Traditionally endodontic bacteria have been studied by means of cultivation based techniques. The past decade has brought many advances in methods based on detection and analysis of microbial nucleic acids. Findings from cultivation based method with regard to the microbiota living in diverse ecosystem have been supplemented and significantly expanded with molecular biology techniques.

Various methods used to examine biofilm are:

A. Culture

B. Microscopy

C. Immunological methods

D. Molecular biology methods

A. Culture8, 9:

Culture is defined as the process of propagating microorganisms in the laboratory and provide them with proper environmental conditions. Ingredients necessary for microbial pathogens can be supplied by living systems or artificial means.

A culture medium or growth medium is a substance in which cells or organisms can grow.

Advantages:

Types of culture medium 10:

2. According to the constituents:

. Simple media-nutrient broth (peptone, meat extract, NaCl and H2O).

. Complex 2% agar

. Synthetic ( peptone water medium)

. Special - a) Enriched media – blood agar, chocolate agar

b) Enrichment media – tetrathionate broth.

3. Aerobic media or anaerobic media:

. Aerobic media : Bower’s thioglycolate broth

. Anaerobic media: fat free minced cooked meat in broth with a layer of sterile Vaseline.

Common ingredients of culture media 10:

Anaerobic bacterial techniques 10 10:

They are important in endodontics since strict aerobic bacteria are not present in the root canals. The safest way to protect anaerobic bacteria is to avoid exposure to O₂ during the lab work.

Two methods have made this possible:

1) The prereduced anaerobically sterilized technique by Hungate. Simplified and further developed by Moore. It is based on production of low reduction oxidation potential by gassing the media with O₂ free gas and affording protection from oxygenation during sterilization and subsequent handling.

2) To use anaerobic glove box. The atmosphere in this box is usually a mixture of N₂-90%, CO₂-10% and H₂O-10%.

Limitations of culture technique:

Reasons for bacterial ‘unculturability:’

B. Microscopy 11:

Direct microscopic examination represents a quick, easy and inexpensive means of screening microbial samples for major morphotypes and staining patterns. However, microscopy has limited senstivity and specificity to detect microorganisms in clinical samples.

C. Immunological methods 12:

Immunological methods employ antibodies that recognize specific microbial antigens to directly detect target species.

Antibodies targeting host immunoglobulins specific to a target species can also be used for indirect detection assays. The reaction can be visualized using a variety of techniques and reaction including direct and indirect immunofluorescence, flow cytometry and ELISA.

Advantages:

Limitations:

D. Molecular biology methods:

The basic strategy for diagnostic molecular microbiology is using microbial DNA or RNA to detect a relatively short sequence of nucleotide bases that is unique to the organism being detected or identified. This is accomplished by using a complementary sequence of nucleotide bases known as the probe or primer.

There are plethora of molecular methods for the study of microorganisms such as:

PCR (Polymerase Chain Reaction) / Genetic Xeroxing 13, 14:

Principle: The PCR method is based on the in vitro replication of DNA through repetitive cycles of denaturation, primer annealing, and extension steps carried out in automated devices called thermocyclers.

Method: There are several methods to check if the intended PCR product was generated. The most commonly used method for detecting PCR product is electrophoresis in an agarose gel.

Uses: PCR has unrivaled sensitivity, it can detect as few as 1 to 10 bacterial cells in a sample, making it atleast 10 to 100 times more sensitive than any other scientific method.

Advantage:

PCR can have remarkable specificity because each distinct microbial species has unique DNA signature sequence.

Derivatives of PCR:

Touchdown PCR 15

Nested PCR 14, 16

Multiplex PCR17

Reverse transcriptase PCR 18

Real-Time PCR 19

PCR based microbial typing 20, 21

Broad-Range PCR 22, 23

Denaturing Gradient Gel Electrophoresis (DGGE):

DNA fragments of the same length but with different nucleotide sequence can be separated in polyacrylamide gels containing a linearly increasing gradient of DNA denaturants.24, 25

Terminal Restriction Fragment Length Polymorphism (T – RFLP):

It provide insight into the structure and function of bacterial communities. T-RLP analysis measures the size polymorphism of terminal restriction fragments from a PCR amplified marker.26, 27

DNA – DNA Hybridization:

It is the process of annealing the complimentary bases of two single stranded DNA molecules. It employs labeled DNA molecules that can locate and bind to a target sequence forming a new duplex molecule. The labeled duplex can then be detected28 .

Types of DNA-DNA hybridization:

Checkerboard DNA-DNA hybridization 29

DNA microarrays 30

FISH (Fluorescence In Situ Hybridization):

This method uses fluorescently labeled rRNA probes and fluorescence microscopy to detect intact microbial cells directly in clinical specimens. It gives information about presence, morphology, number, organization and spatial distribution of microorganisms.31, 32

Almost 700 bacterial species can be found in the oral cavity, with any particular individual harboring 100–200 of these species. Infection progress to apical once the root canal is infected coronally until bacterial products or bacteria themselves got the capability to infect the periapical tissues, which leads to apical periodontitis. Endodontic infections have a polymicrobial nature, with obligate anaerobic bacteria conspicuously dominating the microbiota in primary infections. Intra-radicular and extraradicular infections are associated with many microorganisms which are involved in persistent infection

A. Intraradicular infections:

Primary intraradicular infections are associated with following organisms.

B .melaninogenicus P. intermedia, P. nigrescens, P. tannerae, P. multissacharivorax, P. baroniae, P. denticola, P. endodontalis and P. gingivalis.

Tannerella forsythia D. Pneumosintes , D. invisus , F. nucleatum, F. periodonticum, T. denticola, T. sacranskii, T. parvum, T. maltophilum, T. lecithinolyticum, P. alactolyticus, F. alocis, Actinomyces spp, P. propionicum, Olsenella spp, S. exigua, M. timidum, Eubacterium spp., P. micra (previously called Peptostreptococcus micros or Micromonas micros), Streptococcus spp. which include S. anginosus, S. mitisi, S. sanguinis, E. faecalis.

Other microorganisms in endodontic infections

B. Extraradicular infections:

The extraradicular infections may or may not dependent on intraradicular infection. The dominant microorganisms present are anaerobic bacteria like Actinomyces spp., P. propionicum, Treponema spp., P. endodontalis, Porphyromonas gingivalis, T. forsythia Prevotella spp. and F. nucleatum.

C. Bacteria persisting intracanal disinfection procedures and after root canal treatment:

Microorganisms which are resistant to antimicrobial treatment will survive in the root canal even after biomechanical preparation.

The most common Gram negative anaerobic rods are : F. nucleatum, Prevotella spp. and C. rectus.

The most common Gram positive bacteria are : Streptococci (S. mitis, S. gordonii, S. anginosus, S. oralis), Lactobacilli (L. paracasei , L. acidophilus), Staphylococci, E. faecalis, O. uli, P micra, P. alactolyticus, Propionibacterium spp., Actinomyces spp., Bifidobacterium spp. and Eubacterium spp.

Sometimes, yeasts, commonly C. albicans, are also found in small amounts.

Primary source of biofilm formation within the root canal are the microorganisms present in the oral cavity and the anatomical complexities in the root canal system provide shelter to these microorganisms.

As bacteria in biofilm survive the unfavourable environmental and nutritional conditions, bacterial biofilm can be easily seen beyond the apex of the root. Infectious processes in root canal gain sufficient power to cause subsequent destruction of the pulpal tissues only after biofilm formation. Pulp chamber is invaded by planktonic oral organisms after some tissue breakdown, then only biofilm formation is intiated. At this point, the inflammatory lesion frontage that moves successively towards the apex will provide the fluid vehicle

for the invading planktonic organisms so that these can multiply and continue attaching to the root canal walls. The necrotic pulp tissue becomes a favorable environment for microbial proliferation due to the presence of organic residue or nutrients, which act as substrate or culture medium. Gram-negative bacteria are more frequently associated with biofilm formation than Gram- positive bacteria. Facultative or strict anaerobic microorganisms are more frequent than aerobic microorganisms, and the presence of bacilli and filaments is equivalent to that of cocci.

The infected root canal harbors a polymicrobial population of aerobic, anaerobic, Gram-positive, and Gram-negative bacteria in a biofilm mode of growth. These are commonly isolated by culture, microscopy, immunological methods and molecular biology methods. Pulp chamber is invaded by planktonic oral organisms after some tissue breakdown which initiates biofilm formation.

Biofilm bacteria might not express the drug target as they have been found to be more resistant to amoxicillin, doxycycline, and metronidazole. NaOCl, chlorhexidine, Qmix, IPI, EDTA, citric acid, MTAD, Tetraclean, ozone, Electrochemically activated water, lasers, Photon activated disinfection, antibacterial nanoparticles, herbal alternatives (turmeric, triphala), endovac system, ultrasonic and endoactivator are used for biofilm removal with varying degree of efficacy

Root canal environment is a challenging locate for eliminating surface-adherent biofilm bacteria. Different antimicrobials are being employed in the management of infected root canal systems with varying success. However, sodium hypochlorite has been found to be the most effective irrigant against biofilm. However, further research on endodontic biofilms is required so as to provide a better understanding of its physiology, ecology, pathogenicity, and its response to treatment.