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In Vitro Studies on Sterilization of Carious Dentin. III. Effective Penetration of Germicides into Carious Lesions
IN VITRO STUDIES ON STERILIZATION OF CARIOUS DENTIN. III. EFFECTIVE PENE TRATION OF GERMICIDES INTO CARIOUS LESIONS R o b e r t M . S t e p h a n , M .S ., D.D .S. ; J o h n A . M A l b e r t D o r f m a n , Ph.D., Chicago,
N a previous study of this series, it was shown that o f a great number of germicides, a saturated solution of silver nitrate is by far the most effective for sterilizing carious dentin,' and that even this substance must reach an appre ciable concentration in dentin to destroy all the bacteria.1 In a second study, it was shown that there is nearly always a layer of sterile, partially decalcified den tin between the sound dentin and the infected part of the cavity.2 It was suggested that, in treating deep carious lesions, it m ight be safer to leave the deepest layer of carious dentin rather than expose the pulp, provided the sterility of this dentin could be insured. It therefore seemed desirable to deter mine just how great a thickness of in fected dentin can be sterilized by an application of silver nitrate to a tooth for ,a time that is feasible in routine dental practice. It also seemed desirable to es tablish whether some of the other com monly used germicides could be effec tive for this purpose.
I
m e th o d
In order to demonstrate that a germ i cide has sterilized the dentin in a cari ous lesion to a given depth, it is first necessary to establish that such dentin is infected at this depth.2 This must be From the Chemistry Laboratory of the Walter G. Zoller Memorial Dental Clinic, University of Chicago. Jour. A .D .A ., V ol. 30, December 1, 1943
untz,
111.
M .S ., and
done without making an opening through to the carious surface, as other wise any germicide applied to the cari ous surface would gain direct access to the deeper layers. T h e instrument shown in the accompanying figure was con structed for this purpose. (See page
I907-)
T he principal parts of this instrument are (A) a clamp, mounted on an adjust able ball-and-socket joint, which holds the tooth firmly by the root with the crown fully exposed, and (B) and (C) two dental handpieces that are uni versally adjustable for both position and direction and are mounted in a microm eter screw arrangement, permitting ad vancement or retraction for distances measurable to 0.1 mm. Sterile dental drills used for taking samples of tooth substance at measured distances from the carious surface are held and rotated in these handpieces. T h e rotating shaft of the handpieces can be withdrawn read ily in order to facilitate repeated re moval of the bur from the tooth, as in taking samples for culture. . T h e experiments were performed with sterile technic and were carried out as follow s: T h e depth o f infection in the carious tooth substance was established by taking samples a t measured distances from the carious surface with the hand piece (B). This was done by cutting from a sterilized sound surface into the carious lesion. T h e germicide was then 1905
ig o 6
T
he
J
ournal of t h e
A m e r ic a n D e n t a l A s s o c ia t io n
applied to the carious surface and was washed off after a measured time inter val. A series of samples were then taken with the other handpiece ( C ) , starting at the carious surface and going down to the desired depth. Tubes of culture medium (see page 1895) were inoculated with samples taken in this manner and incubated at 370 C . T h e tubes were observed for bacterial growth on the second, fourth and seventh days. Gram-stained smears and subcultures were made from tubes that were negative or doubtful. O nly experiments in which it had been estab lished that the deepest layers giving neg ative cultures after application o f the
ous experiments showed this to be true.2 A fter treatment with the germicide, it can be seen that organisms did not grow from the 0.5 mm. level, but did grow from the 1.0 mm. level. W e can con clude that sterilization has been achieved to a depth of at least 0.5 mm., but not to 1.0 mm. Table 2 contains a summary of experi ments comparing the ability of saturated T a b l e 2 .— E f f e c t i v e P e n e t r a t i o n o f G e r m ic id e s in t o C a r io u s L e s io n s
Germicidal Solution
T a b l e 1 .— S t e r i l i z a t i o n o p C a r io u s D e n t i n w it h
S a t u r a t e d S il v e r N it r a t e
Silver nitrate (saturated aqueous solution)
A p p l ie d f o r T h r e e M in u t e s
Description of Sample
Before treatment Sound dentin (control) Carious dentin 1.5 mm from surface After treatment Superficial carious dentin Carious dentin 0.5 mm. Carious dentin 1.0 mm. Carious dentin l.S mm.
Days 2 '4 7
GramStained Smear
--------
—
+ + +
+
--------------- + + + + +
— —
+ +
germicide had given positive cultures be fore application o f the germicide were considered valid. These experiments that yielded negative cultures at the test depth in the carious dentin were discarded as non-valid. r esu lts
T h e results o f a typical experiment are given in T able 1. In this experi ment, it will be noted that the sample taken at the 1.5 mm. level before appli cation o f silver nitrate was infected. W e can assume that everything superficial to that level was also infected, since previ
Average Time Depth of Mean Applied Sterili DeviaMinutes zation* tionf Mm. 1
0.1
1°
0.3 0:7 1.3
Ammoniacal silver nitrate (Howe’s solution)
3
0.6
0.4
Hydrogen peroxide (30 percent) (superoxol)
3
0.4
0.4
Alkyldimethylbenzyl ammonium chloride 20 percent (zephiran 1:5) Phenol 95 per cent (liquid phenol)
3
0.3 0.3
10
0.8
0.7
.3
0.0
0.0
3
0.0
0.0
*Each figure is the average of results from at least ten experiments. fWhen the figure for the mean deviation is sub tracted from the figure for the average depth of sterilization, the result closely approximates the minimum depth of sterilization. *
silver nitrate, Howe’s ammoniacal silver nitrate solution, 20 per cent zephiran (alkyl dimethyl benzyl ammonium chlor ide), 30 per cent hydrogen peroxide and 95 per cent phenol to sterilize carious dentin. It will be noted that saturated silver nitrate is effective in sterilizing carious dentin, and that the length of time during which it is applied largely
Steph an , M
u n tz and
D
orfm an —
determines the extent of sterilization. Howe’s solution of ammoniacal silver nitrate was effective, but somewhat less so than saturated silver nitrate. Hydro gen peroxide 30 per cent was still less effective. Its penetration was quite vari able, and it cannot be considered as reliable as silver nitrate for sterilizing infected dentin. Liquid phenol 95 per cent and zephiran 20 per cent were ineffective. In most experiments, they failed to sterilize even the superficial layer. a d d it io n a l e x p e r im e n ts
In order to test the ability of sat-
P e n e t r a t io n
of
G
e r m ic id e s
1907
cotton soaked with a saturated solution of silver .nitrate was applied to the cavity for three minutes. The cavity was then dried with sterile cotton pledgets and a chip blower, and filled with zinc oxideeugenol paste. After placement of the filling, the teeth were put into a humid chamber to prevent drying and incu bated at 370 C. for twenty-four hours. At the end of this period, the filling was removed, with sterile technic. The cari ous floor was cut out, ground in broth and cultured. In addition, samples of the harder dentin beneath the carious floor were taken out and Cultured. All samples were incubated for four days,
Instrument for taking samples of dentin at measured distances from carious surface.
urated silver nitrate to sterilize carious dentin left in a cavity floor over the pulp, the following type of experiment was performed on freshly extracted teeth. Third molars with extensive de cay were selected. A cavity was pre pared for filling by breaking down all undermined enamel and removing the superficial carious dentin, care being taken to leave a floor of leathery dentin from 1 to 2 mm. thick. A pledget of
and bacterial growth was checked with gram-stained smears and by subculture.. Seventeen teeth so treated showed no pulp exposures when the carious floor was removed, and all of these gave nega tive cultures from the floor of carious dentin and the hard dentin beneath this layer. Twelve other teeth showed pulp exposure when the leathery dentin was removed, and, of these, nine gave nega tive cultures and three showed bacterial
T
he
J
o urnal of th e
A m e r ic a n D e n t a l A s s o c ia t io n
growth. O f the three in which sterility was not produced, it could not be estab lished whether the infection came from the dentin or from the exposed pulp. H owever, in view of the uniformly sterile dentin in the seventeen cases in which the pulp was not exposed, it seems reasonable to assume that the infection was from the pulp. d is c u s s io n
These results make it evident that sterilization of infected dentin in carious lesions is possible with a saturated solu tion of silver nitrate, but only if an ade quate procedure is followed. T he time o f application is all important. It seems unreasonable in clinical practice to rely on guesswork to measure the time for application. A clock or other timing device should be used. T h e finding that phenol is of practi cally no value for application as a germi cide if any appreciable thickness of in fected dentin must be sterilized is par ticularly significant in view of the tradi tional use o f this agent for cavity sterili zation. Equally ineffective is a new germ icide of great apparent potency, zephiran (phenol coefficient about 300). T h e results obtained with 30 per cent hydrogen peroxide (superoxol) show m uch greater variation than those ob tained with the other germicides. W hen the hydrogen peroxide was applied to carious dentin, a mass of bubbles quickly formed which apparently interfered with further penetration. Since the enzyme catalase (which decomposes hydrogen peroxide to water and oxygen) is present in carious dentin, the inactivaing influence of this substance must be considered. T h e somewhat greater ef fectiveness of superoxol found in the previous study1 can be explained by the fact that, in the method employing pieces o f carious dentin, the hydrogen peroxide could penetrate from six sur faces instead of only the one, as in this study, and the blocking influence of the
bubbles of oxygen on the further pene tration o f the hydrogen peroxide solu tion would be correspondingly less. These findings confirm the observations made by W . D . M iller,3 who also ob tained inconstant results with hydrogen peroxide. These studies have not determined whether injury to the dental pulp may result from the application of a germi cide. Silver nitrate has been extensively used in the treatment of carious lesions, apparently without any serious pulp re actions. Nevertheless, the possibility of injury to the pulp by application of germicides to carious dentin should not be disregarded. SUM M ARY
A special apparatus was constructed which permitted determination of the depth of sterilization produced by germ icides in carious lesions o f extracted teeth. It was found that a saturated solution of silver nitrate sterilized carious dentin to an average o f 0.3 mm. by a one-minute application, 0.7 mm. by a three-minute application and 1.3 mm. by a ten-minute application. Ammoniacal silver nitrate (Howe’s solution) was slightly less effective. A 30 per cent solution o f hydrogen peroxide (super oxol) was much less effective and very unreliable. A 20 per cent solution of zephiran and 95 per cent phenol were ineffective. B IB L IO G R A P H Y
A .; D o r f m a n , A l f r e d , and R. M.: In Vitro Studies on Steril ization of Carious Dentin: I. Evaluation of Germicides. J . A . D . A . , 29:1893, December r 943 2 . D o r f m a n , A l f r e d ; S t e p h a n , R. M . , a n d M ü n t z , J. A.: In Vitro Studies o n Steriliza tion of Carious Dentin: II. Extent of Infec tion in Carious Lesions. J.A.D.A., 29:1901, December 1943. 3. M i l l e r , W. D.: Comparative Rapidity with Which Different Antiseptics Penetrate Decalcified Dentin: O r What Antiseptic Should Be Used in Sterilizing Cavities Before Filling? D. Cosmos, 33:337, May 1891. 1. M u n t z ,
S te p h a n ,
J.
N a previous study of this series, it was shown that o f a great number of germicides, a saturated solution of silver nitrate is by far the most effective for sterilizing carious dentin,' and that even this substance must reach an appre ciable concentration in dentin to destroy all the bacteria.1 In a second study, it was shown that there is nearly always a layer of sterile, partially decalcified den tin between the sound dentin and the infected part of the cavity.2 It was suggested that, in treating deep carious lesions, it m ight be safer to leave the deepest layer of carious dentin rather than expose the pulp, provided the sterility of this dentin could be insured. It therefore seemed desirable to deter mine just how great a thickness of in fected dentin can be sterilized by an application of silver nitrate to a tooth for ,a time that is feasible in routine dental practice. It also seemed desirable to es tablish whether some of the other com monly used germicides could be effec tive for this purpose.
I
m e th o d
In order to demonstrate that a germ i cide has sterilized the dentin in a cari ous lesion to a given depth, it is first necessary to establish that such dentin is infected at this depth.2 This must be From the Chemistry Laboratory of the Walter G. Zoller Memorial Dental Clinic, University of Chicago. Jour. A .D .A ., V ol. 30, December 1, 1943
untz,
111.
M .S ., and
done without making an opening through to the carious surface, as other wise any germicide applied to the cari ous surface would gain direct access to the deeper layers. T h e instrument shown in the accompanying figure was con structed for this purpose. (See page
I907-)
T he principal parts of this instrument are (A) a clamp, mounted on an adjust able ball-and-socket joint, which holds the tooth firmly by the root with the crown fully exposed, and (B) and (C) two dental handpieces that are uni versally adjustable for both position and direction and are mounted in a microm eter screw arrangement, permitting ad vancement or retraction for distances measurable to 0.1 mm. Sterile dental drills used for taking samples of tooth substance at measured distances from the carious surface are held and rotated in these handpieces. T h e rotating shaft of the handpieces can be withdrawn read ily in order to facilitate repeated re moval of the bur from the tooth, as in taking samples for culture. . T h e experiments were performed with sterile technic and were carried out as follow s: T h e depth o f infection in the carious tooth substance was established by taking samples a t measured distances from the carious surface with the hand piece (B). This was done by cutting from a sterilized sound surface into the carious lesion. T h e germicide was then 1905
ig o 6
T
he
J
ournal of t h e
A m e r ic a n D e n t a l A s s o c ia t io n
applied to the carious surface and was washed off after a measured time inter val. A series of samples were then taken with the other handpiece ( C ) , starting at the carious surface and going down to the desired depth. Tubes of culture medium (see page 1895) were inoculated with samples taken in this manner and incubated at 370 C . T h e tubes were observed for bacterial growth on the second, fourth and seventh days. Gram-stained smears and subcultures were made from tubes that were negative or doubtful. O nly experiments in which it had been estab lished that the deepest layers giving neg ative cultures after application o f the
ous experiments showed this to be true.2 A fter treatment with the germicide, it can be seen that organisms did not grow from the 0.5 mm. level, but did grow from the 1.0 mm. level. W e can con clude that sterilization has been achieved to a depth of at least 0.5 mm., but not to 1.0 mm. Table 2 contains a summary of experi ments comparing the ability of saturated T a b l e 2 .— E f f e c t i v e P e n e t r a t i o n o f G e r m ic id e s in t o C a r io u s L e s io n s
Germicidal Solution
T a b l e 1 .— S t e r i l i z a t i o n o p C a r io u s D e n t i n w it h
S a t u r a t e d S il v e r N it r a t e
Silver nitrate (saturated aqueous solution)
A p p l ie d f o r T h r e e M in u t e s
Description of Sample
Before treatment Sound dentin (control) Carious dentin 1.5 mm from surface After treatment Superficial carious dentin Carious dentin 0.5 mm. Carious dentin 1.0 mm. Carious dentin l.S mm.
Days 2 '4 7
GramStained Smear
--------
—
+ + +
+
--------------- + + + + +
— —
+ +
germicide had given positive cultures be fore application o f the germicide were considered valid. These experiments that yielded negative cultures at the test depth in the carious dentin were discarded as non-valid. r esu lts
T h e results o f a typical experiment are given in T able 1. In this experi ment, it will be noted that the sample taken at the 1.5 mm. level before appli cation o f silver nitrate was infected. W e can assume that everything superficial to that level was also infected, since previ
Average Time Depth of Mean Applied Sterili DeviaMinutes zation* tionf Mm. 1
0.1
1°
0.3 0:7 1.3
Ammoniacal silver nitrate (Howe’s solution)
3
0.6
0.4
Hydrogen peroxide (30 percent) (superoxol)
3
0.4
0.4
Alkyldimethylbenzyl ammonium chloride 20 percent (zephiran 1:5) Phenol 95 per cent (liquid phenol)
3
0.3 0.3
10
0.8
0.7
.3
0.0
0.0
3
0.0
0.0
*Each figure is the average of results from at least ten experiments. fWhen the figure for the mean deviation is sub tracted from the figure for the average depth of sterilization, the result closely approximates the minimum depth of sterilization. *
silver nitrate, Howe’s ammoniacal silver nitrate solution, 20 per cent zephiran (alkyl dimethyl benzyl ammonium chlor ide), 30 per cent hydrogen peroxide and 95 per cent phenol to sterilize carious dentin. It will be noted that saturated silver nitrate is effective in sterilizing carious dentin, and that the length of time during which it is applied largely
Steph an , M
u n tz and
D
orfm an —
determines the extent of sterilization. Howe’s solution of ammoniacal silver nitrate was effective, but somewhat less so than saturated silver nitrate. Hydro gen peroxide 30 per cent was still less effective. Its penetration was quite vari able, and it cannot be considered as reliable as silver nitrate for sterilizing infected dentin. Liquid phenol 95 per cent and zephiran 20 per cent were ineffective. In most experiments, they failed to sterilize even the superficial layer. a d d it io n a l e x p e r im e n ts
In order to test the ability of sat-
P e n e t r a t io n
of
G
e r m ic id e s
1907
cotton soaked with a saturated solution of silver .nitrate was applied to the cavity for three minutes. The cavity was then dried with sterile cotton pledgets and a chip blower, and filled with zinc oxideeugenol paste. After placement of the filling, the teeth were put into a humid chamber to prevent drying and incu bated at 370 C. for twenty-four hours. At the end of this period, the filling was removed, with sterile technic. The cari ous floor was cut out, ground in broth and cultured. In addition, samples of the harder dentin beneath the carious floor were taken out and Cultured. All samples were incubated for four days,
Instrument for taking samples of dentin at measured distances from carious surface.
urated silver nitrate to sterilize carious dentin left in a cavity floor over the pulp, the following type of experiment was performed on freshly extracted teeth. Third molars with extensive de cay were selected. A cavity was pre pared for filling by breaking down all undermined enamel and removing the superficial carious dentin, care being taken to leave a floor of leathery dentin from 1 to 2 mm. thick. A pledget of
and bacterial growth was checked with gram-stained smears and by subculture.. Seventeen teeth so treated showed no pulp exposures when the carious floor was removed, and all of these gave nega tive cultures from the floor of carious dentin and the hard dentin beneath this layer. Twelve other teeth showed pulp exposure when the leathery dentin was removed, and, of these, nine gave nega tive cultures and three showed bacterial
T
he
J
o urnal of th e
A m e r ic a n D e n t a l A s s o c ia t io n
growth. O f the three in which sterility was not produced, it could not be estab lished whether the infection came from the dentin or from the exposed pulp. H owever, in view of the uniformly sterile dentin in the seventeen cases in which the pulp was not exposed, it seems reasonable to assume that the infection was from the pulp. d is c u s s io n
These results make it evident that sterilization of infected dentin in carious lesions is possible with a saturated solu tion of silver nitrate, but only if an ade quate procedure is followed. T he time o f application is all important. It seems unreasonable in clinical practice to rely on guesswork to measure the time for application. A clock or other timing device should be used. T h e finding that phenol is of practi cally no value for application as a germi cide if any appreciable thickness of in fected dentin must be sterilized is par ticularly significant in view of the tradi tional use o f this agent for cavity sterili zation. Equally ineffective is a new germ icide of great apparent potency, zephiran (phenol coefficient about 300). T h e results obtained with 30 per cent hydrogen peroxide (superoxol) show m uch greater variation than those ob tained with the other germicides. W hen the hydrogen peroxide was applied to carious dentin, a mass of bubbles quickly formed which apparently interfered with further penetration. Since the enzyme catalase (which decomposes hydrogen peroxide to water and oxygen) is present in carious dentin, the inactivaing influence of this substance must be considered. T h e somewhat greater ef fectiveness of superoxol found in the previous study1 can be explained by the fact that, in the method employing pieces o f carious dentin, the hydrogen peroxide could penetrate from six sur faces instead of only the one, as in this study, and the blocking influence of the
bubbles of oxygen on the further pene tration o f the hydrogen peroxide solu tion would be correspondingly less. These findings confirm the observations made by W . D . M iller,3 who also ob tained inconstant results with hydrogen peroxide. These studies have not determined whether injury to the dental pulp may result from the application of a germi cide. Silver nitrate has been extensively used in the treatment of carious lesions, apparently without any serious pulp re actions. Nevertheless, the possibility of injury to the pulp by application of germicides to carious dentin should not be disregarded. SUM M ARY
A special apparatus was constructed which permitted determination of the depth of sterilization produced by germ icides in carious lesions o f extracted teeth. It was found that a saturated solution of silver nitrate sterilized carious dentin to an average o f 0.3 mm. by a one-minute application, 0.7 mm. by a three-minute application and 1.3 mm. by a ten-minute application. Ammoniacal silver nitrate (Howe’s solution) was slightly less effective. A 30 per cent solution o f hydrogen peroxide (super oxol) was much less effective and very unreliable. A 20 per cent solution of zephiran and 95 per cent phenol were ineffective. B IB L IO G R A P H Y
A .; D o r f m a n , A l f r e d , and R. M.: In Vitro Studies on Steril ization of Carious Dentin: I. Evaluation of Germicides. J . A . D . A . , 29:1893, December r 943 2 . D o r f m a n , A l f r e d ; S t e p h a n , R. M . , a n d M ü n t z , J. A.: In Vitro Studies o n Steriliza tion of Carious Dentin: II. Extent of Infec tion in Carious Lesions. J.A.D.A., 29:1901, December 1943. 3. M i l l e r , W. D.: Comparative Rapidity with Which Different Antiseptics Penetrate Decalcified Dentin: O r What Antiseptic Should Be Used in Sterilizing Cavities Before Filling? D. Cosmos, 33:337, May 1891. 1. M u n t z ,
S te p h a n ,
J.