Root Canal Microbiology

• For many years it has been a widely held view in Endodontics that cases in which a negative
culture has been obtained prior to root canal filling have a superior long term prognosis to
cases in which the culture result is positive prior to filling.
• A major purpose of the use of the root canal culture is to monitor the effectiveness of your
application of aseptic technique to endodontic therapy.

A culture will be required at two times during endodontic treatment;

1)At the end of the first appointment at which access size is sufficient to permit the taking of a
culture (after irrigation and drying of the canal and prior to medication).
2)At the completion of instrumentation of all canals (after irrigation and drying of the canals
and prior to medication).

• The microbial culture test is the only way to determine the bacteriological status of the root
canal.

Culture must be taken in the following cases:

1- All cases in which the pulp in the root canal is found to be necrotic or partially necrotic.
2-All vital cases where the pulp chamber was exposed to saliva.

Culture need not be taken in the following cases:

1- Cases in which the pulp in the root canal is vital and the pulp chamber has never been exposed
to saliva.
2- Cases for which periapical surgery is planned.

• A greater success rate is achieved in cases with negative cultures than in those with positive
cultures.
• Antibiotic sensitivity testing can be made for organisms isolated from infected root canals.
• Help in determining:
a) Whether or not thorough canal debridement has been performed.
b) Whether or not leakage has occurred between visits.
c) Whether or not an aseptic technique has been followed.

• Portal of entry for microorganisms into the dental pulp:

1-Direct pulp exposure:
This is the most common and obvious and frequent avenue for microorganisms to gain access to
a pulp.

2-Indirect pulp exposure:

Microorganisms may gain entry to the pulp through the dentinal tubules, even though the carious
process has not physically penetrated to the pulp chamber.

3-Lateral canals:
Lateral canals may be found anywhere on the root, and it seems that some organisms may gain
access to the pulp through this pathway.

4-Apical foramen via the periodontal spaces:

If a tooth is dislodged or partially avulsed, organisms may traverse the periodontal spaces and
gain access to the pulp via the apical foramen.
5-Retrogenic infection:
It is the pulp inflammation or death as a result of bacteria from adjacent diseased teeth which
invade the pulp via the apical foramen.

6-Hematogenic anachoresis:
After trauma persistent blood-born bacteria may be attracted to the pulp of a tooth, which has
been traumatized.

Endodontic flora
→ Gram-positive organisms are most frequently isolated, especially Streptococci, which are the
most numerous,isolated type.
→ Some gram negative as Staphylococci,and yeast organisms are expected in smaller number.
→ Some unusual non-resident organisms are occasionally isolated.

Bacterial culturing:
→ It is a method of verifying the sterility of the root canal.
→ Negative culturing does not mean necessarily that the tooth is ready for filling;
→ There may be another factors that contraindicate the obturation of the root canal.
→ While a positive culture definitely indicates that the canal cannot be obturated.

Culture media:
→ The most common contaminants of infected root canals are the streptococcal and
Staphylococcal organisms.
→ Both groups are facultative anaerobes and require a media relatively free of oxygen for
optimal growth.

Various types of acceptable media can be used for the endodontic bacteriological tests as:
1. Brain heart infusion broth with 0.1% agar.
2. Trypticase-soy broth with 0.1% agar.
3. Thioglycollate broth.
4. Glucose ascites broth.
5. Mueller Hinton Agar (MHA) is a culture of e.faecalis

Technique for bacterial culturing

1)The tooth is isolated with the rubber dam; the tooth surfaces and the surrounding dam material
are scrubbed with a suitable bactericidal agent.
2) Canal access is established with sterile burs and a sterile paper point is inserted into the
orifice of each canal.
3) The paper point is allowed to remain into the canal for at least one minute in order to absorb
as much as periapical exudates and microorganisms along the wall of the root canal as possible.
4) The paper points are removed with sterile pliers, the lip of the culture tube is flamed, and the
points are introduced into the medium.
5) The tube is incubated for a minimum of 48 hours at 37ºC in the incubator.
6) The growth of bacteria is checked as indicated by the cloudiness in the tube compared to
noninoculated tubes.
Cloudy tubes are reported as positive culture,
and those remaining clear as negative culture.
Causes of false negative culture:
1)Too dry canal.
2)Insufficient time the paper point was allowed to remain in the canal.
3)Insufficient incubation time.
4)Traces of the antiseptic medicament were carried by the paper point to the media in the
culture tube.
5)Improper culture media.

Causes of false positive cultures:

1) Improper sterilization of the working field and instruments.
2) Leakage through the temporary filling.
3) Infected culture media.

Biofilm
Definition:
Sessile multi-cellular microbial community characterized by cells that are firmly attached to
a surface and enmeshed in a self-produced matrix (Extracellular polymeric substance “EPS”)
- Bacterial cells ±15% by volume
- EPS ±85% by volume

Mechanism of Biofilm resistance

1-Biofilm restrict penetration of antimicrobial agent:
-The matrix in biofilm can bind and retain neutralizing enzymes at concentrations that could
inactivate the antimicrobial agents.
-The antimicrobial agent may adsorb to and even inhibit the bacteria at the surface, but cells
deeply located may remain unaffected.

2-Altered growth rate of biofilm bacteria:

-Bacterial cells present in stationary phase (due to starvation) might represent a general
mechanism of antibiotic resistance.

3-Presence of persister bacteria:

- Increased tolerance of some biofilms to antibiotics may be due to the presence of
subpopulation of specialized survivor cells (persister)

Role of Enterococcus faecalis in secondary infection

1. E. Faecalis may cause secondary infections (44%) that later become persistent.
2. E. Faecalis is able to penetrate dentinal tubules to deep extent to escape the action of
endodontic instruments and irrigants.
3. It has the ability to resist high pH (up to 12 ) values seems to be related to a functioning
proton pump that drives protons into the cell to acidify the cytoplasm.
4. E. Faecalis may colonize root canals in single infection
5. It can enter a so called viable but non cultivable state when exposed to unfavorable
environmental conditions.
6. Can resist 30 days without nutrition ( Resist Starvation ) (starvation tolerance)
7. Can resist high temperature

N.B--- E. Fecalis could only be eradicated from the root canal space using high
concentrations of Sodium Hypochlorite with mechanical activation with
ultrasonic device for the biofilm penetration.