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Cytotoxicity of three endodontic intracanal medicaments
endodontics Editor: WORTH
B. GREGORY,
D.D.S., M.S.D.
American Association of Endodontists School of Dentistry University of North Carolina Chapel Hill, North Carolina 27514
Cytotoxicity of three endodontic intracanal medicaments W. E. Kantz, D.D.S., Ph.D.,* P. J. Ferrillo, D.D.S.,“” and E. R. Zimmermanq D.D.S.,**+ Dallas, Texas BAYLOR
COLLEGE
OF DENTISTRY
Three endodontic intracanal medicaments (camphorated para-monochlorophenol, Cresatin, and Acriphen) were tested for their cytotoxic effect on HeLa cell tissue system. All three drugs showed extreme t0xicit.y to the tissue culture cells even when the concentration of each drug was in a dilution of 1: 1,000.
A
wide variety of nonspecific drugs has been advocated for intracanal medication of root canals. Camphorated para-monochlorophenol has been suggested as the drug of choice for a root canal antiseptiel-* Camphorated para-monochlorophenol has been shown to be cytotoxic to monolayer cultures of baby hamster kidney (BHK-21/4) cells” and diploid human embryonic lung (HEL-199) cells.6 This drug has also been shown to produce severe toxic reactions when placed on the conjunctiva of white rabbits, and tissue necrosis when introduced intradermally into the abdomen of the same type of animah7 Metacresyl acetate (Cresatin) is another of the nonspecific drugs used for medication of root canal systems. Cresatin has previously been suggested for this purpose because of its anodyne properties and its “remarkably low inflammatory potential.“8 Cresatin, however, has been reported to produce a mild irritation in hamster tissue9 and slight inflammation after a 24-hour intradermal injection in the abdomens of rabbits, but no inflammation 3 to 4 hours after the Supported by United States Public Health Service Training Grant DE-00226. *Assistant Professor, Department of Microbiology. ‘*Professor and Chairman, Department of Endodontics. ‘**Professor and Chairman, Department of Oral Pathology.
Volume 38 Number 4
Cytotoxicity
of three intracand
medica,m8ents 601
introduction of 0.51 ml. into the conjunctival sacs of the rabbits.lO Inflammation of pulp tissue has been demonstrated 56 per cent of the time after Cresatin pulpotomy.ll Acriphen, a combination of 9-aminoacridine and benzalkonium chloride, was adopted as an irrigating solution at one dental school because of its antimicrobial properties and its low toxicity.12 The purpose of this study was to determine the toxicity of camphorated paramonochlorophenol, Cresatin, and Acriphen on a tissue culture system. MATERIALS
AND
METHODS
The Gey strain of HeLa* cells was the tissue culture used in this study. The cells were maintained in a monolayer culture in a medium consisting of BME (basal medium Eagle) with Hank’s salts and additions of 5 per cent calf serum, 2 per cent glutamine, 2 per cent penicillin-streptomycin mixture (5,000 units per milliliter) and buffered with sodium bicarbonate (7.5 per cent w/v) solution. The pH was adjusted to 7.4. Camphorated para-monochlorophenol (CPC) ,t metacresyl-acetate (Cresatin) ,$ and 9-aminoacridine 0.01 per cent and benzalkonium chloride 0.02 per cent (Acriphen) $ were the drugs tested. Dilutions of the test drugs were prepared in the stock culture fluid media. Serial dilutions of CPC and Cresatin were made in concentrations of 1: 10, 1: 1,000, and 1: 100,000. Dilutions of Acriphen were 1: 10, 1: 100, and 1: 1,000. The HeLa cells were grown and maintained in 250-ml. tissue culture flasks (Falcon Plastic). After a monolayer became established, the cells were trypsinized for 15 minutes at 37O C. to detach them from the surface of the containers. The cells were placed in sterile 15 ml. conical centrifuge tubes, spun at 1,000 r.p.m. for 5 minutes, and resuspended in growth medium. One half of 1 ml. of the suspension of cells was inoculated into Leighton tubes, along with 1 ml. of stock medium. The tubes were sealed with rubber stoppers and placed in a horizontal position at 37* C. for 72 hours. The cells were allowed to adapt to the glass surface and establish a monolayer. Test procedure
Sterile techniques were used throughout the experimental study. At the end of 72 hours, the liquid medium was decanted from the Leighton tubes, Fortyfive tubes received 1.5 ml. of stock medium and served as controls. Fifteen tubes received 1.5 ml. of the CPC in a dilution of 1: 10 ; fifteen, a 1: 1,000 dilution ; and fifteen, a I: 100,000 dilution. The same procedure was carried out for Cresatin. The same procedure was also carried out for Acriphen, except that the dilutions were 1: 10, 1: 100, and 1: 1,000. The pH of each dilution and the control medium was adjusted to 7.4 prior to placement into the tubes. All tubes were *Microbial Associates, Inc., Bethesda, Mdd. tNovo1 Chemical Mfg. Co., Inc., Brooklyn, N. Y. thlerck, Sharp & Dohme, West Point, Pa. $Obergfel Brothers, Los Angeles, Calif.
602 Kantz, Perrdlo,
Oral Surg. October, 1974
and Zin~mermu~~n
1~~11l:lO
100 ’
--~1:1000 v-7 1: 10000C
90 . 80 ’ ,‘I:
Q$&
50 ’ 40 ’ 30 ’ 20 ’
0 KUR8
12
24
48
CPC Fig. 1. Comparison of cytotoxicity tin, and Acriphen to control.
12
24
48
CRESATIN of camphorated
12
24.
48
ACRIPHEN
para-monochlorophenol
(CPC),
Cresa-
restoppered, replaced in a horizontal position, and incubated at 37O C. At the end of 12, 24, and 48 hours, five tubes of each dilution of the test drug and five control tubes were trypsinized with a 0.25 per cent trypsin solution at 37O C. for 5 minutes. The suspended cells were placed in conical centrifuge tubes and spun for 5 minutes at 1,000 r.p.m. The trypsin solution was decanted off and the cells were resuspended in 0.5 ml. of a 0.5 per cent solution of trypan blue. The cells were exposed to the dye solution for 4 minutes. Aliquots were placed in the counting chamber of a hemacytometer, and the numbers of viable and nonviable cells were established. The nonviable cells stained a dark blue, whereas the viable cells remained clear and unstained when viewed under low power of a light microscope. All statistical analysis was made with the use of the Duncan’s multiple range and multiple F tests.13 RESULTS The average percentages of viable cells are graphically represented in Fig. 1. A comparison of CPC, Cresatin, and Acriphen at various dilutions and time intervals to control cells and to each other is shown in Table I. No statistical differences were found between any of the control viable counts at any time period or between any testing procedures. Statistically significant differences were noted with the following dilutions of drugs when compared to the number of viable cells in the controls : 1. CPC 1:lO at 12, 24, and 48 hours; CPC l:l,OOO at 12, 24, and 48 hours. 2. Cresatin 1: 10 at 12, 24, and 48 hours ; Cresatin 1: 1,000 at 12, 24, and 48 hours. 3. Acriphen 1: 10 at 12, 24, and 48 hours ; Acriphen 1: 100 at 12, 24, and 48 hours; Acriphen 1: 1,000 showed no significant difference at 12 and 24 hours but did show a significant decrease at 48 hours.
Cytotoxicity
Volume 38 Number 4
of three id-ocanal, medicaments
Table I. Comparison of CPC, Cresatin, and *4criphen at various dilutions time intervals to control cells anal each other (all values expressed as mean + S.E. [n-l] per cent viable cells) CPC 10-l 12 hr. 24 hr. 48 hr. 12 hr. 24 hr. 48 hr. 12 hr. 24 hr. 48 hr. *Represents tRepresents val and dilution
o*t 0” 0” Cres 10-l o*t 0* 0* Acr 10-l 22.74 t 1.67* 0” 0* a significant a significant only.
difference difference
CPC 10-s & o*t Cres lo-> 33.87 +_ 0.83’ ;:; Aor lo-” 19.63 + 0.74” 0” 0*
CFC lo-’ 61.47 + 3.37 53.13 2 1.82 56.89 + 2.30 Cres lo-$ 71.92 2 3.60 83.40 f 1.01 76.39 2 1.81 Acr 1O-5 53.39 + 3.70 64.53 2 3.91 34.67 f 6.11”
603 and
Control 68.91 2 2.54 63.45 k 0.46 55.55 ? 2.86 ContToz
71.67 f 3.20 72.42 2 3.76 60.14 ? 2.01 ContTol
63.67 + 9.65 78.52 + 3.38 64.51 2 1.92
between the control and test cells at that time interval. between the toxicity of each drug at same time inter-
Cellular fragments could only be identified from the 1 :lO and 1 :l,OOO dilutions of CPC. Only at a dilution of 1: 100,000 at 12 and 48 hours was no significance of toxicity noted when compared to the viable control cells. Cresatin at a dilution of 1: 10 had similar action by complete destruction of the cells. The cells exposed to the dilution of 1: 1,000 became detached and were nonviable; however, no cellular destruction was noted. Acriphen in dilutions of 1: 10 and 1: 100 was toxic to the tissue culture cells, even though complete cell death was not observed until 24 hours. These cells lost their ability to maintain a monolayer. Acriphen at 1: 1,000 dilution was not toxic to the tissue culture ,cells until after 48 hours. DISCUSSION These data again substantiate the toxicity of the more common root canal disinfectants in an in vitro system and suggest that other medicaments be investigated for this purpose. From the findings in this study and in otherq7-” it would appear that endodontists should rely more on the careful biomechanical cleansing of the root canal system rather than on the use of massive doses of toxic chemicals to eliminate microbial contamination. SUMMARY Three endodontic intracanal medicaments (camphorated para-monochlorophenol, Cresatin, and Acriphen) were tested for their cytotoxic effect on a HeLa cell tissue culture system. Serial dilutions of camphorated para-monochloropheno1 and Cresatin were made in concentrations of 1: 10, 1: 1,000, and 1: 100,000. Dilutions of Acriphen were 1: 10, 1: 100, and 1: 1,000. The tissue cells were exposed to the various dilutions of the drugs for 12, 24, and 48 hours. After trypsinization and resuspension, the nonviable tissue cells were identified by trypan blue staining. The numbers of viable and nonviable cells were determined with the use of a hemacytometer. Although Acriphen was the least toxic of the three medicaments tested, all
Oral Surg. October, 1974
of the ([rugs were cstrcmel~ to& to the tissue culture centration of each tlrug was in a tlilution of 1 : 1,000.
cells even when the con-
REFERENCES
1. Sommer. 12. F.. Ostrxnder. F. If.. and Crowlev. M. C.: Clinical Endodontics. ed. 2. Philadelphia,‘l962, W. B. Saunders Co.; pp. 194-195,” ’ 2. Luebke, R. G.: Symposium of Endodontics, Dent. Clin. North Am., p. 609, November, 1967. 3. Goldman, H. M, Forrest, S. P., Byrd, D. L., and McDonald, R. E.: Current Therapy in Dentistry, St. Louis, 1964, The C. V. Mosby Company, p. 93. St. Louis, 4. Dowson, J, and Garber. F. N.: A Chair-side Manual of Clinical Endodontics, 1967, The ‘C. V. Mosby ‘Company, p. 81. Efficacy and Cytotoxicity of Three 5. VanderWall, G. L, and Shipmnn, C.: Antibacterial Endodontic Drugs, I.A.D.R. 49th general session, March! 1971, p. 176. Efficacy and Cytotoxic6. VxnderWall, G. L., Dowson, J., and Shipman, C.: Antibacterial ity of Three Endodontic Drugs, Oaar, SIJRG. 33: 230-241, 1972. ORAL SURG. of Parachloronhenol, 7. Harrison, J. R., and Madonia, J. V.: The Toxicitr 32: 90-96. 1971. 8. Ingle, J. I.: Endodontics, Philadelphia, 1969, Lea & Febiger, p. 491. 9. Torneck. C. D.: Reaction of Hamster Tissue to Drugs in Sterilization of the Root Canal,
ORAL S~RG. 14: 730-747,1961. 10. Schilder,
H., and Amsterdam,
M.:
inflammatory
Potential
of Root
Canal
Medieaments,
ORAL SURG.12: 211-221, 1959. Response of Dental 11. Sandier, E. S., Frankl, 8. N., and Ruben, M. P.: The Histological Pulp to Cresatin, 5. Dent. Child. 38: 49-58, 1971. 12. Stark, M. H., Hall, N. C., Nicholson, R. J., and Soelberg, K.: 9-Aminoacridine, An Effective Antibacterial Agent With Caries Disclosing Features, ORAL SURG.26: 560-562, 1968. 13. Duncan, D. R.: Multiple Range and Multiple F Tests, Biometrics 11: l-42, 1955.
Reprint requests to: Dr. E. R. Zimmerman Department of Oral Pathology Baylor College of Dentistry Dallas, Texas
B. GREGORY,
D.D.S., M.S.D.
American Association of Endodontists School of Dentistry University of North Carolina Chapel Hill, North Carolina 27514
Cytotoxicity of three endodontic intracanal medicaments W. E. Kantz, D.D.S., Ph.D.,* P. J. Ferrillo, D.D.S.,“” and E. R. Zimmermanq D.D.S.,**+ Dallas, Texas BAYLOR
COLLEGE
OF DENTISTRY
Three endodontic intracanal medicaments (camphorated para-monochlorophenol, Cresatin, and Acriphen) were tested for their cytotoxic effect on HeLa cell tissue system. All three drugs showed extreme t0xicit.y to the tissue culture cells even when the concentration of each drug was in a dilution of 1: 1,000.
A
wide variety of nonspecific drugs has been advocated for intracanal medication of root canals. Camphorated para-monochlorophenol has been suggested as the drug of choice for a root canal antiseptiel-* Camphorated para-monochlorophenol has been shown to be cytotoxic to monolayer cultures of baby hamster kidney (BHK-21/4) cells” and diploid human embryonic lung (HEL-199) cells.6 This drug has also been shown to produce severe toxic reactions when placed on the conjunctiva of white rabbits, and tissue necrosis when introduced intradermally into the abdomen of the same type of animah7 Metacresyl acetate (Cresatin) is another of the nonspecific drugs used for medication of root canal systems. Cresatin has previously been suggested for this purpose because of its anodyne properties and its “remarkably low inflammatory potential.“8 Cresatin, however, has been reported to produce a mild irritation in hamster tissue9 and slight inflammation after a 24-hour intradermal injection in the abdomens of rabbits, but no inflammation 3 to 4 hours after the Supported by United States Public Health Service Training Grant DE-00226. *Assistant Professor, Department of Microbiology. ‘*Professor and Chairman, Department of Endodontics. ‘**Professor and Chairman, Department of Oral Pathology.
Volume 38 Number 4
Cytotoxicity
of three intracand
medica,m8ents 601
introduction of 0.51 ml. into the conjunctival sacs of the rabbits.lO Inflammation of pulp tissue has been demonstrated 56 per cent of the time after Cresatin pulpotomy.ll Acriphen, a combination of 9-aminoacridine and benzalkonium chloride, was adopted as an irrigating solution at one dental school because of its antimicrobial properties and its low toxicity.12 The purpose of this study was to determine the toxicity of camphorated paramonochlorophenol, Cresatin, and Acriphen on a tissue culture system. MATERIALS
AND
METHODS
The Gey strain of HeLa* cells was the tissue culture used in this study. The cells were maintained in a monolayer culture in a medium consisting of BME (basal medium Eagle) with Hank’s salts and additions of 5 per cent calf serum, 2 per cent glutamine, 2 per cent penicillin-streptomycin mixture (5,000 units per milliliter) and buffered with sodium bicarbonate (7.5 per cent w/v) solution. The pH was adjusted to 7.4. Camphorated para-monochlorophenol (CPC) ,t metacresyl-acetate (Cresatin) ,$ and 9-aminoacridine 0.01 per cent and benzalkonium chloride 0.02 per cent (Acriphen) $ were the drugs tested. Dilutions of the test drugs were prepared in the stock culture fluid media. Serial dilutions of CPC and Cresatin were made in concentrations of 1: 10, 1: 1,000, and 1: 100,000. Dilutions of Acriphen were 1: 10, 1: 100, and 1: 1,000. The HeLa cells were grown and maintained in 250-ml. tissue culture flasks (Falcon Plastic). After a monolayer became established, the cells were trypsinized for 15 minutes at 37O C. to detach them from the surface of the containers. The cells were placed in sterile 15 ml. conical centrifuge tubes, spun at 1,000 r.p.m. for 5 minutes, and resuspended in growth medium. One half of 1 ml. of the suspension of cells was inoculated into Leighton tubes, along with 1 ml. of stock medium. The tubes were sealed with rubber stoppers and placed in a horizontal position at 37* C. for 72 hours. The cells were allowed to adapt to the glass surface and establish a monolayer. Test procedure
Sterile techniques were used throughout the experimental study. At the end of 72 hours, the liquid medium was decanted from the Leighton tubes, Fortyfive tubes received 1.5 ml. of stock medium and served as controls. Fifteen tubes received 1.5 ml. of the CPC in a dilution of 1: 10 ; fifteen, a 1: 1,000 dilution ; and fifteen, a I: 100,000 dilution. The same procedure was carried out for Cresatin. The same procedure was also carried out for Acriphen, except that the dilutions were 1: 10, 1: 100, and 1: 1,000. The pH of each dilution and the control medium was adjusted to 7.4 prior to placement into the tubes. All tubes were *Microbial Associates, Inc., Bethesda, Mdd. tNovo1 Chemical Mfg. Co., Inc., Brooklyn, N. Y. thlerck, Sharp & Dohme, West Point, Pa. $Obergfel Brothers, Los Angeles, Calif.
602 Kantz, Perrdlo,
Oral Surg. October, 1974
and Zin~mermu~~n
1~~11l:lO
100 ’
--~1:1000 v-7 1: 10000C
90 . 80 ’ ,‘I:
Q$&
50 ’ 40 ’ 30 ’ 20 ’
0 KUR8
12
24
48
CPC Fig. 1. Comparison of cytotoxicity tin, and Acriphen to control.
12
24
48
CRESATIN of camphorated
12
24.
48
ACRIPHEN
para-monochlorophenol
(CPC),
Cresa-
restoppered, replaced in a horizontal position, and incubated at 37O C. At the end of 12, 24, and 48 hours, five tubes of each dilution of the test drug and five control tubes were trypsinized with a 0.25 per cent trypsin solution at 37O C. for 5 minutes. The suspended cells were placed in conical centrifuge tubes and spun for 5 minutes at 1,000 r.p.m. The trypsin solution was decanted off and the cells were resuspended in 0.5 ml. of a 0.5 per cent solution of trypan blue. The cells were exposed to the dye solution for 4 minutes. Aliquots were placed in the counting chamber of a hemacytometer, and the numbers of viable and nonviable cells were established. The nonviable cells stained a dark blue, whereas the viable cells remained clear and unstained when viewed under low power of a light microscope. All statistical analysis was made with the use of the Duncan’s multiple range and multiple F tests.13 RESULTS The average percentages of viable cells are graphically represented in Fig. 1. A comparison of CPC, Cresatin, and Acriphen at various dilutions and time intervals to control cells and to each other is shown in Table I. No statistical differences were found between any of the control viable counts at any time period or between any testing procedures. Statistically significant differences were noted with the following dilutions of drugs when compared to the number of viable cells in the controls : 1. CPC 1:lO at 12, 24, and 48 hours; CPC l:l,OOO at 12, 24, and 48 hours. 2. Cresatin 1: 10 at 12, 24, and 48 hours ; Cresatin 1: 1,000 at 12, 24, and 48 hours. 3. Acriphen 1: 10 at 12, 24, and 48 hours ; Acriphen 1: 100 at 12, 24, and 48 hours; Acriphen 1: 1,000 showed no significant difference at 12 and 24 hours but did show a significant decrease at 48 hours.
Cytotoxicity
Volume 38 Number 4
of three id-ocanal, medicaments
Table I. Comparison of CPC, Cresatin, and *4criphen at various dilutions time intervals to control cells anal each other (all values expressed as mean + S.E. [n-l] per cent viable cells) CPC 10-l 12 hr. 24 hr. 48 hr. 12 hr. 24 hr. 48 hr. 12 hr. 24 hr. 48 hr. *Represents tRepresents val and dilution
o*t 0” 0” Cres 10-l o*t 0* 0* Acr 10-l 22.74 t 1.67* 0” 0* a significant a significant only.
difference difference
CPC 10-s & o*t Cres lo-> 33.87 +_ 0.83’ ;:; Aor lo-” 19.63 + 0.74” 0” 0*
CFC lo-’ 61.47 + 3.37 53.13 2 1.82 56.89 + 2.30 Cres lo-$ 71.92 2 3.60 83.40 f 1.01 76.39 2 1.81 Acr 1O-5 53.39 + 3.70 64.53 2 3.91 34.67 f 6.11”
603 and
Control 68.91 2 2.54 63.45 k 0.46 55.55 ? 2.86 ContToz
71.67 f 3.20 72.42 2 3.76 60.14 ? 2.01 ContTol
63.67 + 9.65 78.52 + 3.38 64.51 2 1.92
between the control and test cells at that time interval. between the toxicity of each drug at same time inter-
Cellular fragments could only be identified from the 1 :lO and 1 :l,OOO dilutions of CPC. Only at a dilution of 1: 100,000 at 12 and 48 hours was no significance of toxicity noted when compared to the viable control cells. Cresatin at a dilution of 1: 10 had similar action by complete destruction of the cells. The cells exposed to the dilution of 1: 1,000 became detached and were nonviable; however, no cellular destruction was noted. Acriphen in dilutions of 1: 10 and 1: 100 was toxic to the tissue culture cells, even though complete cell death was not observed until 24 hours. These cells lost their ability to maintain a monolayer. Acriphen at 1: 1,000 dilution was not toxic to the tissue culture ,cells until after 48 hours. DISCUSSION These data again substantiate the toxicity of the more common root canal disinfectants in an in vitro system and suggest that other medicaments be investigated for this purpose. From the findings in this study and in otherq7-” it would appear that endodontists should rely more on the careful biomechanical cleansing of the root canal system rather than on the use of massive doses of toxic chemicals to eliminate microbial contamination. SUMMARY Three endodontic intracanal medicaments (camphorated para-monochlorophenol, Cresatin, and Acriphen) were tested for their cytotoxic effect on a HeLa cell tissue culture system. Serial dilutions of camphorated para-monochloropheno1 and Cresatin were made in concentrations of 1: 10, 1: 1,000, and 1: 100,000. Dilutions of Acriphen were 1: 10, 1: 100, and 1: 1,000. The tissue cells were exposed to the various dilutions of the drugs for 12, 24, and 48 hours. After trypsinization and resuspension, the nonviable tissue cells were identified by trypan blue staining. The numbers of viable and nonviable cells were determined with the use of a hemacytometer. Although Acriphen was the least toxic of the three medicaments tested, all
Oral Surg. October, 1974
of the ([rugs were cstrcmel~ to& to the tissue culture centration of each tlrug was in a tlilution of 1 : 1,000.
cells even when the con-
REFERENCES
1. Sommer. 12. F.. Ostrxnder. F. If.. and Crowlev. M. C.: Clinical Endodontics. ed. 2. Philadelphia,‘l962, W. B. Saunders Co.; pp. 194-195,” ’ 2. Luebke, R. G.: Symposium of Endodontics, Dent. Clin. North Am., p. 609, November, 1967. 3. Goldman, H. M, Forrest, S. P., Byrd, D. L., and McDonald, R. E.: Current Therapy in Dentistry, St. Louis, 1964, The C. V. Mosby Company, p. 93. St. Louis, 4. Dowson, J, and Garber. F. N.: A Chair-side Manual of Clinical Endodontics, 1967, The ‘C. V. Mosby ‘Company, p. 81. Efficacy and Cytotoxicity of Three 5. VanderWall, G. L, and Shipmnn, C.: Antibacterial Endodontic Drugs, I.A.D.R. 49th general session, March! 1971, p. 176. Efficacy and Cytotoxic6. VxnderWall, G. L., Dowson, J., and Shipman, C.: Antibacterial ity of Three Endodontic Drugs, Oaar, SIJRG. 33: 230-241, 1972. ORAL SURG. of Parachloronhenol, 7. Harrison, J. R., and Madonia, J. V.: The Toxicitr 32: 90-96. 1971. 8. Ingle, J. I.: Endodontics, Philadelphia, 1969, Lea & Febiger, p. 491. 9. Torneck. C. D.: Reaction of Hamster Tissue to Drugs in Sterilization of the Root Canal,
ORAL S~RG. 14: 730-747,1961. 10. Schilder,
H., and Amsterdam,
M.:
inflammatory
Potential
of Root
Canal
Medieaments,
ORAL SURG.12: 211-221, 1959. Response of Dental 11. Sandier, E. S., Frankl, 8. N., and Ruben, M. P.: The Histological Pulp to Cresatin, 5. Dent. Child. 38: 49-58, 1971. 12. Stark, M. H., Hall, N. C., Nicholson, R. J., and Soelberg, K.: 9-Aminoacridine, An Effective Antibacterial Agent With Caries Disclosing Features, ORAL SURG.26: 560-562, 1968. 13. Duncan, D. R.: Multiple Range and Multiple F Tests, Biometrics 11: l-42, 1955.
Reprint requests to: Dr. E. R. Zimmerman Department of Oral Pathology Baylor College of Dentistry Dallas, Texas