Dark Field Microscopy in Periodontal Microbiology

Dark field microscopy and phase contrast microscopy are valuable techniques in microbiological studies, particularly in the field of periodontal research. These methods allow for the direct observation of bacteria in plaque samples, providing insights into their morphology and motility. This lecture will discuss the principles of dark field microscopy, its applications in periodontal disease assessment, and its limitations.

Dark Field Microscopy

• Definition: Dark field microscopy is a technique that enhances the contrast of unstained, transparent specimens, allowing for the visualization of live microorganisms in their natural state.
• Principle: The method uses a special condenser that directs light at an angle, creating a dark background against which the specimen appears bright. This allows for the observation of motility and morphology without the need for staining.

Applications in Periodontal Microbiology

1. Alternative to Culture Methods:

   • Dark field microscopy has been suggested as a rapid alternative to traditional culture methods for assessing bacterial populations in periodontal plaque samples. It allows for immediate observation of bacteria without the time-consuming process of culturing.

2. Assessment of Morphology and Motility:

   • The technique enables direct and rapid assessment of the morphology (shape and structure) and motility (movement) of bacteria present in plaque samples. This information can be crucial for understanding the dynamics of periodontal disease.

3. Indication of Periodontal Disease Status:

   • Dark field microscopy has been used to indicate the status of periodontal disease and the effectiveness of maintenance programs. By observing the presence and activity of specific bacteria, clinicians can gain insights into the health of periodontal tissues.

Limitations of Dark Field Microscopy

1. Analysis of Major Periodontal Pathogens:

   • While dark field microscopy can visualize motile bacteria, it is important to note that many major periodontal pathogens, such as Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Bacteroides forsythus, Eikenella corrodens, and Eubacterium species, are motile. However, the technique may not provide detailed information about their specific characteristics or pathogenic potential.

2. Differentiation of Treponema Species:

   • Dark field microscopy cannot differentiate between species of Treponema, which is a limitation when identifying specific pathogens associated with periodontal disease. This lack of specificity can hinder the ability to tailor treatment based on the exact microbial profile.

3. Limited Quantitative Analysis:

   • While dark field microscopy allows for qualitative observations, it may not provide quantitative data on bacterial populations, which can be important for assessing disease severity and treatment outcomes.

Keratinized Gingiva and Attached Gingiva

The gingiva is an essential component of the periodontal tissues, providing support and protection for the teeth. Understanding the characteristics of keratinized gingiva, particularly attached gingiva, is crucial for assessing periodontal health.

Keratinized Gingiva

1. Definition:

   • Keratinized gingiva refers to the gingival tissue that is covered by a layer of keratinized epithelium, providing a protective barrier against mechanical and microbial insults.

2. Areas of Keratinized Gingiva:

   • Attached Gingiva:
     • Extends from the gingival groove to the mucogingival junction.
   • Marginal Gingiva:
     • The free gingival margin that surrounds the teeth.
   • Hard Palate:
     • The roof of the mouth, which is also covered by keratinized tissue.

Attached Gingiva

1. Location:

   • The attached gingiva is the portion of the gingiva that is firmly bound to the underlying alveolar bone.

2. Width of Attached Gingiva:

   • The width of attached gingiva varies based on location and can increase with age and in cases of supraerupted teeth.

3. Measurements:

   • Greatest Width:
     • Found in the incisor region:
       • Maxilla: 3.5 mm - 4.5 mm
       • Mandible: 3.3 mm - 3.9 mm
   • Narrowest Width:
     • Found in the posterior region:
       • Maxillary First Premolar: 1.9 mm
       • Mandibular First Premolar: 1.8 mm

Clinical Significance

• Importance of Attached Gingiva:

  • The width of attached gingiva is important for periodontal health, as it provides a buffer zone against mechanical forces and helps maintain the integrity of the periodontal attachment.
  • Insufficient attached gingiva may lead to increased susceptibility to periodontal disease and gingival recession.

• Assessment:

  • Regular assessment of the width of attached gingiva is essential during periodontal examinations to identify potential areas of concern and to plan appropriate treatment strategies.

Anatomy and Histology of the Periodontium

Gingiva (normal clinical appearance): no muscles, no glands; keratinized

• Color: coral pink but does vary with individuals and races due to cutaneous pigmentation
• Papillary contour: pyramidal shape with one F and one L papilla and the col filling interproximal space to the contact area (col the starting place gingivitis)
• Marginal contour: knife-edged and scalloped
• Texture: stippled (orange-peel texture); blow air to dry out and see where stippling ends to see end of gingiva
• Consistency: firm and resilient (push against it and won’t move); bound to underlying bone
• Sulcus depth: 0-3mm
• Exudate: no exudates (blood, pus, water)

  Anatomic and histological structures

Gingival unit: includes periodontium above alveolar crest of bone

a. Alveolar mucosa: histology- non-keratinized, stratified, squamous epithelium, submucosa with glands, loose connective tissue with collagen and elastin, muscles.  No epithelial ridges, no stratum granulosum (flattened cells below keratin layer)

b. Mucogingival junction: clinical demarcation between alveolar mucosa and attached gingiva

c. Attached gingiva: histology- keratinized, stratified, squamous epithelium with epithelial ridges (basal cell layer, prickle cell layer, granular cell layer (stratum granulosum), keratin layer); no submucosa

• Dense connective tissue: predominantly collagen, bound to periosteum of bone by Sharpey fibers
• Reticular fibers between collagen fibers and are continuous with reticulin in blood vessels

d. Free gingival groove: demarcation between attached and free gingiva; denotes base of gingival sulcus in normal gingiva; not always seen

e. Free gingival margin: area from free gingival groove to epithelial attachment (up and over ® inside)

• Oral surface: stratified, squamous epithelium with epithelial ridges
• Tooth side surface (sulcular epithelium): non-keratinized, stratified, squamous epithelium with no epithelial ridges (basal cell and prickle cell layers)

f. Gingival sulcus: space bounded by tooth surface, sulcular epithelium, and junctional epithelium; 0-3mm depth; space between epithelium and tooth

g. Dento-gingival junction: combination of epithelial and fibrous attachment

• Junctional epithelium (epithelial attachment): attachment of epithelial cells by hemi-desmosomes and sticky substances (basal lamina- 800-1200 A, DAS-acid mucopolysaccharides, hyaluronic acid, chondroitin sulfate A, C, and B), to enamel, enamel and cementum, or cementum depending on stage of passive eruption.  Length ranges from 0.25-1.35mm.
• Fibrous attachment: attachment of collagen fibers (Sharpey’s fibers) into cementum just beneath epithelial attachment; ~ 1mm thick

h. Nerve fibers: myelinated and non-myelinated (for pain) in connective tissue.  Both free and specialized endings for pain, touch pressure, and temperature -> proprioception.  If dentures, rely on TMJ.

i.Mesh of terminal argyophilic fibers (stain silver), some extending into epithelium

ii  Meissner-type corpuscles: pressure sensitive sensory nerve encased in CT

iii.Krause-type corpuscles: temperature receptors

iv. Encapsulated spindles

i. Gingival fibers:

i.  Gingivodental group:

• Group I (A): from cementum to free gingival margin
• Group II (B): from cementum to attached gingiva
• Group III (C): from cementum over alveolar crest to periosteum on buccal and lingual plates

ii.  Circular (ligamentum circularis): encircles tooth in free gingiva

iii. Transeptal fibers: connects cementum of adjacent teeth, runs over interdental septum of alveolar bone.  Separates gingival unit from attachment apparatus.

Transeptal and Group III fibers the major defense against stuff getting into bone and ligament.

2.  Attachment apparatus: periodontium below alveolar crest of bone

Periodontal ligament: Sharpey’s fibers (collagen) connecting cementum to bone (bundle bone).  Few elastic and oxytalan fibers associated with blood vessels and embedded in cementum in cervical third of tooth.  Components divided as follows:

i. Alveolar crest fibers: from cementum just below CEJ apical to alveolar crest of bone

ii.Horizontal fibers: just apical to alveolar crest group, run at right angles to long axis of tooth from cementum horizontally to alveolar bone proper

iii.Oblique fibers: most numerous, from cementum run coronally to alveolar bone proper

iv. Apical fibers: radiate from cementum around apex of root apically to alveolar bone proper, form socket base

v. Interradicular fibers: found only between roots of multi-rooted teeth from cementum to alveolar bone proper

vi. Intermediate plexus: fibers which splice Sharpey’s fibers from bone and cementum

vii. Epithelial Rests of Malassez: cluster and individual epithelial cells close to cementum which are remnants of Hertwig’s epithelial root sheath; potential source of periodontal cysts.

viii. Nerve fibers: myelinated and non-myelinated; abundant supply of sensory free nerve endings capable of transmitting tactile pressure and pain sensation by trigeminal pathway and elongated spindle-like nerve fiber for proprioceptive impulses

Cementum: 45-50% inorganic; 50-55% organic (enamel is 97% inorganic; dentin 70% inorganic)

i.  Acellular cementum: no cementocytes; covers dentin (older) in coronal ½ to 2/3 of root, 16-60 mm thick

ii. Cellular cementum: cementocytes; covers dentin in apical ½ to 1/3 of root; also may cover acellular cementum areas in repair areas, 15-200 mm thick

iii. Precementum (cementoid): meshwork of irregularly arranged collagen in surface of cementum where formation starts

iv. Cemento-enamel junction (CEJ): 60-65% of time cementum overlaps enamel; 30% meet end-to-end; 5-10% space between

v. Cementum slower healing than bone or PDL.  If expose dentinotubules ® root sensitivity.

Alveolar bone: 65% inorganic, 35% organic

i. Alveolar bone proper (cribriform plate): lamina dura on x-ray; bundle bone receive Sharpey fibers from PDL

ii. Supporting bone: cancellous, trabecular (vascularized) and F and L plates of compact bone

Blood supply to periodontium

i. Alveolar blood vessels (inferior and superior)

A) Interalveolar: actually runs through bone then exits, main supply to alveolar bone and PDL

B) Supraperiosteal: just outside bone, to gingiva and alveolar bone

C) Dental (pulpal): to pulp and periapical area

D) Terminal vessels (supracrestal): anastomose of A and B above beneath the sulcular epithelium

E) PDL gets blood from: most from branches of interalveolar blood vessels from alveolar bone marrow spaces, supraperiosteal vessels when interalveolar vessels not present, pulpal (apical) vessels, supracrestal gingival vessels

ii. Lymphatic drainage: accompany blood vessels to regional lymph nodes (esp. submaxillary group)

Dental Calculus

Dental calculus, also known as tartar, is a hard deposit that forms on teeth due to the mineralization of dental plaque. Understanding the composition and crystal forms of calculus is essential for dental professionals in diagnosing and managing periodontal disease.

Crystal Forms in Dental Calculus

1. Common Crystal Forms:

   • Dental calculus typically contains two or more crystal forms. The most frequently detected forms include:
     • Hydroxyapatite:
       • This is the primary mineral component of both enamel and calculus, constituting a significant portion of the calculus sample.
       • Hydroxyapatite is a crystalline structure that provides strength and stability to the calculus.
     • Octacalcium Phosphate:
       • Detected in a high percentage of supragingival calculus samples (97% to 100%).
       • This form is also a significant contributor to the bulk of calculus.

2. Other Crystal Forms:

   • Brushite:
     • More commonly found in the mandibular anterior region of the mouth.
     • Brushite is a less stable form of calcium phosphate and may indicate a younger calculus deposit.
   • Magnesium Whitlockite:
     • Typically found in the posterior areas of the mouth.
     • This form may be associated with older calculus deposits and can indicate changes in the mineral composition over time.

3. Variation with Age:

   • The incidence and types of crystal forms present in calculus can vary with the age of the deposit.
   • Younger calculus deposits may have a higher proportion of brushite, while older deposits may show a predominance of hydroxyapatite and magnesium whitlockite.

Clinical Significance

1. Understanding Calculus Formation:

   • Knowledge of the crystal forms in calculus can help dental professionals understand the mineralization process and the conditions under which calculus forms.

2. Implications for Treatment:

   • The composition of calculus can influence treatment strategies. For example, older calculus deposits may be more difficult to remove due to their hardness and mineral content.

3. Assessment of Periodontal Health:

   • The presence and type of calculus can provide insights into a patient’s oral hygiene practices and periodontal health. Regular monitoring and removal of calculus are essential for preventing periodontal disease.

4. Research and Development:

   • Understanding the mineral composition of calculus can aid in the development of new dental materials and treatments aimed at preventing calculus formation and promoting oral health.

Localized Aggressive Periodontitis and Necrotizing Ulcerative Gingivitis

Localized Aggressive Periodontitis (LAP)

Localized aggressive periodontitis, previously known as localized juvenile periodontitis, is characterized by specific microbial profiles and clinical features.

• Microbiota Composition:
  • The microbiota associated with LAP is predominantly composed of:
    • Gram-Negative, Capnophilic, and Anaerobic Rods.
  • Key Organisms:
    • Actinobacillus actinomycetemcomitans: The main organism involved in LAP.
    • Other significant organisms include:
      • Porphyromonas gingivalis
      • Eikenella corrodens
      • Campylobacter rectus
      • Bacteroides capillus
      • Spirochetes (various species).
  • Viral Associations:
    • Herpes viruses, including Epstein-Barr Virus-1 (EBV-1) and Human Cytomegalovirus (HCMV), have also been associated with LAP.

Necrotizing Ulcerative Gingivitis (NUG)

• Microbial Profile:
  • NUG is characterized by high levels of:
    • Prevotella intermedia
    • Spirochetes (various species).
• Clinical Features:
  • NUG presents with necrosis of the gingival tissue, pain, and ulceration, often accompanied by systemic symptoms.

Microbial Shifts in Periodontal Disease

When comparing the microbiota across different states of periodontal health, a distinct microbial shift can be identified as the disease progresses from health to gingivitis to periodontitis:

1. From Gram-Positive to Gram-Negative:

   • Healthy gingival sites are predominantly colonized by gram-positive bacteria, while diseased sites show an increase in gram-negative bacteria.

2. From Cocci to Rods (and Later to Spirochetes):

   • In health, cocci (spherical bacteria) are prevalent. As the disease progresses, there is a shift towards rod-shaped bacteria, and in advanced stages, spirochetes become more prominent.

3. From Non-Motile to Motile Organisms:

   • Healthy sites are often dominated by non-motile bacteria, while motile organisms increase in number as periodontal disease develops.

4. From Facultative Anaerobes to Obligate Anaerobes:

   • In health, facultative anaerobes (which can survive with or without oxygen) are common. In contrast, obligate anaerobes (which thrive in the absence of oxygen) become more prevalent in periodontal disease.

5. From Fermenting to Proteolytic Species:

   • The microbial community shifts from fermentative bacteria, which primarily metabolize carbohydrates, to proteolytic species that break down proteins, contributing to tissue destruction and inflammation.

Classification of Embrasures

1. Type I Embrasures:

   • Description: These are characterized by the presence of interdental papillae that completely fill the embrasure space, with no gingival recession.
   • Recommended Cleaning Device:
     • Dental Floss: Dental floss is most effective in cleaning Type I embrasures. It can effectively remove plaque and debris from the tight spaces between teeth.

2. Type II Embrasures:

   • Description: These embrasures have larger spaces due to some loss of attachment, but the interdental papillae are still present.
   • Recommended Cleaning Device:
     • Interproximal Brush: For Type II embrasures, interproximal brushes are recommended. These brushes have bristles that can effectively clean around the exposed root surfaces and between teeth, providing better plaque removal than dental floss in these larger spaces.

3. Type III Embrasures:

   • Description: These spaces occur when there is significant loss of attachment, resulting in the absence of interdental papillae.
   • Recommended Cleaning Device:
     • Single Tufted Brushes: Single tufted brushes (also known as end-tuft brushes) are ideal for cleaning Type III embrasures. They can reach areas that are difficult to access with traditional floss or brushes, effectively cleaning the exposed root surfaces and the surrounding areas.