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INTRODUCTION: Antony Van Leeuwenhock (1632-1723) was the first to describe bacteria in the root canal. He took the decayed stuff out of the hollows in the roots and mixed it with clean rainwater and set it before magnifying glass. He saw that the whole stuff seemed to be alive. Then it was Miller who worked in Robert Koch’s laboratory

applied

all

the

current

bacteriological

techniques to studies of the oral microbiota. Koch had developed method of staining bacteria in smears and in 1881 he introduced the solid media technique, which made it possible to obtain pure culture from mixed infections. Miller observed a wide variety of bacteria form including spirochetes. He also observed that teeth with open pulp chambers had bacteria in the pulp chamber that were different from those present in the root canals. When strict anaerobic techniques were introduced it was

found

that

the

obligatory

anaerobic

bacteria

dominated in the infected root canals (90% of the flora). Before we go further it is very important to know about the oral Ecosystem from which the bacteria flora originate. Oral Ecosystem: Four

major

Ecosystems

on

the

basis

of

the

distribution of indigenous flora and the site. 1


1. Buccal epithelium. 2. Dorsum of tongue. 3. Supragingival tooth surface. 4. Crevicular and epithelial surfaces. Root canal flora, which we are concerned of Classification of Bacteria: O 2 is the key factor; O 2 can be lethal to bacteria that cannot cope with some metabolic products formed in its presence. Two such substances in particular are Superoxide

H 2 O 2 (cause DNA damage)

Radical

These 2 react to form Hydroxyl radical

All of these substances are harmful for cell. Obligate Aerobes (includes bacillus, pseudomonas

1)

and some other bacilli. -

These require O 2 for their growth

-

They possess both enzymes

→ Catalase

→ Superoxide dismutase Facultative Anaerobes (includes enteric bacteria

2)

and staphylococcus). -

These organisms grow in the presence / absence of O2.

2


-

They

produce

both

catalase

and

superoxide

dismutase. 3) Microaerophilic – (includes lactic acid bacteria and streptococcus) -

These grow in an O 2 environment but desire their energy for fermentative pathways that occur in the absence of O 2 .

-

These bacteria grow well in low O 2 tensions.

-

Microaerophilic

organisms

contain

superoxide

dismutase but no catalase. 4) Obligate

Anaerobes:

fusobacterium,

(includes

bacteroids,

peptococcus

and

peptostreptococcus). -

These bacteria grow only in the absence of O2.

-

All function only at low O2 reduction potentials.

-

These organisms generally lack both superoxide dismutase and catalase.

In infected root canal we find only anerobic bacterias that can be listed as:

3


Recognized Microbial Genera and Species in infected root canals: Gram positive bacteria Aerobic and facult Anaerobic anaerobic Cocci: Streptococcus Streptococcus S.mille ri S.consllatus S.mitios S.intermedius S.mutans S.morbillorum S.sanguis S.tuccalis Peptostreptococcus P.anae robins P.me gnus P.micros P.pre voti Rods: Actinomycos Actinomyces A.naeslundi A.israelii A.viscosus A.meyeri A.odontolyticus Arachnia A.propionica Eubacterium E.alactolyticum E.brachy E.lentum E.nodatum E.timidum Lactobacillus L.eatenaforms L.minutus Proprionibacterium P.acnes

Gram negative bacteria Aerobic and facult anaerobic

Anaerobic Veillonella V. parvula

Capnocytophaga C. Ochrace a Eikenella E.corrodens Campylobacks C.sputorum

Bacteroides B.buccae B.de nticola B.e ndodontalis B.gingivalis B.inte rme dius B.loe sche i B.oralis B.oris B.ure olyticus Fusobacterium F.nucle atum Selenomonas S.sputige na Wolinella W.re cta W.curva

Routes of Microorganisms Ingress: 1.

Through the open cavity – caused by dental caries / fracture restoration.

4


2.

Through the dentinal tubules ↓

Either by salivary

Through adjacent carious

Contamination

lesions/microleakage

↓ During cavity preparation through tubules ↓ Microorganism of low virulence enters So pulp protection is necessary during restorations. 3.

Through the gingival sulcus or periodontal pocket. -

Microorganisms from the periodontal ligament can enter the pulp through.

-

Vessels

passing

through

apical

foramen

or

any

auxiliary foramina. -

Also auxiliary canals may be present some distance from the apex of the root towards the crown.

-

Microorganisms in the gingival sulcus can also gain entry into the pulp if periodontal disease destroys soft

tissue

or

bone

and

exposes

the

canal

(resorption). 4.

Through

the

blood

stream:

(ANACHORESIS)

Robinson and Boiling in 1941 5


Anachoresis – mechanism by which bacteria present in the systemic blood is attracted to the dental pulp following trauma or operative procedures that produced inflammation without causing exposure. 5.

Through extension of periapical infection from adjacent infected teeth. There is a considerable question as to whether

bacteria from a periapical area will enter an adjacent, non infected tooth. -

Large

periapical

radiolucencies

may

appear

to

encompass multiple teeth, yet be caused by pulpal necrosis of a single tooth. -

Only the causative tooth is treated endodontically and the entire radiolucency heals.

-

If a pulpitis trauma severely affects a tooth its neighbours does have an infected periapical area, the microorganisms may easily reach the newer problem by the interlacing blood and lymph systems either by: • Physical extension. • Pressure. Similar to anachoretic effect.

Microorganisms in the pulpal space: 1.

In 1919 Henrici and Hortzell identified bacteria in the pulpal space as: 65% streptococci 20% staphylococci Remaining corinybacteria and yeasts 6


2.

1952 – Grossman 77% gm +ve cocci 16% yeasts 5% gm –ve rods

3.

Another

study

demonstrated

75%

of

the

microorganisms isolated from the pulp were either streptococci or staphylococci. These were the studies lacking anaerobic culturing techniques. 4.

1974 – Kantz and Henry used anaerobic isolation and found obligate anaerobes in 27% of their samples. -

Actinomyces.

-

Bacteroids.

-

Eubacterium.

-

Fusobacterium.

-

Peptococcus

-

Veillonella.

Root canal space: Another study investigated the root canals of 40 teeth with pulp necrosis. 80% of sample yielded +ve gm for culture and majority were +ve for oblique anaerobes. 1976

Sundqvist

studied

bacterial

content

in

traumatized but intact teeth with necrotic pulps. Cultures anaerobically

were found

obtained the

both

presence

aerobically of

and

bacteroides

melaninogenicus. 7


Sundquist

later

focused

on

the

role

of

black-

pigmented bacteroides in root canal infection. B. intermedius

Common isolates

B. Endodontalis

“

Establishment of the Root canal Flora or survival: Root canal contains anaerobes mainly studies show that

the

observed

decrease

of

facultative

anaerobic

bacteria and concomitant increase of anaerobic bacteria with time may depend on the consumption of O 2 and development of low reduction oxidation potential in the environment of most bacteria, even oral streptococci. This low reduction-oxidation potential may cause faster growth of anaerobic bacteria. The nutritional environment is also important for the establishment of the microbial community in the root canal. A nutrient to support the growth of very fastidious organisms is present in the tissue fluid and valuable nutrients may also be available when connective tissue cells disintegrate. Bacteria can utilize products from other bacteria as nutrients. Carboxylic acids, ammonia and hydrogen sulfide are produced when amino acids and peptides are used as energy sources.

8


Another factor, which may be important to the bacterial ecology, is the production of bacteriocins. A

bacteriocin

is

a

protein,

produced

by

microorganisms, which has the capacity to inhibit the growth of limited number of other strains. Bacteria in the periradicular space: -

Bacteria then march down the root canal into the periradicular space.

1.

1951 Hedman evaluated periradicular areas of the teeth with necrotic pulp found 68% of these associated with bacteria.

2.

Baumgartner isolated 50 strains of bacteria from apical

5mm

of

root

canal

in

teeth

with

carious

exposures and periradicular – 68% strict anaerobes. 3.

Wayman – isolated 50 different species. 65.4% anaerobes. 27.5% facultative aerobes. 6.8% aerobes.

4.

A recent study by Fukushima and Associates in which

root

apex

and

periradicular

region

were

evaluated for the presence of bacteria showed: -

Eubacterium

-

Bacteroids

-

Peptococcus

-

Peptostreptococcus

Non pigmented (cause abcesses)

Types of bacteroides: -

Bile resistant.

-

Black pigmented. 9


-

Bile sensitive (saccharostatic).

-

Bile sensitive (osaccharolytic).

Culture Media: Definition: Media essential to grow the microorganisms from infected material to identify the cause of infection.

Indications: To demonstrate the bacteria: -

In case of persistent symptoms.

-

In medically compromised patients.

Patient undergoing immunosuppressive therapy High-risk patients for endocarditis. Patients with heart valve prosthesis. -

As a procedural check.

-

Antibiotic sensitivity test.

Classification of C.M. I] According to the consistency: -

Liquid media.

-

Semi-solid media.

-

Solid media.

II] According to the constituents: -

Simple media-nutrient broth (peptone, meat extract, NaCl and H 2 O).

-

Complex 2% agar

-

Synthetic (defined – peptone water medium).

-

Semi defined.

-

Special 10


Enriched media – blood agar, chocolate agar.

Enrichment media – tetrathionate broth.

Selective – desonycholate.

Indicator / differential.

Sugar – fermentable e.g. pentose, xylose.

Transport media – Stuart’s medium (transport delicar microta).

III] Aerobic media, anaerobic media: Anaerobic media: fat free minced cooked meat in broth with a layer of sterile Vaseline. e.g. Robertson’s cooked meat medium Special

-

properties

like

urease

production

and

miscellaneous tests for special properties. For identifying prepared media, a color code is

-

adopted. Aerobic:

Bower’s

thioglycolate

broth

for

endodontic

culturing. Common ingredients of culture media: -

Water. Agar. Casein hydrolysate. Accurate methods for sampling are mandatory in

order to isolate the bacteria from infected root canals and

to

study

their

characteristics

and

pathogenic

potential. The results of earlier studies are often misleading.

11


The drawback is that the initial cultivation of the sample in many studies was made in broth. This increases the likelihood of recovering only the fast

growing

bacteria

when

the

sample

originally

contained a mixture of bacterial species. Anaerobic bacterial techniques are important in endodontics since strict aerobic bacteria are not present in the root canals. Anaerobic

bacteria

cannot

tolerate

much

of

O2

exposure. The

toxic

effect

of

O2

depends

mostly

on

the

formation of H 2 O 2 , superoxide radicals and hydroxyl radicals, which are more toxic than oxygen. So the safest way is to protect anaerobic bacteria is to avoid exposure to O 2 during the lab work. Two methods have made this possible: 1.

The prereduced anaerobically sterilized technique by Hungate. -

Simplified and further developed by Moore.

-

Based on production of low reduction oxidation potential by gassing the media with O 2 free gas and affording

protection

from

oxygenation

during

sterilization and subsequent handling. 2.

Another method is to use anaerobic glove box. The atmosphere in this box is usually a mixture of N 2 -90%, CO 2 -10% and H 2 O-10%. 12


Brelbs developed a classical and skill very useful aerobic culture. The media supplies nutrients and conditions when incubated with O 2 present to suppose the growth of aerobes commonly found in endodontic infections. The ingredients of Bower’s thioglycolate broth and their uses. Constituent

Function Enrichment

Trypticase

Essential amino acid

Cystine

Carbohydrate

Dextrose

Vit. And growth factor

Yeast extract

Keeps solution isotonic.

Sodium chloride

Reducing agent – keeps

Na thioglycolate

O 2 level low

Resazurin

Dye that shows O 2

Agar

presence with red band Prevents O 2 diffusion.

Several

other

media

satisfactory

for

culturing

material from root canals are such as: -

Brain heart infusion broth with 0.1% agar.

-

Trypticase soy broth with 0.1% agar (TSA).

-

Glucose ascites broth.

13


Moller investigated the influence of water quality, various salts, organic materials, reducing agents, and different methods of obtaining oxygen free environment. Found

viability

medium

for

growth

(VMG)

and

Stuart’s transport medium both equally effective for most species of organisms. Muller developed a base culture medium containing veal, veal heart, peptone products in an agar gel, and certain supplements and found that: Better growth was obtained than with commercially dehydrated media. Studies

revealed

the

following

microorganism

growing in this medium. Streptococci. Lactobacilli. Anaerobes

Gram

positive

cocci

(peptococci

&

streptococci. Gram positive rods Eucabacteria Lactobacilli Corynebacteria. Taking the culture The dressing from the previous visit is removed and discarded.

14


A sterile absorbent point is inserted into the canal, with a wiping motion to cleanse the canal surface of any trace of medicament (which can inhibit bacterial growth). A fresh sterile absorbent point is now inserted into the apical foramen and is allowed to remain there for at least 1 minute to absorb the periapical exudates and microorganisms from the root canal as possible. The absorbent point is removed with sterilized cotton pliers held with the thumb, index and middle fingers, while the plug or cap of the test tube is removed with the little finger and palm of the same hand. The test tube is held in the other hand and is tilted slightly to prevent air contamination. The absorbent point is dropped into the medium, the lip of the tube is flamed, the plug or cap is replaced, and the culture tube is incubated properly. Anaerobic culturing: Culturing obligate anaerobes requires special equipment and media used in a temperature-controlled oxygen-free environment. Periradicular sample: Using an aseptic technique, insert the sterile needle of a Luer Lok syringe into the periradicular space (i.e. swelling), aspirate fluid, eject any air inside the syringe barrel immediately, insert the needle through the rubber septum stopper of an Anaport vial, and eject the fluid. The Anaport vial 10ml vial with a rubber septum stopper from which the gas has been removed, should be transported to any anaerobic culturing depot, usually located in a hospital or health care institution within 4 hours. 15


Root canal sample: inject a few drops of prereduced, anaerobically sterilized medium (chopped meat glucose broth was used by Sundquist) into the chamber, pump the medium into the root canal with a sterile endodontic file. Aspirate the fluid, eject any air insert the needle through the rubber stopper of an Anaport vial and eject the fluid. Transport within 4 hours.

Culture reversal: Grossman examined approximately 1000 cases and found that 2% of the cultures were –ve after 48 hours of incubation, but they turned positive when incubated for 10 days. It is advisable to allow more than 48 hours between taking the culture and filling the root canal, preferably 96 or more hours and it is recommended that the culture tube be reexamined immediately before obturating a canal to make sure that no evidence of growth is present. Recent Advances in Culturing 1. Immunofluorescence. Pantera et al demonstrated this for the detection of bacteriodes species (porphygromonas and prevotella) in the dental pulp. Advantages: -

Less time consuming.

-

Less expensive analytical method.

2. DNA finger printing: The most accurate method available for bacterial identification by Glickman. Uses a genetic code analysis application for bacterial identification. (DNA from one infection can 16


be compared with DNA from another and relationships can be confirmed). REFERENCES: 1. Endodontic Practice by Grossman. 2. Textbook of Endodontology by Samuel Seltzer. 3. Textbook of Microbiology by Anantnarayan. 4. Pathways of the Pulp by Stephen Cohen.

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Root canal flora and culture media/ dental implant courses by Indian dental academy  

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