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The bactericidal efficiency of sodium hypochlorite as an endodontic irrigant
The bactericidal efficiency of sodium hypochlorite as an endodontic irrigant Ming Shih, B.M.D., M.S., P. James Marshall, Samuel Rosen, Ph.D.,** Columbus, Ohio OHIO
STATE
UNIVERSITY
COLLEGE
OF
D.M.D.,
M.S.,” and
DENTISTRY
I
ngle and Zeldowl and Nicholls2 suggested that the reduction in bacterial population due to the cleansing of a contaminated root canal was to someextent associated with the antiseptic effect of the fluid used for irrigation. Sodium hypochlorite is one of the most popularly used root canal irrigants. It was proved to be a powerful germicide, effective against a wide spectrum of microorganisms,3 and a solvent for necrotic tissue.4-oIn the absenceof organic matter, all vegetative bacteria were killed when exposed to a solution containing 0.15 to 0.25 ppm available chlorine for 30 seconds at pH 9 and 25O C.? The spore-forming organisms were about 10 to 1,000 times more resistant to chlorine than vegetative bacterial forms. The pulp tissue solvent properties were reported by Grossman.8,Q However, the relative efficiency of sodium hypochlorite in sterilizing and cleansing root canals is not known. The present study was designed to investigate the bactericidal efficiency of sodium hypochlorite as a root canal irrigant. METHODS AND MATERIALS Clorox t was used in this study as a source of sodium hypochlorite. Streptococcus faecalis and Staphylococcus aureus were chosen as the test organisms. The two organisms were tested separately in all the experiment,s. This article is abstracted from a thesis submitted by the senior author in partial fulfillment of the requirements for the degree of Master of Science in the Graduate School, Ohio State University. *Professor and Head, Department of Endodontics. **Professor, College of Dentistry and Academic Faculty of Microbial and Cellular Biology. tClorox, a product of The Clorox Company, Oakland, Calif., contains: Sodium hypochlorite 5.25% 0.207% Sodium carbonate 4.07& Sodium chloride Free sodium hydroxide 0.005-0.015~
613
614
Shih, Marshall,
and Rosen.
Oral April,
Surg. 1970
Periodically throughout the study Gram stains and streaked blood agar plates were made to ensure the viability of the organism, the continuation of the original pure cultures, and the absence of contamination. Two experiments were undertaken. The first tested the germicidal efficiency of Clorox by serial tube dilution studies, and the second (using extracted human teeth) tested the germicidal efficiency of Clorox as a root canal irrigant. Experiment
1
A series of tenfold serial dilutions of Clorox were prepared with sterile distilled water. Five milliliters of each solution (full-strength Clorox, Clorox diluted with sterile distilled water 1 :lO, 1 :lOO, 1 :l,OOO, 1 :lO,OOO, or sterile distilled water as a control) was dispensed aseptically into two sets of six tubes each. Five milliliters each of a 24-hour culture of Str. faecalis and S. aureus was centrifuged. After the supernatant was discarded, each sediment was resuspended in 5 ml. of Bacto-as&tic fluid. * Next 0.1 ml. of Str. faecalis suspension was added to one set of six tubes and 0.1 ml. of S. aureus suspension was added to the other set of six tubes. All tubes were agitated during the experiment to ensure thorough mixing of the hypochlorite solution and bacteria. Samples were taken from these inoculated solutions at 30 seconds, 1, 2, 3, 4, 5, 10, and 20 minutes, and 24 hours. They were placed in tubes containing 15 ml. trypticase soy broth with 0.1 per cent agart and incubated at 3’7OC. for 72 hours under aerobic conditions. Cloudiness of the culture medium at the end of the incubation period was used as the criterion of the presence of bacterial growth. Experiment
2
The root canals of 120 freshly extracted single-rooted human teeth were prepared by standard endodontic techniques. The occlusal openings were accentuated. Water was used during preparation to remove any dentine shavings from the canals. The root canals were reamed and filed through the apex, and the canals were enlarged to the size of a No. 80 file. The enlarged apices were sealed with an epoxy resin.$ The prepared teeth were then placed in polyethylene bags containing water, double sealed, and sterilized by exposure to cobalt-60 radiation at 1.27 to 1.61 x lo5 r per hour for 48 hours. After sterilization all teeth were seated in presterilized mounting boards with crowns exposed. Sixty sterilized teeth were then inoculated with Str. faecalG and sixty were inoculated with S. aureus. The inoculums were introduced into the apical thirds of the root canals with the aid of tuberculin syringes and straight platinum wires. These 120 teeth, still mounted in boards, were placed in a sterile container and incubated for 48 hours at 37’ C. They were then divided into three groups of forty teeth for treatment, Twenty teeth were inoculated with Str. faecazis and twenty teeth were inoculated with S. aureus. Each group was treated with procedures described below using varying concentrations of Clorox. “Difco Laboratories, Detroit, Mich. tBalt.imore Biological Laboratories, Baltimore, $Carter’s General Purpose Epoxy, Carter’s Ink
Md. Co., Cambridge,
Mass.
Volume Number
29 4
Bactericidal
eficiency
of sodium
hypochlorite
615
Two pilot studies suggested that 5 ml. of full-strength Clorox or 5 ml. oi Clorox diluted 1 :lO with water should be used to irrigate each root canal in this portion of the study. Group I teeth were irrigated with full-strength Clorox, Group II with Clorox diluted l:lO, and Group III wit,h sterile distilled water as a control. Preirrigation culture samples were taken from all inoculated root canals in the group, using a wet-point culturing techniquelO modified so that 15 ml. of trypticase soy broth with 0.1 per cent agar was used. The root canals were then irrigated with the following test solutions, using a technique that simulated clinical procedures: 5 ml. of full-strength Clorox, 5 ml. of Clorox diluted 1 :lO, or 5 ml. of sterile distilled water. Each solution was introduced slowly into the root canal by means of a 5 ml. disposable syringe and a 11/b inch 25 gauge needle. The irrigating needle was inserted as far as possible, without binding, into the canal. The solution was injected into the canal at l-minute intervals and the full amount was dispensed within 3 minutes. A sterile No. 15 reamel was used during this period to stir the solution in the canal. The overflow of the solution was caught and removed with a suction device attached to a dental unit. After irrigation, each canal was washed with 2 ml. of sterile distilled water. A postirrigation culture sample was taken from each canal. The canals were then filled with culture medium and reincubated at 37’ C. and 100 per cent humidity for 7 days. Two additional culture samples were taken from each canal-one 2 days and one 7 days following the treatment. The root canals were kept moist during this period of incubation by the addition of culturcL medium at 2-day intervals. All culture tubes were examined a.fter they had been incubated aerobically at 37’ C. for 72 hours. RESULTS Experiment
1
The tube dilution tests (Table I) showed that Str. faeculis and S. aureus suspended in 0.1 ml. of Bacto-a&tic fluid were completely killed in 30 seconds when they were added to 5 ml. of full-strength Clorox, and Clorox diluted l:lO, 1 :lOO, or l:l,OOO. A Clorox-water dilution of l:lO,OOO, however, did not inhibit the bacteria1 growth, even after contacting the organism for 24 hours. Experiment
2
The results of the extracted-tooth experiment (Table II) showed that the preirrigation culture samples were positive in 119 out of 120 teeth. All culture samples taken from the forty teeth inoculated with either Str. faecalis or S. aureus after irrigation with full-strength Clorox showed no growth in culture tubes immediately after the irrigation. When teeth were irrigated with Clorox diluted 1 :lO, seven of the twenty teeth inoculated with Str. faecalis and two of the twenty teeth inoculated with 8. aureus showed growth when cultured immediately after the irrigation. Two days postirrigation, the group treated with full-strength Clorox gave positive cultures in eight of twenty teeth inoculated with S. faecalis and five of
616
Shih, Marshall,
Table
Oral April,
and Rosen
Surg. 1970
I. Tube dilution studies of bacterial inhibition by Clorox at various times Streptococcus Time
faecalis
of exposzlre
St,aphylococoas Time
(nzinzctes)
0.5
1
2
3
4
5
10
a0
1,440 (24 hrs.)
diluted
-
-
-
_
-
-
-
_
Clorox diluted 1:lOO
-
-
-
-
-
-
-
-
Clorox diluted l:l,OOO
-
-------
_
Clorox diluted 1: 10,000
+
+++++++
Water
+
t
azcrezcs
of exposure
0.5
1
2
3
-
-
-
-
-
-
-
-
+
+
++++++t
t
+
+
4
(minutes) 1,440 ($4 hrs.)
5
10 20
_
-
-
-
-
_
-
-
-
-
Clorox full strength (Jorox 1:lO
(control)
+
+
+
+
+
+
+
t +
+
+
+
+
t
- = No growth. t = Growth.
II. Incidence of positive cultures following clinical treatment Table
irrigations
that simulate
Concentration Water Streptococcus
St’aphylococcus
*Numerator: Denominatk-:
faecalis Preirrigation Immediately postirrigation 2 days postirrigation 7 days postirrigation
20/20* o/20 s/20 16/20
“i:z 13/20 15/20
20/20 20/20 19/20 19/2,0
Preirrigation Immediately postirrigation 2 days postirrigation 7 days postirrigation
2’0/20 o/20 5/20 11/20
20/20 2/20 14/20 16,‘20
19/20 18/20 18/20 19/20
aweus
Number Total
of positive cultures. number of teeth used.
twenty teeth inoculated with S. aureus. In the group treated with Clorox diluted 1 :lO, thirteen of twenty teeth inoculated with Str. faecalis and fourteen of twenty teeth inoculated with S. aureus produced culture reversals. Seven days postirrigation the group treated with full-strength Clorox showed positive cultures in sixteen of twenty teeth inoculated with Str. faecalis and eleven of twenty teeth inoculated with S. aureus. In the group treated with Clorox l:lO, fifteen of the twenty teeth inoculated with Str. faecalis and sixteen of the twenty teeth inoculated with S. aureus sho,wedresidual “infection.” Inoculated root canals washed mechanically only with sterile distilled water
Volume 2f, P\iumber 4
BactericidaZ
eficiency
of sodium
hypochlo~ite
617
showed growth in eighteen or nineteen of twenty teeth for either microorganisnl in all test periods. DISCUSSION
Tube dilution studies showed that Cloros had germicidal activity against faecalis and S. aureus, even at a dilution of l:l,OOO. The continuation studies involving a simulated clinical procedure on extracted human teeth showed that only full-strength Clorox had an apparent immediate sterilizing effect in root canals of extracted human teeth inoculated with Str. faecalis or S. aweus. In the tube dilution studies there was a greater possibility of carry-over of Clorox than in the extracted-tooth studies, and thus perhaps a greater sterilizing effect was achieved for the tube dilution studies. Limited contact of the germicidal solution with the root canal contents ma! also contribute to the differences between these studies. Although 5 ml. of test solution was used to irrigate each canal, only a small portion of the solution was in actual contact with the root canal contents per unit time because of the limited size and the irregular shape of a root canal. Irregularities in the main canal or untreated lateral canals may contain organic tissue not mechanically removed. This tissue could readily bind the chlorine and neutralize the germicidal effect of small amounts of contacting Clorox. On the other hand, the organic contents of the test solution in the tube study (0.1 ml. of Bacto-ascitic fluid) may have been insufficient to neutralize the effect of the Clorox. The microorganisms supported by the untreated organic material could remain viable and thus serve as a residual source of infection. High reversal rates 2 days following the treatment, 40 per cent (eight out of twenty) in the Xtr. faecnlis group and 25 per cent (five out of t.wentyj in the 8. aweus group, suggests that the culturing technique used here and clinically in endodontic treatment rnav not be sensitive enough to detect, small amounts of residual bacteria left in a root canal. The paper point used for sampling is not in direct contact with the accessory canal contents. If the cnltl~ring technique uses a “dry” paper point, there is even less chance of obtaining an inoculum. Thus, a negative posttreatment report may suggest that only the main root canal is sterile or that it contains a highly reduced bacterial population. II substrate is available or becomes available, the organisms may bc re-establish& in high numbers in the main root canal during the interim. Seven-day reversal rates in all groups were found to be higher than 2-d;&> reversal rates. An increased time period may have increased the chance for microorganisms to overcome the shock of treatment and to come into contact with additional nutrients. The reversal rate following one negative culture, 40 per cent for Str. faecdis and 25 per cent for S. azcreus, was much higher than those reported by Myers’l (25.9 per cent) or Bender and Seltze? (16.6 per cent). Nutrients were added to canals after irrigation in this study and not in the others. This could have: helped residual organisms to proliferate. Xo medicament was used betwt*tn treatments to suppress bacterial growth. A wet-point culturing technique (not Str.
618
Shih,
Marshall,
and Rosen
Oral April,
Surg. 1970
used by Myers or by Bender and Seltzer) was a more sensitive technique for obtaining inoculums. The carry-over effect of sodium hypochlorite was minimized in our study by washing the treated canals with 2 ml. sterile distilled water, but there could have been a carry-over of medicament in the studies of MyersI and Bender and Seltzer.lz All these factors could have helped to increase our reversal rate. The results from the control group agree with findings of Ingle and Zedlowl and Nicholls2 that mechanical flushing of the root canal with sterile distilled water alone does not eliminate root canal infection. The results of this study suggest that sodium hypochlorite irrigating solutions at a concentration of 5.25 per cent (full-strength Clorox) may be required to reduce effectively the microbial population of an infected root canal. The reversal rates also suggest that many canals with negative cultures have residual bacteria at the time of root canal filling. A negative culture does not justify the assumption that the canal is sterile at the time of insertion of a root canal filling. The use of an intracanal medicament would seem to be desirable to aid in establishing the sterility of the canal. S. mreus has been used in many standard methods to test the germicidal power of various antiseptics. This microorganism is the most common cause of suppuration and is a highly resistant non-spore-forming infectious agent. It was used in this study because of its frequent presence in infected root canals. Str. faecalk was used as the other test organism because of its prevalence in infected root canals and because of its high resistance to routine medication in root canal therapy. Myersll reported that it comprised 20.8 per cent of all the organisms found in root canals at the time of filling, and EngstromX3 found it the most difficult to remove from an infected root canal. The effectiveness of irrigation with Clorox, as tested here on two organisms individually, may be different when the organisms are tested together. Bacterial growth potential and resistance to irrigation may be quite different in a mixed culture in vivo than in separate cultures in vitro. Laboratory tests of any kind are only the first steps in a study of the effectiveness of antiseptics. The ultimate test of the effectiveness of an antiseptic is the demonstration of its germicidal efficiency under conditions of practical application. Further studies should include the effect of sodium hypochlorite on extracted human teeth inoculated with mixed cultures containing a variety of oral microorganisms. This should be followed by a clinical study for a final assessment of the bactericidal efficiency of sodium hypochlorite as a root canal irrigant. SUMMARY
A laboratory study was undertaken to appraise the bactericidal efficiency of sodium hypochlorite used as a root canal irrigant. Clorox (5.25 per cent sodium hypochlorite, pH 11 to 11.5) was the source of sodium hypochlorite. A laboratory tube dilution study showed sodium hypochlorite to be a powerful germicide. Str. faecdis and S. aureus suspended in 0.1 ml. of Bacto-as&tic
Volume 29 Number 4
Bactericidal
eficiency
of sodium
hypochlorite
619
fluid were completely killed in 30 seconds when added to 5 ml. of Clorox diluted 1 :l,OOO (0.00525 per cent sodium hypochlorite) . Experimental studies with a simulated clinical irrigating procedure showed that only full-strength Clorox had a 100 per cent sterilizing effect in root canals inoculated with Str. faecalis and 8. aweus. Two-day and ‘I-day postirrigation culture results revealed high reversal rates in the group irrigated with fullstrength Clorox or in the group irrigated with CIorox diluted 1 :lO. The ?-day reversal rates were greater than the 2-day rates. CONCLUSION
1. The bactericidal efficiency of sodium hypochlorite in tube dilution studies is not related to the effect of sodium hypochlorite used in extracted huma,n teeth. 2. It is necessary to use Clorox at full strength (5.25 per cent sodium hypochlorite) to obtain an immediate sterilizing effect in root canals of extracted human teeth inoculated with Str. faecalis and 8. aureus. Irrigation with fullstrength Clorox does not ensure the lasting sterility of an inoculated root canal. 3. The holding action of an intracanal medicament between appointments is necessary to control the root canal microbial population. 4. A negative culture report after treatment indicates that the bacterial population in the root canal may be highly reduced, not that the canal is sterile. 5. Mechanical flushing with sterile distilled water is not effective in removing all bacteria from root canals. 6. Further studies should consider the effect of sodium hypochlorite on extracted human teeth inoculated with mixed cultures containing various oral microorganisms. 7. A clinical study should be carried out for a final assessment of the bactericidal efficiency of sodium hypochlorite. REFERENCES
1. Ingle,
J. I., and Zedlow, B. J.: An Evaluation of Mechanical Instrumentation and the Negative Culture in Endodontic Therapy, J. Amer. Dent. Ass. 57: 471-474, 1958. 2. Nicholls, E.: The Efficiency of Cleansing of the Root Canal,. Brit. Dent. J. 112: IBS170, 1962. 3. Less, M. A.: Hypochlorite as Sanitizers, Soap t Sanit. Chem. 25: 119-125, 139, 1949. 4. Dakip, H. D.: The Antiseptic Action of Hypochlorites, Brit. Med. J. 2: 889-910, 191.5. 5. Austin, J. H., and Taylor, H. D.: Behavior of Hypochlorite and of Chloramine-T Solutions in Contact With Necrotic and Normal Tissues In Vivo, J. Exp. Med. 27: 627-633, 1918. 6. Taylor, H. D., and Austin, J. II.: The Solvent Action of Antiseptics on Necrotic Tissue, J. Exp. Med. 27: 155-164, 1918. 7. Snow, W. B.: Recommended Chlorine Residual for Military Water Supplies, J.A.W.\V. A. 48: 1510-1514. 1956. 8. Grossman, L. I.; and Meiman, B. W.: Solution of Pulp Tissue by Chemical Agents, J. Amer. Dent. Ass. 28: 223-225, 1941. 9. Grossman, L. 1.: Irrigation of Root Canals, J. Amer. Dent. Ass. 30: 1915-1917, 1943. IO. Marshall, F. J., and Savoie, F. L.: Efficiency of Endodontic Culturing Procedure Using Wet and Dry Paper Points, ORAL BURG. 23: 806~810,1967. 11. Myers, J. W.: The Microflora of Root Canals at the Time of Filling, Thesis, Ohio State University, School of Dentistry, 1968. 12. Bender, I. B., and Seltzer, S.: To Culture or Not To Culture, ORAL SURG. 18: 527-539,
1964.
13. Engstrom, 15: 87-106,
B.: The 1964.
Significance
of Enterococci
in
Root
Canal
Treatment,
Odont.
Rev.
STATE
UNIVERSITY
COLLEGE
OF
D.M.D.,
M.S.,” and
DENTISTRY
I
ngle and Zeldowl and Nicholls2 suggested that the reduction in bacterial population due to the cleansing of a contaminated root canal was to someextent associated with the antiseptic effect of the fluid used for irrigation. Sodium hypochlorite is one of the most popularly used root canal irrigants. It was proved to be a powerful germicide, effective against a wide spectrum of microorganisms,3 and a solvent for necrotic tissue.4-oIn the absenceof organic matter, all vegetative bacteria were killed when exposed to a solution containing 0.15 to 0.25 ppm available chlorine for 30 seconds at pH 9 and 25O C.? The spore-forming organisms were about 10 to 1,000 times more resistant to chlorine than vegetative bacterial forms. The pulp tissue solvent properties were reported by Grossman.8,Q However, the relative efficiency of sodium hypochlorite in sterilizing and cleansing root canals is not known. The present study was designed to investigate the bactericidal efficiency of sodium hypochlorite as a root canal irrigant. METHODS AND MATERIALS Clorox t was used in this study as a source of sodium hypochlorite. Streptococcus faecalis and Staphylococcus aureus were chosen as the test organisms. The two organisms were tested separately in all the experiment,s. This article is abstracted from a thesis submitted by the senior author in partial fulfillment of the requirements for the degree of Master of Science in the Graduate School, Ohio State University. *Professor and Head, Department of Endodontics. **Professor, College of Dentistry and Academic Faculty of Microbial and Cellular Biology. tClorox, a product of The Clorox Company, Oakland, Calif., contains: Sodium hypochlorite 5.25% 0.207% Sodium carbonate 4.07& Sodium chloride Free sodium hydroxide 0.005-0.015~
613
614
Shih, Marshall,
and Rosen.
Oral April,
Surg. 1970
Periodically throughout the study Gram stains and streaked blood agar plates were made to ensure the viability of the organism, the continuation of the original pure cultures, and the absence of contamination. Two experiments were undertaken. The first tested the germicidal efficiency of Clorox by serial tube dilution studies, and the second (using extracted human teeth) tested the germicidal efficiency of Clorox as a root canal irrigant. Experiment
1
A series of tenfold serial dilutions of Clorox were prepared with sterile distilled water. Five milliliters of each solution (full-strength Clorox, Clorox diluted with sterile distilled water 1 :lO, 1 :lOO, 1 :l,OOO, 1 :lO,OOO, or sterile distilled water as a control) was dispensed aseptically into two sets of six tubes each. Five milliliters each of a 24-hour culture of Str. faecalis and S. aureus was centrifuged. After the supernatant was discarded, each sediment was resuspended in 5 ml. of Bacto-as&tic fluid. * Next 0.1 ml. of Str. faecalis suspension was added to one set of six tubes and 0.1 ml. of S. aureus suspension was added to the other set of six tubes. All tubes were agitated during the experiment to ensure thorough mixing of the hypochlorite solution and bacteria. Samples were taken from these inoculated solutions at 30 seconds, 1, 2, 3, 4, 5, 10, and 20 minutes, and 24 hours. They were placed in tubes containing 15 ml. trypticase soy broth with 0.1 per cent agart and incubated at 3’7OC. for 72 hours under aerobic conditions. Cloudiness of the culture medium at the end of the incubation period was used as the criterion of the presence of bacterial growth. Experiment
2
The root canals of 120 freshly extracted single-rooted human teeth were prepared by standard endodontic techniques. The occlusal openings were accentuated. Water was used during preparation to remove any dentine shavings from the canals. The root canals were reamed and filed through the apex, and the canals were enlarged to the size of a No. 80 file. The enlarged apices were sealed with an epoxy resin.$ The prepared teeth were then placed in polyethylene bags containing water, double sealed, and sterilized by exposure to cobalt-60 radiation at 1.27 to 1.61 x lo5 r per hour for 48 hours. After sterilization all teeth were seated in presterilized mounting boards with crowns exposed. Sixty sterilized teeth were then inoculated with Str. faecalG and sixty were inoculated with S. aureus. The inoculums were introduced into the apical thirds of the root canals with the aid of tuberculin syringes and straight platinum wires. These 120 teeth, still mounted in boards, were placed in a sterile container and incubated for 48 hours at 37’ C. They were then divided into three groups of forty teeth for treatment, Twenty teeth were inoculated with Str. faecazis and twenty teeth were inoculated with S. aureus. Each group was treated with procedures described below using varying concentrations of Clorox. “Difco Laboratories, Detroit, Mich. tBalt.imore Biological Laboratories, Baltimore, $Carter’s General Purpose Epoxy, Carter’s Ink
Md. Co., Cambridge,
Mass.
Volume Number
29 4
Bactericidal
eficiency
of sodium
hypochlorite
615
Two pilot studies suggested that 5 ml. of full-strength Clorox or 5 ml. oi Clorox diluted 1 :lO with water should be used to irrigate each root canal in this portion of the study. Group I teeth were irrigated with full-strength Clorox, Group II with Clorox diluted l:lO, and Group III wit,h sterile distilled water as a control. Preirrigation culture samples were taken from all inoculated root canals in the group, using a wet-point culturing techniquelO modified so that 15 ml. of trypticase soy broth with 0.1 per cent agar was used. The root canals were then irrigated with the following test solutions, using a technique that simulated clinical procedures: 5 ml. of full-strength Clorox, 5 ml. of Clorox diluted 1 :lO, or 5 ml. of sterile distilled water. Each solution was introduced slowly into the root canal by means of a 5 ml. disposable syringe and a 11/b inch 25 gauge needle. The irrigating needle was inserted as far as possible, without binding, into the canal. The solution was injected into the canal at l-minute intervals and the full amount was dispensed within 3 minutes. A sterile No. 15 reamel was used during this period to stir the solution in the canal. The overflow of the solution was caught and removed with a suction device attached to a dental unit. After irrigation, each canal was washed with 2 ml. of sterile distilled water. A postirrigation culture sample was taken from each canal. The canals were then filled with culture medium and reincubated at 37’ C. and 100 per cent humidity for 7 days. Two additional culture samples were taken from each canal-one 2 days and one 7 days following the treatment. The root canals were kept moist during this period of incubation by the addition of culturcL medium at 2-day intervals. All culture tubes were examined a.fter they had been incubated aerobically at 37’ C. for 72 hours. RESULTS Experiment
1
The tube dilution tests (Table I) showed that Str. faeculis and S. aureus suspended in 0.1 ml. of Bacto-a&tic fluid were completely killed in 30 seconds when they were added to 5 ml. of full-strength Clorox, and Clorox diluted l:lO, 1 :lOO, or l:l,OOO. A Clorox-water dilution of l:lO,OOO, however, did not inhibit the bacteria1 growth, even after contacting the organism for 24 hours. Experiment
2
The results of the extracted-tooth experiment (Table II) showed that the preirrigation culture samples were positive in 119 out of 120 teeth. All culture samples taken from the forty teeth inoculated with either Str. faecalis or S. aureus after irrigation with full-strength Clorox showed no growth in culture tubes immediately after the irrigation. When teeth were irrigated with Clorox diluted 1 :lO, seven of the twenty teeth inoculated with Str. faecalis and two of the twenty teeth inoculated with 8. aureus showed growth when cultured immediately after the irrigation. Two days postirrigation, the group treated with full-strength Clorox gave positive cultures in eight of twenty teeth inoculated with S. faecalis and five of
616
Shih, Marshall,
Table
Oral April,
and Rosen
Surg. 1970
I. Tube dilution studies of bacterial inhibition by Clorox at various times Streptococcus Time
faecalis
of exposzlre
St,aphylococoas Time
(nzinzctes)
0.5
1
2
3
4
5
10
a0
1,440 (24 hrs.)
diluted
-
-
-
_
-
-
-
_
Clorox diluted 1:lOO
-
-
-
-
-
-
-
-
Clorox diluted l:l,OOO
-
-------
_
Clorox diluted 1: 10,000
+
+++++++
Water
+
t
azcrezcs
of exposure
0.5
1
2
3
-
-
-
-
-
-
-
-
+
+
++++++t
t
+
+
4
(minutes) 1,440 ($4 hrs.)
5
10 20
_
-
-
-
-
_
-
-
-
-
Clorox full strength (Jorox 1:lO
(control)
+
+
+
+
+
+
+
t +
+
+
+
+
t
- = No growth. t = Growth.
II. Incidence of positive cultures following clinical treatment Table
irrigations
that simulate
Concentration Water Streptococcus
St’aphylococcus
*Numerator: Denominatk-:
faecalis Preirrigation Immediately postirrigation 2 days postirrigation 7 days postirrigation
20/20* o/20 s/20 16/20
“i:z 13/20 15/20
20/20 20/20 19/20 19/2,0
Preirrigation Immediately postirrigation 2 days postirrigation 7 days postirrigation
2’0/20 o/20 5/20 11/20
20/20 2/20 14/20 16,‘20
19/20 18/20 18/20 19/20
aweus
Number Total
of positive cultures. number of teeth used.
twenty teeth inoculated with S. aureus. In the group treated with Clorox diluted 1 :lO, thirteen of twenty teeth inoculated with Str. faecalis and fourteen of twenty teeth inoculated with S. aureus produced culture reversals. Seven days postirrigation the group treated with full-strength Clorox showed positive cultures in sixteen of twenty teeth inoculated with Str. faecalis and eleven of twenty teeth inoculated with S. aureus. In the group treated with Clorox l:lO, fifteen of the twenty teeth inoculated with Str. faecalis and sixteen of the twenty teeth inoculated with S. aureus sho,wedresidual “infection.” Inoculated root canals washed mechanically only with sterile distilled water
Volume 2f, P\iumber 4
BactericidaZ
eficiency
of sodium
hypochlo~ite
617
showed growth in eighteen or nineteen of twenty teeth for either microorganisnl in all test periods. DISCUSSION
Tube dilution studies showed that Cloros had germicidal activity against faecalis and S. aureus, even at a dilution of l:l,OOO. The continuation studies involving a simulated clinical procedure on extracted human teeth showed that only full-strength Clorox had an apparent immediate sterilizing effect in root canals of extracted human teeth inoculated with Str. faecalis or S. aweus. In the tube dilution studies there was a greater possibility of carry-over of Clorox than in the extracted-tooth studies, and thus perhaps a greater sterilizing effect was achieved for the tube dilution studies. Limited contact of the germicidal solution with the root canal contents ma! also contribute to the differences between these studies. Although 5 ml. of test solution was used to irrigate each canal, only a small portion of the solution was in actual contact with the root canal contents per unit time because of the limited size and the irregular shape of a root canal. Irregularities in the main canal or untreated lateral canals may contain organic tissue not mechanically removed. This tissue could readily bind the chlorine and neutralize the germicidal effect of small amounts of contacting Clorox. On the other hand, the organic contents of the test solution in the tube study (0.1 ml. of Bacto-ascitic fluid) may have been insufficient to neutralize the effect of the Clorox. The microorganisms supported by the untreated organic material could remain viable and thus serve as a residual source of infection. High reversal rates 2 days following the treatment, 40 per cent (eight out of twenty) in the Xtr. faecnlis group and 25 per cent (five out of t.wentyj in the 8. aweus group, suggests that the culturing technique used here and clinically in endodontic treatment rnav not be sensitive enough to detect, small amounts of residual bacteria left in a root canal. The paper point used for sampling is not in direct contact with the accessory canal contents. If the cnltl~ring technique uses a “dry” paper point, there is even less chance of obtaining an inoculum. Thus, a negative posttreatment report may suggest that only the main root canal is sterile or that it contains a highly reduced bacterial population. II substrate is available or becomes available, the organisms may bc re-establish& in high numbers in the main root canal during the interim. Seven-day reversal rates in all groups were found to be higher than 2-d;&> reversal rates. An increased time period may have increased the chance for microorganisms to overcome the shock of treatment and to come into contact with additional nutrients. The reversal rate following one negative culture, 40 per cent for Str. faecdis and 25 per cent for S. azcreus, was much higher than those reported by Myers’l (25.9 per cent) or Bender and Seltze? (16.6 per cent). Nutrients were added to canals after irrigation in this study and not in the others. This could have: helped residual organisms to proliferate. Xo medicament was used betwt*tn treatments to suppress bacterial growth. A wet-point culturing technique (not Str.
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used by Myers or by Bender and Seltzer) was a more sensitive technique for obtaining inoculums. The carry-over effect of sodium hypochlorite was minimized in our study by washing the treated canals with 2 ml. sterile distilled water, but there could have been a carry-over of medicament in the studies of MyersI and Bender and Seltzer.lz All these factors could have helped to increase our reversal rate. The results from the control group agree with findings of Ingle and Zedlowl and Nicholls2 that mechanical flushing of the root canal with sterile distilled water alone does not eliminate root canal infection. The results of this study suggest that sodium hypochlorite irrigating solutions at a concentration of 5.25 per cent (full-strength Clorox) may be required to reduce effectively the microbial population of an infected root canal. The reversal rates also suggest that many canals with negative cultures have residual bacteria at the time of root canal filling. A negative culture does not justify the assumption that the canal is sterile at the time of insertion of a root canal filling. The use of an intracanal medicament would seem to be desirable to aid in establishing the sterility of the canal. S. mreus has been used in many standard methods to test the germicidal power of various antiseptics. This microorganism is the most common cause of suppuration and is a highly resistant non-spore-forming infectious agent. It was used in this study because of its frequent presence in infected root canals. Str. faecalk was used as the other test organism because of its prevalence in infected root canals and because of its high resistance to routine medication in root canal therapy. Myersll reported that it comprised 20.8 per cent of all the organisms found in root canals at the time of filling, and EngstromX3 found it the most difficult to remove from an infected root canal. The effectiveness of irrigation with Clorox, as tested here on two organisms individually, may be different when the organisms are tested together. Bacterial growth potential and resistance to irrigation may be quite different in a mixed culture in vivo than in separate cultures in vitro. Laboratory tests of any kind are only the first steps in a study of the effectiveness of antiseptics. The ultimate test of the effectiveness of an antiseptic is the demonstration of its germicidal efficiency under conditions of practical application. Further studies should include the effect of sodium hypochlorite on extracted human teeth inoculated with mixed cultures containing a variety of oral microorganisms. This should be followed by a clinical study for a final assessment of the bactericidal efficiency of sodium hypochlorite as a root canal irrigant. SUMMARY
A laboratory study was undertaken to appraise the bactericidal efficiency of sodium hypochlorite used as a root canal irrigant. Clorox (5.25 per cent sodium hypochlorite, pH 11 to 11.5) was the source of sodium hypochlorite. A laboratory tube dilution study showed sodium hypochlorite to be a powerful germicide. Str. faecdis and S. aureus suspended in 0.1 ml. of Bacto-as&tic
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fluid were completely killed in 30 seconds when added to 5 ml. of Clorox diluted 1 :l,OOO (0.00525 per cent sodium hypochlorite) . Experimental studies with a simulated clinical irrigating procedure showed that only full-strength Clorox had a 100 per cent sterilizing effect in root canals inoculated with Str. faecalis and 8. aweus. Two-day and ‘I-day postirrigation culture results revealed high reversal rates in the group irrigated with fullstrength Clorox or in the group irrigated with CIorox diluted 1 :lO. The ?-day reversal rates were greater than the 2-day rates. CONCLUSION
1. The bactericidal efficiency of sodium hypochlorite in tube dilution studies is not related to the effect of sodium hypochlorite used in extracted huma,n teeth. 2. It is necessary to use Clorox at full strength (5.25 per cent sodium hypochlorite) to obtain an immediate sterilizing effect in root canals of extracted human teeth inoculated with Str. faecalis and 8. aureus. Irrigation with fullstrength Clorox does not ensure the lasting sterility of an inoculated root canal. 3. The holding action of an intracanal medicament between appointments is necessary to control the root canal microbial population. 4. A negative culture report after treatment indicates that the bacterial population in the root canal may be highly reduced, not that the canal is sterile. 5. Mechanical flushing with sterile distilled water is not effective in removing all bacteria from root canals. 6. Further studies should consider the effect of sodium hypochlorite on extracted human teeth inoculated with mixed cultures containing various oral microorganisms. 7. A clinical study should be carried out for a final assessment of the bactericidal efficiency of sodium hypochlorite. REFERENCES
1. Ingle,
J. I., and Zedlow, B. J.: An Evaluation of Mechanical Instrumentation and the Negative Culture in Endodontic Therapy, J. Amer. Dent. Ass. 57: 471-474, 1958. 2. Nicholls, E.: The Efficiency of Cleansing of the Root Canal,. Brit. Dent. J. 112: IBS170, 1962. 3. Less, M. A.: Hypochlorite as Sanitizers, Soap t Sanit. Chem. 25: 119-125, 139, 1949. 4. Dakip, H. D.: The Antiseptic Action of Hypochlorites, Brit. Med. J. 2: 889-910, 191.5. 5. Austin, J. H., and Taylor, H. D.: Behavior of Hypochlorite and of Chloramine-T Solutions in Contact With Necrotic and Normal Tissues In Vivo, J. Exp. Med. 27: 627-633, 1918. 6. Taylor, H. D., and Austin, J. II.: The Solvent Action of Antiseptics on Necrotic Tissue, J. Exp. Med. 27: 155-164, 1918. 7. Snow, W. B.: Recommended Chlorine Residual for Military Water Supplies, J.A.W.\V. A. 48: 1510-1514. 1956. 8. Grossman, L. I.; and Meiman, B. W.: Solution of Pulp Tissue by Chemical Agents, J. Amer. Dent. Ass. 28: 223-225, 1941. 9. Grossman, L. 1.: Irrigation of Root Canals, J. Amer. Dent. Ass. 30: 1915-1917, 1943. IO. Marshall, F. J., and Savoie, F. L.: Efficiency of Endodontic Culturing Procedure Using Wet and Dry Paper Points, ORAL BURG. 23: 806~810,1967. 11. Myers, J. W.: The Microflora of Root Canals at the Time of Filling, Thesis, Ohio State University, School of Dentistry, 1968. 12. Bender, I. B., and Seltzer, S.: To Culture or Not To Culture, ORAL SURG. 18: 527-539,
1964.
13. Engstrom, 15: 87-106,
B.: The 1964.
Significance
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