Plaque

basic and ultrastructural desription of dental biofilm

1. 1 GOOD MORNING

2. 2 Plaque Guided by: Dr. Anita Panchal Dr. Sachin K. Dr. Bhaumik Nanavati Dr. Rahul Shah Dr. Mansi Pathak Presented by: Ganesh Nair

3. Contents: Introduction Definition Structure and composition of dental plaque Plaque as a biofilm Contents of plaque Plaque formation at the ultra structural level Colonization and Plaque Maturation Growth Dynamics of Dental Plaque Physiologic Properties of Dental Plaque Quorum sensing 3

4. 4 Association of Plaque Microorganisms with Periodontal Disease Microbial Specificity of Periodontal Diseases Microorganisms associated with Specific Periodontal Diseases Key Characteristics of Specific Peripathogens Future Advances in Periodontal Microbiology Conclusion Bibliography

5. Introduction: The colonization of the oral cavity also starts within a few hours and by 2 years the entire human micro flora is formed by complex collection of approximately 10 14 consisting of 400 different species. After tooth eruption more complex oral flora is established. It is considered as an “ open growth system ” with uninterrupted ingestion and removal of microorganisms and their nutrients. In this system a dynamic equilibrium exists between the adhesion forces of microorganisms and a variety of removal forces originating from:- 1) Swallowing, mastication and blowing the nose; 2) Tongue and oral hygiene implements; 3) Washout effect of the salivary, nasal, and crevicular fluid; 4) Active motion of ciliae (nasal and sinus wall). 5

6. Definition 6 WHO defines plaque as a specific but a highly variable structural entity consisting of an array of strains and species of organism seen on the tooth surface, restoration and other parts of the oral cavity. It also consists of salivary components such as mucin , desquamated epithelial cells, food debris all embedded in an extracellular gelatinous matrix . An organized mass, consisting mainly of microorganisms, that adheres to teeth, prostheses, and oral surfaces and is found in the gingival crevice and periodontal pockets. Other components include an organic, polysaccharide-protein matrix consisting of bacterial by-products such as enzymes, food debris, desquamated cells, and inorganic components such as calcium and phosphate. Glossary of periodontal terms

7. 7 Dental plaque is defined clinically as a structured, resilient, yellow- grayish substance that adheres tenaciously to the intraoral hard surfaces, including removable and fixed restorations. Carranza’s clinical periodontology 10 th edition .

8. Structure and composition of dental plaque:- Dental plaque is mainly composed of microorganisms. 1 gm of plaque ( wet weight ) contains approximately 10 11 bacteria. 8

9. The apical border of the plaque mass is separated from the junctional epithelium by a layer of host leucocytes {the plaque free zone }, and bacteria of this apical tooth associated region show an increased concentration of gram-negative rods. 9 Supra –gingival plaque Sub-gingival plaque Present above gingival margin Present below gingival margin Always tooth associated May be tooth or tissue associated Primary source of nutrition for bacteria is saliva and ingested food Primary source of nutrition is gingival crevicular fluid Mainly responsible for gingivitis (marginal plaque) or calculus formation and dental caries Responsible for calculus formation and root caries (tooth associated plaque) and destruction of soft tissue (tissue associated plaque)

10. Plaque as a biofilm Nutrient component penetrate this fluid medium by molecular diffusion. Steep diffusion gradients, especially for oxygen, exist in more compact lower regions of biofilm , which further explains changes in microbial composition. 10

11. The biofilm matrix acts as a barrier . Substances produced by the bacteria within the biofilm are retained and essentially concentrated, which fosters metabolic interactions among the different bacteria. Basic biofilm Properties: Cooperating community of various types of microoraganisms , Protective matrix by micro colonies formation, Differing environment within the biofilm , Primitive communication system , Resistance against antibiotics. 11

12. Contents of plaque 12

13. Plaque formation at the ultra structural level The process of plaque formation is divided into three major phases: The formation of pellicle on the tooth surface, Initial adhesion and attachment of bacteria, and Colonization and plaque maturation. 13 Image courtesy- Carranza 10 th edition.

14. Formation of a pellicle:- All surfaces of the oral cavity are coated with a pellicle. Within nanoseconds after vigorous polishing the teeth, a thin saliva derived layer called the acquired pellicle , covers the tooth surface. The pellicle consists of numerous components, including glycoproteins ( mucins ), proline rich proteins, phosphoproteins , histidine rich proteins and enzymes and other molecules that can function as adhesion sites for bacteria. Absolom et al even observed a clear relationship between the type of protein adsorbed in the pellicle and the substratum surface. Busscher et al observed that the detachment of adhering bacteria might occur through the cohesive failure in the conditioning film between the bacteria and the pellicle. 14

15. Initial adhesion and attachment of bacteria:- This concept approaches the microbial adhesion to the surface in an aquatic environment . This diagram of events would also clarify the importance of hard tissue characteristics of the plaque. Phase 1 : Transport to the surface :- the initial transport of the bacterium to the tooth surface occur by random contacts eg via the Brownian motion ( avg displacement 40µm/hour), through sedimentation of microorganism, through liquid flow or through active microbial movement. 15

16. 2. Phase 2 : Initial adhesion :- in initial it is reversible adhesion of bacteria, initiated by the interaction between the bacterium and the surface, from certain distance( 50nm ), through long range and short range forces, including van der waal attractive forces and electrostatic repulsive forces. 16 Image courtesy- Lindhe 6 th edition.

17. 17 3. Phase 3 : Attachment :- after initial adhesion, a firm anchorage between bacterium and surface will established by specific interactions On a rough surface, bacteria are better protected from shear forces so that a change from reversible to irreversible bonding occurs more easily and more frequently. Image courtesy- Lindhe 6 th edition.

18. The bonding between the bacteria and the pellicle is mediated by specific extracellular proteinaceous components of the organism and complementary receptors on the surface and is species specific for eg . Streptococcus and Actinomyces strain bind with specific salivary molecules. Some bacteria also attach at the areas of conformational changes within the proline rich proteins eg . A. viscosus . Such convenient parts for adhesion of the bacteria are referred to as cryptitopes (cryptic, hidden; topo , place). 18

19. Colonization and Plaque Maturation Atleast 18 genera from the oral cavity have shown from the oral cavity have shown some form of coaggregation . Most coaggregation that occurs are among strains from different genera are mediated by lectin like adhesions and can be inhibited by lactose and other galactosides . The initial colonizers( streptococci and actinomyces ) are facultative anaerobes, and doubling times for microbial populations during the first 4 hours of development are less than 1 hour . 19

20. These two group of primary colonizers are taught to prepare a favourable environment for the attachment of secondary colonizers. Special forms of coaggregation are the corncob and testtube brush formation. 20 Image courtesy- Carranza 10 th edition.

21. 21 Image courtesy- Internet.

22. Growth Dynamics of Dental Plaque : the first 2 – 8 hrs, the adherent pioneering streptococci saturate the salivary pellicular binding sites and thus cover 3-30% of enamel surface . After 1 day, the term biofilm is fully deserved because organization takes place within it. As the bacteria density approaches approximately 2-6 million bacteria/mm 2 on the enamel surface, a marked increase in growth rate can be observed to about 32 million bacteria/mm 2 . The thickness of plaque increases slowly with time, increasing to about 20-30µm after 3 days . Adamson m, carlson j: lactoperoxidase and thiocyanate protect bacteria from hydrogen peroxide, infect immun 35:20,1982. 22

23. There is an ecologic shift within the biofilm , there is a transition from the early aerobic environment characterized by gram positive facultative species to a highly oxygen deprived environment in which gram negative anaerobic microorganism predominate. Carranza’s clinical perodontology , 10:146 An early undisturbed plaque formation on teeth follows an exponential growth curve when measured planimetrically . The slow start of the plaque growth curve can be explained by a colony of bacteria needing to reach a certain size before it can clinically detected. 23

24. The lengthening of the microbial generation time(1 hour for initial plaque to 12 hrs for 3 days old plaque), on the other hand, explains the leveling of the slope from day 4 onwards. Weiger r,netuschil l, von ohle c, et al: microbial generation time during the early phase of supragingival dental plaque formation, oral micribiol immunol 10:93,1995. During the night , plaque growth is reduced to about 50% as the supragingival plaque obtains its nutrients mainly from the saliva , which plays a more significant role than the antimicrobial activity of saliva. 24 Image courtesy- Carranza 10 th edition.

25. Topography of supragingival plaque: Early plaque formation on the teeth follow a typical topographic pattern, with the initial growth along the gingival ,margin and from the interdental space. Scanning electron microscopy studies clearly reveal early colonization of the enamel surface starts from the surface irregularities, where bacteria escape shear force , allowing the time needed to change from reversible to irreversible binding. 25

26. Surface microroughness Rough intraoral surfaces accumulate and retain more plaque and calculus in term of thickness, area, and colony forming units. Smoothning an intraoral surface decreases the rate of plaque formation, however smoothning the surface below a surface roughness of 0.2µm does not result in additional reduction of plaque formation . 26

27. Individual Variables Influencing Plaque Formation: A distinction is often made as heavy and light plaque formers . A multiple regression analysis by Simmonsson et al showed that the clinical wettability of the tooth surfaces , the saliva induced aggregation of oral bacteria, and the relative salivary flow conditions around the sampled teeth explained 90% of the variation. Simmonson t, ronstrom a, rudenberg j, et al:rate of plaque formation: some clinical and biochemical chaeacteristics of “heavy” and “light” plaque formers, scand j dent res 95:97,1987 . 27

28. Zee et al found that after day 1 of undisturbed plaque formation, the heavy plaque formers showed more plaque with more complex supragingival structure . From 3-14 days, however there was no discernible differences between both groups, except for a more prominent intermicrobial matrixmin the group of fast growers. Zee et al in another study also concluded the presence of higher proportions of gram negative rods (35% vs. 17%) in 14 day old plaque . Zee ky , samaranayake lp , attstorm r: scanning electron microscopy of microbial colonization of “rapid” and “slow” dental plaque formers in vivo, arch oral biol 42:735,1997 28

29. Inter-subject variation in plaque formation can be explained by factors such as diet, chewing fibrous food, smoking, the presence of copper amalgam, tongue and palate brushing, the colloid stability of bacteria in the saliva, the chemical composition of pellicle and retention depths of dentogingival areas. Variation with dentition: Plaque formation occurs faster in In the lower jaw compared to the upper jaw, In molar areas, On the buccal surfaces as compared to other surfaces, In the interdental regions compared to strict buccal or oral surfaces. 29

30. Impact of gingival inflammation: Studies suggest that the increase in crevicular fluid production enhances the formation of plaque, some substances ( eg . Mineral, protein, carbohydrates) favour the initial adhesion and the growth of early colonizing bacteria. Impact of patients age: No significant differences. Spontaneous tooth cleaning: No significant differences. Winkel eg , abbas f, van der velden u, et al: experimental gingivitis in relation to age in individuals not susceptible to periodontal destruction, j clin periodontol 14:499,1987 30

31. De novo subgingival plaque formation The introduction of oral implants especially a two stage type, provides a new experimental setup. Oral implants have been used as a model to study the impact of surface roughness on subgingival plaque formation, smooth abutments ( avg roughness < 0.2µm) were found to harbour 25 times less bacteria than the rough ones, with a higher density of coccoid cells. Quiryen m, bollen cm, papaioannou w, et al: the influence of titanium abutment surface roughness on plaque accumulation and gingivitis: short term observations, int j oral maxillofac implants 11,:169, 1996 . 31

32. Physiologic Properties of Dental Plaque: The transition of gram positive to gram negative microorganism is observed as a part of structural development. The early colonizers use oxygen and lower the redox potential to the environment, which then favour the growth of anaerobic species. Gram positive species use sugar as an energy source and saliva as carbon source . The bacteria that predominate in mature plaque are anaerobic in nature and asaccharolytic and use amino acids and small peptides as an energy source . 32

33. Special Bacterial Behaviour in Biofilms : Bacteria growing in microbial communities adherent to a surface do not behave the same as bacteria growing suspended in a liquid environment ( planktonic or unattached state) as shown by few properties of the biofilm like antibiotic resistance. It is generally accepted that the resistance of bacteria to antibiotics is affected by their nutritional status, growth rate, temperature, pH, and prior exposure to the antibiotic. The biofilm matrix, although not a significant barrier in itself to the diffusion antibiotics, does have certain properties that can resist diffusion . 33

34. Quorum sensing: Quorum sensing or quenching in bacteria “ involves the regulation of specific genes through the accumulation of signalling compounds that mediate intercellular communication ”.(Prosser 1999) Specific genes from a few cells express signal compounds in the extracellular matrix which when reaches the threshold level( quorum cell density ) gene expression can be activated. It occurs more rapidly in microcolonies as the concentration would be higher in the proximity. 34

35. Principle of Bacterial Transmission, Translocation, or Cross infection: Bacteria “ fingerprinting ” clearly illustrates that periodontal pathogens are transmissible within members of a family . Bacterial transmission occurs between subjects even from animals and humans (contagious involves the occurrence of disease); translocation or cross infection is the transmission of bacteria from one niche to another. The existence of intraoral transmission of bacteria was first examined in cariology . 35

36. Microbiology of implants in partially edentulous patients: It has been suggested that, at least in partially edentulous patients, teeth might acts as a reservoir for the colonization of the subgingival area around the implant. Translocation and Guided Tissue Regenration : Nowzari et al evaluated and concluded that the healthy group showed significantly less membrane contamination both immediately after insertion as well as removal after 6 weeks. 36

37. Translocation and mechanical debridement: To reduce the chance for an intraoral translocation, a treatment strategy called one stage, full-mouth disinfection was introduced by the Leuven et al in the 1990’s. The one stage, full mouth disinfection consists of the following therapeutic efforts:- ( Quiryen M et al ) Full mouth scaling and root planning within 24 hours to reduce the number of subgingival pathogenic organisms. Subgingival irrigation of all pockets with a 1% chlorhexidine gel to kill the remaining bacteria. Tongue brushing with an antiseptic to suppress the bacteria in the niche. Mouth rinsing with an antiseptic to reduce the bacteria in the saliva and on the tonsils. 37

38. Association of Plaque Microorganisms with Periodontal Disease: Current concepts of periodontitis etiology considers three groups of factors:- A susceptible host, The presence of pathogenic species, The absence or a small amout of “beneficial” bacteria. Slots j, rams te : new views on periodontal microbiota in special patient categories, j clin periodontol 18:411,1991 38

39. The susceptible host is partially hereditary but can be enfluenced by environment and behaviour factors, such as smoking, stress, and diabetes. Several studies have been conducted and have concluded the influence of host response and environment in the progress of periodontal disease like: Increased interlukin-1 production in periodontitis susceptibility, Early onset of periodontitis in hereditary high susceptibility, Direct and linear response between periodontitis and smoking (by Grossi et al), Higher risk of periodontal infection in diabetes , Stressful conditions aggrevating periodontal destruction . 39

40. The second essential factor for disease initiation and progression is the presence of one or more pathogens , of the susceptible clonal type and in sufficient in number . The mere presence of putative periodontal pathogens in the gingival crevice is not sufficient to cause periodontal inflammation. An elevation in the relative proportion or the number of these pathogens to reach critical mass seems more crucial to mount an effective tissue damaging process. 40

41. The role of “beneficial” bacteria of the host is less obvious in the progression of disease. They help by: Passively occupying a niche that may otherwise be colonized by pathogens, Actively limiting a pathogens ability to adhere to appropriate tissue surface, Adversely affecting the vitality or the growth of pathogen , Affecting the ability of the pathogen to produce virulence factors, Degrading virulence factors produced by the pathogen. 41

42. Microbial Specificity of Periodontal Diseases: Traditional Non Specific Plaque Hypothesis : - Periodontal diseases were believed to result from an accumulation of plaque over time, eventually in conjunction with a diminished host response and increased host susceptibility with age. It is termed as non specific plaque hypothesis because of epidemiological studies that showed correlation between patient’s age and amount of plaque with evidence of periodontitis . The non specific plaque hypothesis maintains that periodontal disease results from the “ elaboration of noxious products by the entire plaque flora ”. Loesche wj : chemotherapy of dental plaque infections, oral sci rev 9:65, 1976 42

43. Specific Plaque Hypothesis : - The hypothesis states that only certain plaque is pathogenic , and its pathogenicity depends on the presence of or increase in specific microorganisms. The result was of tremendous increase in the ability to isolate periodontal microorganisms and considerable refinement in bacterial taxonomy . Acceptance of the specific plaque hypothesis was spurred in recognition of A.actinomycetemcomitans as a pathogen in localized aggressive periodontitis . Slots j: subgingival microflora of advanced periodontitis , j clin periodontol 6:351, 1977 43

44. 44 Updated Non specific Plaque hypothesis Theilade’s statement ( 1986) that “ any microbial colonization of sufficient quantity in the gingival crevice causes at least gingivitis ” It was supported by the fact that a non-pathogenic plaque (i.e., not causing gin- givitis in the absence of oral hygiene) had never been observed.

45. Ecological plaque hypothesis : - (1994) Some researchers consider the pathologic flora as a result of environmental perturbations to the habitat, the “ecological plaque hypothesis”. The hypothesis reconciles the key elements of the key elements of the specific and non specific hypothesis: The selection of “pathogenic” bacteria is directly coupled to changes in the environment. Diseases need not have a specific etiology ; any species with relevant traits can contribute to the disease process. Marsh pd: oral ecology and its impact on oral microbial diversity. In kuramitsu hk , ellen rp , editors: oral bacterial ecology: the molecular basis, norfolk , va , 2000, horizon scientific press, p12 45

46. 46 Keystone Pathogen Hypothesis (Kph) – (2012) George Hajishengallis and colleagues believed that certain species have an effect on their environment that is disproportional relative to their over all abundance by proposing “The Keystone- PathogenHypothesis ”. The KPH was developed by observing the properties of the “ red complex ”. The role of the host-immune system is critical in the KPH. P. gingivalis can inhibit the production of IL-8 which delays the recruitment of neutrophils preventing proper neutrophil wall formation, of which was proposed that it could facilitate initial microbial colonization of the periodontium . This mechanism is called the local chemokine paralysis. Darveau,R.P.,Belton,C.M.,Reife,R.A.,andLamont,R.J .(1998).Local chemokineparalysis,anovelpathogenicmechanismfor Porphyromonasgingi - valis . Infect.Immun . 66, 1660–1665.

47. Complicating factors : - Periodontitis is considered as a mixed infection, which has significant impact on both its diagnosis and its treatment. For the diagnosis the clinician must evaluate the presence of up to 10 species, and it is still unclear whether some combinations of species are more pathogenic than other. Because several species might be involved, the use of antimicrobials is extremely difficult, because not all periopathogens are susceptible to the same antibiotic and also increases the chance of severe side effects.   47

48. Criteria for Identification of Periodontal Pathogens : - In 1870 , Robert Koch developed the criteria by which the microorganism can be judged to be a causative agent in human infections. Criteria is known as the Koch’s postulates, which are: Must be routinely identified in diseased individual, Must be grown in pure culture in the laboratory, Must produce a similar disease when inoculated into susceptible laboratory animals, Must be recovered from the lesion in the diseased laboratory animal. 48

49. In case of periodontology there are three major problems: The inability to culture all the microorganism that have been associated with the disease, The difficulties inherent in defining and culturing sites of active disease, The lack of good animal model system for the study of periodontitis . 49

50. Sigmund Socransky , proposed a criteria by which periodontal microorganisms may b judged to be a potential pathogens. The criterion is as follows:- Must be associated with disease, as evident by increases in the number of organisms at diseased sites. Must be eliminated or decreased in the sites that demonstrate clinical resolution of the disease with treatment . Must demonstrate host response , in the form of an alteration in the host cellular or humoral immune response. Must be capable of causing disease in experimental animal models. Must demonstrate virulence factors responsible for enabling the microorganism to cause destruction of periodontal tissue. 50

51. Microorganisms associated with Specific Periodontal Diseases:- It appears that bacteria from periodontally healthy sites consists predominantly of gram positive facultative rods and cocci ( approx. 75%). These decrease are accompanied by increases in proportions of gram negative rods, from 13% in health to 40% in gingivitis and 74% in advanced periodontitis . 51

52. Periodontal health : - The bacteria associated with periodontal health are primarily gram positive facultative species and members of the genera Streptococcus and Actinomyces . Small portions of gram negative species are also found, most frequently P. intermedia , F. nucleatum , and Capnocytophaga , Neisseria , and Veillonella species. Certain bacteria have been proposed to be “beneficial” to the host including S. sanguis , Veillonella parvula , C. ocharaceus . 52

53. Gingivitis : - The development of gingivitis has been extensively studied in model system referred to as experimental gingivitis. The microbiota of dental plaque induced gingivitis consists approximately equal proportions of gram positive(56%) and gram negative(44%) species, as well as facultative(59%) and anerobic (41%) microorganisms. Predominant gram positive organisms are S. sanguis , S. mitis , S. intermedius , S. oralis , A. viscosus , A. naeslundii , P. micros. The gram negative are F. nucleatum , P. intermedia , and V. Parvula , as well as haemophilus , capnocytophaga , and campylobacter species. 53

54. Chronic periodontitis : - Microscopic examinations of plaque from the sites of chronic periodontitis has consistently revealed elevated proportions of spirochetes . Cultivation of plaque microorganisms from the sites of chronic periodontitis reveals high percentages of anaerobic bacteria (90%) and gram negative (75%) bacterial species. When periodontally active sites were examined in comparision with inactive sites, C. rectus, P. gingivalis , P. intermedia , F. nucleatum , and T. forsythia were found to be elevated in active sites. 54

55. Recent studies have documented an association between chronic periodontitis and viral microorganisms of the herpesvirus group, most notably Epstein-Barr virus-1 and human cytomegalo virus. Microbial Shift during Disease : - From gram positive to gram negative , From cocci to rods ( later to spirochetes), From non motile to motile , From facultative anaerobes to obligate anaerobes, From fermenting to proteolytic species. 55

56. Localized Aggressive Periodontitis : - The microbiota associated with localized aggressive periodontitis is predominantly composed of gram negative, capnophilic , anaerobic rods. Microbiological studies reveal almost all the diseases sites harbour A. actinomycetemcomitans , which may compose as much as 90% of the total cultivable microbiota . A. actinomycetemcomitans is generally accepted as the primary eitiological agent in most, but not all cases of localized aggressive periodontitis . Socransky ss , haffa ad: the bacterial etiology of destructive periodontal disease: current concepts, j periodontol 63:322,1992 56

57. Necrotizing Periodontal Diseases : - Microbiological studies indicate that high level of P. intermedia , and especially of spirochetes, are found in necrotizing ulcerative gingivitis lesion. Spirochetes are found to penetrate necrotic tissue and apparently unaffected connective tissue. Listgarten MA, Socransky SS: Ultrastructural characteristics of a spirochete in the lesion of acute necrotizing ulcerative gingivostomatitis (Vincent’s infection), Arch Oral Biol 16:95, 1964. 57

58. Abscess of the periodontium : - Periodontal abscess also may occur in the absence of periodontal disease, eg impaction of foreign substance within the gingival pocket. Investigation reveal that bacteria recognized as periodontal pathogen are typically found in significant number in periodontal abscesses. These microorganisms include F. nucleatum , P.gingivalis , P. micros, and T. forsythia. Herrera D, Roldan S, Gonzalez I, et al: The periodontal abscess. I. Clinical and microbiological findings, J Clin Periodontol 27:387, 2000. 58

59. Periodontitis as a Manifestation of Systemic Disease : - Recent studies have demonstrated that some cases of severe periodontitis are associated with a mutation in the cathepsin C gene in affected children. Periimplantitis : - This inflammatory process has been associated with a microbiota comparable to that of periodontitis with high proportions of anaerobic gram negative rods, motile organism, and spirochetes. Certain species like Pseudomonas aeruginosa , enterobacteriaceae , Candida albicans and staphylococci, are also detected around implants. Alcoforado GA, Slots J: Actinobacillus actinomycetemcomitans and black-pigmented bacteroides in advanced periodontitis in man: theoretical and practical considerations, Rev Port Estomatol Cir Maxilofac 31:89, 1990. 59

60. 60 Key Pathogen Motility and shape Gram Forms Culture & identification(mostly blood agar) Virulence factors A.actinomycetemcomitans Straigh rod like, non motile Negative Subtypes from a to e White translucent culture, star shaped internal structures Lipopolysaccharides , leukotoxins . T. forsythia spindle shaped, highly pleomorphic rod, non motile Negative anaerobic proteolytic enymes P. gingivalis non motile, pleomorphic rod Negative different genotypes basis of capsule type. anaerobic dark pigmentation protease hemolysin collagenase

61. 61 Prevotella spp. short, round ended, non motile Negative P. intermedia and P. nigrescens common dark pigmentation, anaerobic less pathogenic less proteolytic than P. Gingivalis C. rectus motile short rod, curved or helical, polar flagellum Negative dark pigment when sulphide is added , anaerobic leukotoxins is less virulent F. nucleatum cigar shaped bacillus pointed ends Negative Several subtypes anaerobic induces apoptotic cell coaggregate trigger release of cytokines Spirochetes spiral, motile specific medium, anaerobic

62. Future Advances in Periodontal Microbiology :- DNA based methodology for the identification and detection of specific bacteria and viruses offer remarkable advantages in time and cost savings compared with culturing techniques. Finally the recognition of the beneficial activity of several groups of commensal species, such as probiotics , might open new stratergies for periodontal therapy. 62

63. Conclusion: 63 In general, once established, the microbial composition of the biofilm at a site remains stable over time, unless a major perturbation. Such perturbations can drive shifts in the balance of the microbiota , which can increase the risk of disease. New research and are to be done in isolation and identification of principle causative organism.

64. Bibliography: 64 Microbiology Of Periodontal Disease, Carranza’s Clilical Periodontology , 10 th Edition. Dental Biofilms , Clinical Periodontology And Implant Dentistry, 6 th Edition. Weiger R,netuschil L, Von Ohle C, Et Al: Microbial Generation Time During The Early Phase Of Supragingival Dental Plaque Formation, Oral Micribiol Immunol 10:93,1995 Quiryen M, Bollen Cm, Papaioannou W, Et Al: The Influence Of Titanium Abutment Surface Roughness On Plaque Accumulation And Gingivitis: Short Term Observations, Int J Oral Maxillofac Implants 11,:169, 1996.

65. 65 Marsh Pd: Oral Ecology And Its Impact On Oral Microbial Diversity. In Kuramitsu Hk , Ellen Rp , Editors: Oral Bacterial Ecology: The Molecular Basis, Norfolk, Va , 2000, Horizon Scientific Press, P12 Slots J, Rams Te: New Views On Periodontal Microbiota In Special Patient Categories, J Clin Periodontol 18:411,1991 Zee Ky , Samaranayake Lp , Attstorm R: Scanning Electron Microscopy Of Microbial Colonization Of “Rapid” And “Slow” Dental Plaque Formers In Vivo, Arch Oral Biol 42:735,1997 Winkel Eg , Abbas F, Van Der Velden U, Et Al: Experimental Gingivitis In Relation To Age In Individuals Not Susceptible To Periodontal Destruction, J Clin Periodontol 14:499,1987 Historicalandcontemporaryhypothesesonthedevelopmentoforaldiseases:arewethereyet?REVIEW ARTICLE published: 16July2014 doi : 10.3389/ fcimb

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