque volume. It consists of microbial substances, salivary material and gingival exudates. The organic portion aids in adherence and co- aggregation. These include poly saccharides, glyco proteins. The inorganic components include calcium, P, K, Mg and Na which increase on calculus formation. Types of plaque: According to site  Supra gingival: Fissure Approximal Smooth Surface  Sub Gingival  Denture related Formation of plaque:
  12. 12. Stage I Acquired pellicle formation: It involves the adsorption of salivary proteins to apatite surfaces via electrostatic ionic interactions Stage 2: Transport and Adherence of pioneering organisms: The transition between pellicle to plaque is rapid. The first constituents are cocci with small numbers of epithelial cells and PMNL’s which initially adhere via electrostatic interactions these long range interactions facilitate a reversible adhesion. A little later specific short range interactions occur between adhesins on microbial surface and receptors in the pellicle and results in irreversible adhesions. S. mutans and S. Sanguis produce glycans in the presence of sucrose which aids in adherence to pellicle and also promotes adherence to other micro organisms Within 8-10 hours, about 10,000 cells/ mm2 are deposited. The pioneer ent. An extra-cellular matrix develops consisting of polysaccharides, salivaryorganisms multiply to produce micro colonies which with time become conflu glycoproteins. Stage 3: Co-Aggregation (Within 1-3 days): The metabolic products of the pioneering organisms alter the immediate environment such as creating conditions with a low redox potential suitable for anaerobes. Other organisms become incorporated into the plaque with a resulting gradual increase in microbial complexity, biomass and thickness Stage 4: Multiplication: The proliferation of the attached micro-organisms and further aggregation produces a confluent growth and a biofilm. Stage 5: Seeding: Detachment of cells form this bio film into the saliva results in colonization of fresh sites. Growth And Accumulation Of Supra-Gingival Plaque: The total plaque mass that develops is mainly determined by the multiplication of the attached bacteria as well as cohesion of bacterial cells. Thus, growth is by: -adhesion - cohesion
  13. 13. Factors which determine the ultimate composition and pathogencity of plaque are: Bacterial factors o Extra-cellular products: eg.glucans produced by S.mutans are sticky and help in co- aggregation o Bacterial interactions: are important for bacteria that cannot attach directly to the tooth.Eg. Violonella which of incapable of direct attachment accumulates on A.viscosis. o Plaque ecology: formation and growth of plaque is an orderly sequence of ‘replacement community’ with each community modifying the local environment of that site. This process of mutual change of community and its environment is called ‘ecological succession’. Host factors: ⇒ Oral cleansing mechanism such as salivary flow, movements of tongue and cheek control the plaque formation rate. ⇒ Saliva influences • The plaque pH by its buffering action and acid neutralization. • Inhibition of adherence by coating the surface receptors • Inhibition of adherence via promotion of bacterial agglutination ⇒ Immune response: Main sources of immune components in oral cavity are: • IgA and antibodies in saliva which compete with bacterial adherence and influences their metabolism, growth and accumulation. • Crevicular fluid which contains antibodies, leukocytes, complement factors etc. Structure of plaque: 1) Bacterial cells near to the enamel surface tend to have a reduced cytoplasm:cell wall ratio, indicating metabolic inactivity.
  14. 14. 2) In some areas (esp. outer surface) cocci attach and grow on the surface of filamentous microorganisms giving a ‘corn-cob’ appearance. 3) There is a tendency for filamentous bacteria to orient themselves at right angles to the enamel surface producing a ‘palisade effect’. 4) Bacteria containing glycogen-like storage granules intra-cellularly are seen indicating that these bacteria are relying on themselves for nutrition. Sub gingival plaque: Supra-gingival plaque can induce inflammatory changes in the gingiva, which leads to edematous enlargement of the gingiva. This leads to an increase in the capacity of the sub-gingival area for bacterial colonization. Since this area is protected from normal cleaning mechanisms, has increased crevicular fluid and has desquamated epithelial cells, it favors a new ecological environment favoring anaerobic bacteria. Thus, Gram +ve filaments, Gram –ve cocci, rods and spirochetes predominate here. This flora is associated with root caries periodontitis, gingivitis etc. MICROBIOLOGY OF DENTAL CARIES: Over the years, there has been a debate whether one or more specific bacteria are principally involved in the initiation of caries or if this disease is caused by a non – specific mixture of bacteria. Given the variations found in the microbial composition, it seems unlikely that the initiation and progression of all carious lesions are associated with identical or even similar plaques. However, there is evidence that some bacteria – S. mutans, Lactobacilli and actinomyces- are more important than others. S. mutans: The feature that supports its role as cariogenic organisms are its: - Rapid generation time - Acidogenic nature - Production of extra cellular polysaccharides from sucrose which aids on adherence and acts as a nutrient
  15. 15. - Isolated in high numbers in caries active mouth in incipient lesions - Capacity to attain critical pH for enamel demineralization more rapidly than other plaque bacteria. S.mutans is able to utilize dietary sucrose to enhance colonization. S. mutans can produce glucan by using glucosyl transferase enzyme.It is this glucans which help in the attachment of the bacteria to the tooth surface. S. Mutans also produces dextran using enzyme dixtransucrase which helps in clumping ofbacteria. Once caries initiates, the local environment alters and other bacteria arrive and take up the process of progression of the caries. Notable among these are the lactobacilli. Lactobacilli for many years was believed to be the causative agent of dental caries as - High numbers were obtained in most enamel caries - Production of caries in germ-free rats - Able to synthesis extra cellular and intra cellular polysaccharides form glucose. - They produces lactic acid at pH < 5 Although these properties seemed valuable to a cariogenic organism, it was also seen that their affinity for the tooth surface and their number prior to development of caries was low. They were, in fact, absent from incipient lesions while present is significant numbers in developed caries. Thus, they were categorized as secondary invaders which caused progression of caries due to their acidogenic and aciduric properties. Other Cariogenic micro organisms: Actinomyces species: A.viscous, some other Gram +ve bacilli are involved in the initiation of lesions on root surface. Role played by a large number of other bacteria isolated from caries such as Arachnia, Eubacterium, Rothia etc are not yet clearly known. Veillonella: Is a species which is supposed to be antagonistic to the caries process. Its protective role is contributed to its ability to decrease the acidity within the plaque.
  16. 16. MICRO BIOLOGY OF PERIODONTAL DISEASE: PD disease occurs in all parts of the world and few individuals live out their natural life span without getting affected. The main PD diseases believed to have a microbial etiology are: AUG(Acute Ulcerative Gingivitis) Acute Abscess  Gingivitis Chronic Acute-abscess  Periodontitis Adult Chronic Early onset -juvenile -pre-pubertal - rapidly progressive  Acute herpetic stomatitis The microbial composition during health is mainly Gram +ve cocci and few spirochaetes In AUG, treponemes and bacteroide are present while in chronic gingivitis actinomyces and bacteroides are present. In LJP, 65% are Gram –ve especially A.actinomycetemcomitans while in chronic adult periodontitis 75% are gram –ve with predominant species being spirochactes eg. B. melaninogenicus .
  17. 17. Periodontal abscess cultures reveal colonies of B. gingivalis, Fusobacterium and Capnocytophaga Dental calculus: Are calcifying or calcified deposit on teeth which can be either supra- gingival or sub-gingival. Calculus is preceded by dental plaque which subsequently gets calcified. The plaque matrix gets calcified first followed b the bacteria themselves. The degenerating bacteria can act as seeding agents for mineralisation. The outer surface of calculus is covered by a thin viable dental plaque. It is this plaque which has toxic and irritant potential rather than the calculus; of course, calculus does cause mechanical trauma and its rough surface serves as a nidus for continuous accumulation of plaque and keeps the latter in close proximity to the gingiva. MICROBIOLOGY OF ENDODONTIC INFECTIONS: A large number of bacteria and some fungi can cause infection in the dental pulp as well as periapical tissues. Dental pulp is a unique formative organ with limited capacity to withstand bacterial, mechanical and chemical onslaughts. When bacteria enter the pulp it is unusual for the host defenses to completely eliminate them. Healing is uncommon and necrosis results With pulpal degeneration, antigens collect within the root canal system and move to the periapical tissues. Inflammatory or immunological responses ensue and can lead to formation of abscess, granulomas or cysts. Sources Of Infection: - Infected carious lesion / iatrogenic exposure - Via periodontal tissues through exposed dentinal tubules, lateral accessory canals or apical foramen. - By the lymphatic / hematogeneous route i.e anachoresis which is defined as the localization of transient bacteria in the blood into an inflamed area, such as a traumatized / inflamed pulp.
  18. 18. - Traumatic displacement / fracture can also become a route for microbial ingress to the pulp. Once the disease sets in, these tissues can act as source for spreading infection to various organs of body through blood resulting in septicemia. Disease occurring secondary to endodontic treatment include - Infective endocarditis - Cavernous sinus thrombosis - Bacterial myocarditis - Cerebral abscess Various lesions result as sequelae to pulp necrosis such as granulomas, abscesses, actinomycosis, cellulites, osteomylitis etc. The organisms most commonly encountered in endodontic infections include – • Bacteroides especially B.endodontalis and B. gingivalis. • Streptococci mainly S.milleri • Enterococci • Lactobacilli • Actinomyces • Yeasts It is interesting to note that flare-ups during endodontic therapy are associated with facultative Gram +ve cocci especially, S. salivarius, S. mutans and enterococci and no anaerobes are present. Dento-alveolar infections are associated with mainly streptococci, Bacteroids, Fusobacterium STUDY OF MICROBES: - Direct examination under Microscopy using: • Phase Contrast • Gram Stain • Acid fast stain -Sampling
  19. 19.  Culture  Biochemical tests  Antigenic structures study  Typing  Animal pathogenecity  Antibiotic sensitivity test SUMMARY AND CONCLUSION: “PREVENTION IS BETTER THAN CURE” A thorough knowledge concerning the microorganisms is essential for not only providing safe and aseptic means of treatment for the patient but also for keeping ourselves safe from any communicable diseases. Some new organism is discovered almost everyday. So, the next time you decide to treat a patient without a mouth mask or a pair of gloves, remember that there is a microorganism just waiting round the corner to make you its next victim!