SCIENTIFIC ARTICLES

E f f e c t of e n d o d o n t i c p r o c e d u r e s o n t h e p o p u l a t i o n of v i a b l e m i c r o o r g a n i s m s in the infected root canal E. S. A k p a t a , BCkD, FDS, L a q o s , N i g e r i a

To d e t e r m i n e q u a n t i t a t i v e l y t h e m i c r o f l o r a of t h e i n f e c t e d root c a n a l , 20 f r e s h l y e x t r a c t e d p u l p ] e s s h u m a n teeth w e r e t r e a t e d e n d o d o n t i c a l l y a n d t h e n c r u s h e d , c u l t u r e d , a n d e v a l u a t e d . In a d d i t i o n , a c o m p a r i s o n w a s m a d e w i t h root c a n a l c u l t u r e s o b t a i n e d from p a p e r p o i n t i n o c u l a t i o n s . A f t e r m e c h a n i c a l i n s t r u m e n t a t i o n a n d i r r i q a t i o n of t h e root c a n a l , t h e m e a n total v i a b l e c o u n t of m i c r o o r q a n i s m s o b t a i n e d from s o l i d a q a r p l a t e s w a s a p p r o x i m a t e l y 200 p e r tooth. After m e c h a n i c a l i n s t r u m e n t a tion, irriqation, a n d m e d i c a t i o n w i t h 35% c a m p h o r a t e d p a r a c h l o r o p h e n o l , t h e c o u n t w a s r e d u c e d to a p p r o x i m a t e l y 50 p e r tooth. N e q a t i v e r o o t c a n a l c u l t u r e s d i d not n e c e s s a r i l y i n d i c a t e sterility of t h e r o o t c a n a l .

Sterilization of the root canal is one of the aims of endodontic therapy. Conventionally, the bacteriologic status of the infected root canal is determined by cultures based on paper point inoculations. Although the reliability of such root canal cultures has been questioned 1-3 and no known endodontic drug is capable of sterilizing the root canal, 4 some endodontists insist on negative cultures before obturating the root canal. If negative cultures cannot be achieved, the extraction of the involved tooth has been advised.5 Since prognosis for repair is least when infection is present, 6 elimination of microorganisms from the root canal, or at least reduction of their number, is indicated. The suc-

cess of this total or partial reduction of microorganisms is best evaluated by quantitative methods. The purpose of this study was to estimate the total viable count of microorganisms in the infected root canal after biomechanical preparation and antiseptic dressing, and to evaluate the reliability of conventional root canal culturing techniques.

Methods and M a t e r i a l s Twenty pulpless single-rooted human teeth that were clinically tender to percussion and had either a carious or traumatic exposure of the pulp were selected for study. After extraction, the teeth were made clinically cariesfree by means of a spoon ex-

cavator and a low speed no. 3 or no. 5 round bur. To ensure that the zone of bacterial invasion was totally excavated, some apparently normal dentin adjacent to the zone was removed. It was necessary to cut the anatomical crown off four of the teeth while making the teeth cariesfree. After the teeth had been washed under running tap water, they were dried with sterile gauze. Each tooth was held in a sterile vise and an access cavity was prepared in the conventional manner. The outer surface of each tooth, including its access cavity, was then sterilized by an ultraviolet ray lamp according to a technique described by Akpata. 7 After excavation and sterilization

369

JOURNAL OF ENDODONTICS 1 VOL 2, NO 12, DECEMBER 1976

each root canal was instrumented through the apex and enlarged four sizes greater than the size of the instrument that began to bind in the unprepared canal. The operator ensured that the tip of each instrument used to prepare the canal was just visible at the root apex. The root canal was irrigated with 2 ml sterile normal saline after the use of each instrument and the final irrigation was with 4 ml normal saline. In the initial phase of this experiment, microorganisms obtained from infected root canals after instrumentation and irrigation were tested by the disk diffusion method for sensitivity to 35% camphorated parachlorophenol. After instrumentation and irrigation with normal saline, an absorbent paper point saturated with 35% camphorated parachlorophenol was placed in the root canals of 10 of the 20 teeth. Then a cotton pellet saturated with the antiseptic was packed into the pulp chamber and the access cavity was sealed with fastsetting zinc oxide-eugenol cement. Each of the ten teeth was wrapped in moist sterile gauze and cotton wool. The wrapped tooth was stored in a petri dish and incubated at 37 C for two 'to three days. After incubation, the canals were reinstrumented with the last size instrument used previously and irrigated with 10 ml sterile normal saline. In each root canal of the 20 teeth, cultures were taken immediately after pulp extirpation (A1, A2, A3). Three other cultures were taken after instrumentation and irrigation (B1, B2, B3). In the ten teeth with ~he camphorated parachlorophenol dressings, three additional cultures were taken after the final irrigation with normal saline (C1, C2, C 3 ) . In each instance the paper point was allowed to make contact with each moist root canal for one minute and was inoculated in 10 ml of 0.5% glucose broth (pH of 7.4). As a control, another paper point from 370

the same pack was used to inoculate the sterile glucose broth. The cultures were incubated at 37 C and examined for turbidity after 48 hours and after 65 hours. To ascertain whether the presence of microorganisms in the incubated glucose broth is always indicated by turbidity, blood agar plates were streaked with the 48-hour root canal cultures. After incubation at 37 C for 65 hours, the plates were examined for growth of microorganisms. After the initial phase of this study, the outer surfaces of the treated teeth were again exposed to the ultraviolet ray light to sterilize them. A blood agar plate was then streaked with each of the exposed teeth. If there was growth of microorganisms on the agar plate after incubation at 37 C for 65 hours, the data obtained from that experimental tooth were discarded. After crushing each of the 20 teeth in a diamond mortar, 20 ml of nutrient broth was added to the tooth particles. As recommended by Akpata, r this tooth suspension was sonicated with the tip of a Cavitron ultrasonic scaler at 25,000 cycles per second and an amplitude of 0.0025 m m for two minutes. Eight blood agar plates, four tomato juice agar plates, and four Sabouraud's agar plates were inoculated with 1-ml aliquots of the sonicared tooth mixture. F o u r of the blood agar plates were incubated anaerobically and the remaining 12 agar plates were incubated aerobically at 37 C for 65 hours. Blood agar, tomato juice agar, and Sabouraud's agar plates inoculated with 1 ml blank nutrient broth were used as controls. Both the pour plate and surface plate methods were followed; half of the number of each solid media was used in each of the methods. After incubation, the number of colonies growing in the agar plates was counted and the total viable counts of microorganisms in the root canals were calculated. Two autoclaved teeth were analyzed

similarly to assess the level of contamination during plating and the manipulation of the tooth suspension. The predominant contaminant on the control plates was Bacillus subtilis. Therefore all agar plates contaminated with this organism were discarded throughout the experiment. A blood agar plate was inoculated with the residual tooth particles (pour plate) and incubated at 37 C for 65 hours. Results

After instrumentation of the root canal and irrigation with sterile normal saline, the mean total viable count of microorganisms in the infected root canal obtained in the solid agar media was reduced to 200 microorganisms per tooth. This is statistically significant (Wilcoxon paired analysis, P = 0 . 0 3 4 ) when compared with the total viable count for the infected dental pulp which is 106 per tooth3 Nevertheless, none of the teeth were sterile after instrumentation and irrigation (Table 1). When the root canals were dressed with 35% camphorated parachlorophenol, the mean total viable count obtained from the blood agar plates was reduced to approximately 50 or less per tooth, which is statistically significant (Wilcoxon paired analysis, P = 0 . 0 0 6 ) . The counts from the tomato juice agar and Sabouraud's agar were even fewer. Two of the ten teeth with the antiseptic dressing were sterile (Table 2). The incidence of negative cultures was higher in the root canals containing fewer viable microorganisms (Tables 1 and 2). A negative culture was obtained even when the total viable count of microorganisms in the root canal was 500 per tooth (Table 1). Three successive negative cultures (C1, C2, C3) were obtained from some of the root canals containing up to approximately 50 viable organisms per tooth (Table 2).

JOURNAL OF ENDODONTICS I VOL 2, N O 12, DECEMBER 1976

T a b l e 1 - Total v i a b l e counts a n d root c a n a l cultures after instrumentation a n d l n t g a t i o n w i t h n o r m a l aallne.

Counts per tooth Blood a g a r Aerobic Tooth* 1 2 3 4 5 6

7 8 9

10 Mean Standard UO!lg!AOp

Anaerobic

Pour plate

Surface plate

80 990 790 0 80 80 50 60 0 0 213

60 900 600 0 120 140 40 40 40 0 194

361.0

272.3

Pour plate 220 1,410 210 0 20 80 40 50 0 0 203 432.9

T o m a t o juice a g a r S a b o u r a u d ' s a g a r

Surface plate

Pour plate

Surface plate

Pour plate

80 1,400 400 0

10 870 760 30

40 800 500 20

20 730 600 10

Surface plate

Root canal culturest B1

B2

B3

0 600 740 10

+ -

+ + -

+ + + +

30

0

0

0

0

+

--

-

100 30 40 0 0 208

20 0 60 20 0 177

10 20 70 0 0 146

70 60 20 0 70 158

40 100 20 20 120 165

+ +

+

+

--

--

--

+ -

-+

+ +

373.6

353.6

273.1

267.1

250.9

*1, m a x i l l a r y c e n t r a l incisor; 2, m a x i l l a r y canine; all o t h e r s are p r e m o l a r s . r A f t e r i n c u b a t i o n period o f 65 h o u r s .

Table 2"

Total

viable

counts a n d root c a n a l cultures after instrumentation, c a m p h o r a t e d p a r a c h l o r o p h e n o l dressinq.

irrigation,

and

C o u n t s per t o o t h Blood a g a r Aerobic

Anaerobic

Tooth* 1

10

0

0

10

10

40

0

2

0 0 0 140 50 0 240 90 0 53

40 0 0 40 60 0 220 60 10 43

30 0 0 0 0 0 30 160 10 23

0 0 0 60 0 20 20 1O0 10 22

0 0 0 0 0 10 0 0 0 2

0 0 0 0 0 40 0 0 0 8

0 0 0 0 20 20 0 0 10 5

20.1

10.7

3 4 5 6 7 8 9 10 Mean Standard deviation

99.5

Surface plate

T o m a t o juice a g a r S a b o u r a u d ' s a g a r

Pour plate

60.5

Pour plate

Surface plate

Pour plate

Surface plate

39.6

28.5

6.2

Pour plate

Surface plate

Root canal culturest CI

C2

C3

10

-

-

-

0 0 0 0 40 20 0 0 10 8

+ -+ -

--

+ --

1.8

*1 and 2, m a x i l l a r y central incisors; all o t h e r s a r e p r e m o l a r s . r a f t e r i n c u b a t i o n period o f 65 h o u r s .

371

JOURNAL

All the 60 root canal cultures (A1, A2, A3) taken soon after pulp extirpation were positive after incubation at 37 C for 65 hours. After instrumentation and irrigation, 38.4% of the cultures (B1, B2, B3) were positive (Table 3). However, after antiseptic dressing, only 10% of the root canal cultures (CI, C2, C3) were positive (Table 2). In only one case did all the three successive postinstrumentation and irrigation cultures (B1, B2, B3) yield three successive negative results (Table 1). Table 3 shows that the first culture yielded positive results more frequently ( 4 5 % ) than the second or third cultures ( 3 5 % ) . I n two cases, the 48-hour culture resulted in microbial growth on the plated blood agar, even though the inoculated glucose broth was not turbid after incubation at 37 C for 65 hours (Table 3). However, these two glucose broth cultures became turbid after incubation at 37 C for one week. When the sensitivity of intracanal microorganisms to 35% camphorated parachlorophenol was tested by the disk-diffusion method, there was a zone of inhibition of at least 10 mm. No growth was obtained in blood agar inoculated with the tooth particles where the corresponding total viable count of microorganisms was zero. However, in cases where viable microorganisms were obtained from the root canals, the tooth particles either yielded no growth in the inoculated blood agar or there was only one colony in most cases. No glucose broth in the control group turned turbid after incubation at 37 C for 65 hours. Discussion

Previous methods of evaluating the effect of endodontic procedures on the microbiota of infected root canals have been qualitative. These have included serial tube dilutions,s agar 372

Table

3 9 T u r b i d i t y of r o o t c a n a l

OF ENDODONT|CS

cultures

on solid blood aqar

[ VOL

after incubation

2, N O

for 4 8 a n d

plated with 48-hour qlucose

1.2, D E C E M B E R

~5 h o u r s a n d

1576

qrowth

broth cultures.

Turbidity* B1 Tooth 1

48 hr

B2

65 hr

48 hr

--

+

--

B3

65 hr

48 hr

+

65 hr

B1

B2

--

+

--

+

+

+

+ +++

-+

-+

+ + +

2 3

. +++

4

.

5 6 7

+++ ++ ++

8

.

9

+++

+++

--

--

--

10

-

-

-

+

-

11 12 13 14

. . . .

. . . .

15

--

+ + +

16 17

-++ . .

+ +++ +++ . .

18 19

20

. . . +++ +++ .

.

.

. +++

.

+++ ++ +++

+ + .

.

. -

. . +++

.

.

.

. . . .

.

-

--

+

--

--

. +

+++

+ +

+

+

+++

+

--

+

-

+

+

.

. .

. .

+ ++

. .

. + +

+ +

. .

. .

++'+

++ +++ +++ . . . . . . . . .

B3

.

.

. .

Growth on plated blood agar

. .

. .

. .

.

.

. .

+

--

--

+

+

--

-.

--

+ +

+ +

--

+

--

--

. .

. .

. .

-

. .

Note: +, slightly turbid; + +, moderately turbid; + + +, very turbid. plates, 9 and the number of visits to obtain negative cultures. 1~ The technique used in this study is quantitative and offers a new approach to the problem. This quantitative technique has the advantage of taking into account those living microorganisms in the root canals, accessory canals, and the lateral canals as well as in the surrounding dentin walls. Germicides used in endodontics may inhibit microbial growth when they are transferred into the culture medium by the paper point, l~,le The transference of the antiseptic was kept to a m i n i m u m in the current study by diluting the residual antiseptic in the root canal with the irrigant (10 ml sterile normal saline). I n addition, the small amount of the diluted residual antiseptic that was carried by the paper points was further diluted by the 10 mI glucose broth culture medium. Similarly, when estimating the

total viable counts, the diluted residual antiseptic in the root canal was further diluted by the 20 ml nutrient broth added to the crushed tooth particles. In addition, the 1-ml aliquots of the sonicated tooth mixtures were diluted by the volume of the agar media in the pour plates. The close correlation between the turbidity of the glucose broth after incubation for 65 hours and the growth on the blood agar plated with 48-hour glucose broth culture (Table 3) substantiated the fact that the amount of antiseptic carried over to the culture medium was insignificant. Vander Wall, Dowson, and Shipman 9 showed that 35% camphorated parachlorophenol is bactericidal only when it is in contact with the bacteria and concluded that the drug should be applied on moist paper points in the canal to be effective. Even if their recommended technique is followed,

JOURNAL OF ENDODONTICS ] VOL 2, NO 12, DECEMBER 1976

the antiseptic may fail to reach the microorganisms lodged in the dentinal tubules, accessory canals, and lateral canals. 13 This may explain the presence of microorganisms in most of the teeth after treatment with the antiseptic in this study. Instrumentation and irrigation alone will not sterilize the root canal, but these procedures may significantly reduce the microbial population of the infected root canal such that negative cultures ensue. Despite the presence of some microorganisms after instrumentation and irrigation, one of the ten teeth yielded three successive negative cultures (Table 1 ). The reliability of root canal cultures has been questioned by several endodontists. 1,2,~4 This investigation shows clearly that a negative culture does not necessarily mean that the root canal together with the surrounding dentin wall is sterile. Rather, a negative culture may indicate either the presence of few or no microorganisms. It is only in this light that the culture technique in endodontics is justifiable. Conflicting reports exist concerning the relationship of the results of root canal cultures and the prognosis of endodontics.15-1s In the current study, after instrumentation, irrigation, and antiseptic dressing, the viable microorganisms obtained from the infected root canal were either reduced to a very low level or eliminated (Table 2). The presence of a few viable microorganisms in the root canal is not accurately monitored by conventional root canal culture and the residual microorganisms in the root canal tend to die out in time after root canal filling.4 Therefore, persisting residual living microorganisms following instrumentation, irrigation, and medication may be nonvirulent or too few in number to cause infection that would influence the prognosis of root canal therapy.

Summary Twenty single-rooted human pulpless teeth were studied to determine quantitatively the microflora of the infected root canal. After extraction and excavation, cultures were obtained of the root canal before mechanical instrumentation and irrigation, and after intracanal medication with 35% camphorated parachlorophenol. Then the teeth were crushed, plated, and incubated both anaerobically and aerobically. The number of microorganisms within a root canal was significantly reduced after mechanical instrumentation and irrigation. With the use of 35% camphorated parachlorophenol as an intracanal medicament, the number of microorganisms was reduced even further. Negative root canal cultures did not necessarily indicate sterility of the root canal. After root canal filling, the living residual microorganisms may be too few to cause infection or influence the prognosis of endodontic therapy. The author thanks the department of microbiology and the Division of Dentistry, University of Lagos, for their cooperation, Mr. S. F. Lawal for his technical assistance, and Dr. J. R. Ana for the loan of the diamond mortar. Dr. Akpata is senior lecturer in the department of restorative dentistry, College of Medicine, University of Lagos. Requests for reprints should be directed to Dr. Akpata at the Department of Restorative Dentistry, University of Lagos, P.M.B. 12003, Surulere, Lagos, Nigeria. References

1. Ingle, J.I., and Zeldow, B.J. Evaluation of mechanical instrumentation and the negative culture in endodontic therapy. JADA 57:471 Oct 1958. 2. Seltzer, S., and Bender, I.B. Cognitive dissonance in endodontics. Oral Surg 20:505 Oct 1965. 3. Hobson, P. The bacteriological problems of root-canal therapy. Dent

Pract (Bristol) 16:43 Oct 1965. 4. Matsumiya, S., and Kitamura, M. Histopathological and histobacteriological studies of the relation between the condition of sterilization of the interior of the root canal and the healing process of periapical tissues in experimentally infected root canal treatment. Bull Tokyo Dent Coll 1:1 Oct 1960. 5. Filgueiras, J. Importance of bacteriological tests in root-canal therapy. JADA 29:2211 Dec 1942. 6. Torneck, C.D. Reaction of rat connective tissue to polyethylene tube implants. II. Oral Surg 24:674 Nov 1967. 7. Akpata, E.S. Total viable count of microorganisms in the infected dental pulp. J Dent Res 53:1330 Nov-Dec 1974. 8. Harrison, J.W., and Madonia, J.V. Antimicrobial effectiveness of parachlorophenol. Oral Surg 30:267 Aug 1970. 9. Vander Wall, G.L.; Dowson, J.; and Shipman, C., Jr. Antibacterial efficacy and cytotoxicity of three endodontic drags. Oral Surg 33:230 Feb 1972. 10. Atkinson, A.M., and Hampson, E.L. Sterilization of root canals. An evaluation of mixtures containing aminoacridine, cetrimide, and chlorhexidine. Br Dent J 117-526 Dec 15, 1964. 11. Grossman, L.I. Endodontic Practice, ed 6. Philadelphia, Lea & Febiger, 1965, p 324. 12. Seltzer, S. Endodontology; biologic considerations in endodontic procedures, New York, McGraw-Hill Inc., 1971, p 296. 13. Rubbo, S.D.; Reich, J.; and Dixson, S. Use of a combination of neomycin, bacitracin and polymyxin in endodontia. Oral Surg 11:878 Aug 1958. 14. Maisto, O.A. Present status of bacteriologic control in endodontics. Dent Abs 1:468 Aug 1956. 15. Oliet, S. Evaluation of culturing in endodontic therapy. A prelim,inary clinical report. Oral Surg 15:727 June 1962. 16. Engstrom, B.; Hard Af Segerstad, L.; Ramstrom, G.; and Frostell, G. Correlation of positive cultures with the prognosis for root canal treatment. Odontol Revy 15:257, 1964. 17. Bender, I.B.; Seltzer, S.; and Turkenkopf, S. To culture or not ~o culture? Oral Surg I8:527 Oct 1964. 18. Seltzer, S.; Turkenkopf, S.; Vito, A.; Green, D.; and Bender, I.B.H.istologic evaluation of periapical repair following positive and negative root canal cultures. Oral Surg 17:507 April 1964. 373