Bacteriologic study of a basic fuchsin caries-disclosing dye

Bacteriologic study of a basic fuchsin caries-disclosing dye

COMPOSITE RESIN POROSITY materials that showed increases in small voids with syringed samples were three of the four materials that had the largest...

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COMPOSITE

RESIN

POROSITY

materials that showed increases in small voids with syringed samples were three of the four materials that had the largest areas occupied by large voids for the hand-inserted materials.

Table III. Comparison of total mean porosity areas of hand and syringe inserted composite resins (lo4 Km*) Hand Material

Mean

Concise Durafill Phaseafill Isopast Silar Extra Smooth Finesse Adaptic Vertical (Duncan

27 28 55

bars connect test).

Syringe SD

Mean

SD

4 7 8

3 5 5

8 31

12

19

11

30 35

19 23

291 18 / 26j

65

37

67 106

34 51

120 129

170 185

means nor significantly

dilferent

4

at 0.05 level

CONCLUSIONS The composite resin syringe is an effective instrument for reducing porosity in clinical composite resin restoration. The manufacturer must still strive to provide materials that are inherently pore-free. The pore content of even light-cured resins is reduced by syringe insertion. REFERENCES I. 2.

are two possible explanations. First, the shear stresses induced by syringing may break large pores into smaller pores. If this were true, an increase in medium-size pores would be expected also, but none was observed. In addition, such shear reduction would be expected to increase the small voids in all materials rather than just in three of eight. The other possible explanation is that the large voids simply mask surface area that otherwise would have been observed to be covered with small voids. This effect would be most marked in samples with the greatest area occupied by large voids. Indeed, the

3. 4. 5.

Dragan WH: ,2 simplilied method f'or the precision placement of composite resins and cements. J Acad Gen Dent 19:l 1, 1071. Fischel IIF, Tay WM. ElTect of manipulative tec,hniques on porosity in composite resins. J Dent Res 56(Abstr No. 432) 1977. Finger W, Jorgensen KD: Porosity in composite resttrratix resins. J Drm Res 56(Abstr No. 433) 1977. Gjerdet NR, Hegdahl 7’: Porosity of resin filling materials. Acta Odontol Srand 36~303, 1978. Zinck JH: Composite resin systems: A comparison. Dent Student 60:s 1, 1987.

Bacteriologic study of a basic fuchsin caries-disclosing dye Maxwell H. Anderson, Gerald T. Charbeneau, University

of Michigan,

School

D.D.S., M.S.,* Walter D.D.S.; M.S.;** of Dentistry,

Ann

Arbor,

T. Loesche, D.M.D., -

The views expressed herein are those of the authors and in no way reflect the views of the Navy Dental Corps or the Department of the Navy. *Operative Dentistry Consultant, Naval Dental Clinic, Naval Station, San Diego, Calif. **Professor, Department of Microbiology. ***Professor and Chairman, Department of Operative Dentistry. JOURNAL

OF PROSTHETIC

DENTISTRY

and

Mich

lreparation of teeth to receive restorations normally involves the removal of all carious tooth tissue. The adequacy of caries removal is a subjective judgment based on discoloration and/or the texture of dentin. It

THE

Ph.D.,**

may be based also on more objective criteria. Various dye techniques have been developed and tested in vivo and in vitro.‘-’ They purport to differentially stain cariously affected dentin while leaving potentially sound dentin unstained.8-‘0 The most thoroughly investigated dye is 0.5% basic fuchsin in propylene glycol.*-‘3 The clinical significance of residual dye-stainable dentin has not been determined. However, in terms of residual caries, if the stainable material is infected as well as softened, the implication of leaving this material in completed cavity preparations is apparent. 51

ANDERSON,

KKzD SAMPLE ---

l.Oml

l.Oml

I1

I-

10.0 ml RTF with EDTA

@jg

9.0 ml RTF with EDTA

@

@

Fig. 1. Dentin sample dilution sequence.

Table I. Data from part 1’ Sample 1s NS 2s NS 3s NS 4s NS 5s NS

Weight (MG)

Log 10 CFU/mg

2.05 1.65 1.15 1.95 2.05 1.75 0.80 1.75 1.85 2.30

7.5694 7.1238 7.7566 7.7679 7.4669 7.8756 6.1038 6.3579 6.6031 6.1732

S = Stained sample; NS = nonstained sample. *Means: S = 7.1000; NS = 7.0597; t = 0.08; p = 0.93

Bacteriologic experiments have been performed to determine whether viable organisms remain on the dentinal surface at the termination of routine cavity preparation and to determine the probable fate of residual organisms in the presenceof various medicaments.‘4-23 Besic16showed that bacteria left in a cavity preparation could survive for periodslonger than a year, but he also noted that there was no indication of caries progressionduring the observation period. Schouboeand Macdonaid” found that fermentative organisms remained viable under nonantiseptic restorations for as long as 139 days. They, too, could demonstrate no adverseeffects generated by these bacteria. Leung et al-l8 found that bacteria in grossly softened carious dentin, sealed from the oral environment and isolated from medicaments,showed a dramatic increase in the number of colony-forming units per milligram (CFU/mg) of carious dentin. The number of CFU/mg could be decreasedwith the use of a proprietary calcium hydroxide product. ” However, to date there have been no reports on the bacteriologic aspectsof dye-stainable vs. nonstainabledentin. The purposeof this study was to 52

LOESCH.

AND

CHARBENEAC

determine the relative concentration of bacteria in fuch. sin-stainable and nonstainable dentin.

MATERIAL

AND

METHODS

Two separate bacteriologic experiments were performed. The first was designedto evaluate the effect of ;+ sterile 0.5% basicfuchsin in propylene glycol solution on the viability of bacteria indigenousto advancing dentinal caries. The secondexperiment was designedto detcrmine if there was a difference in the number of bacteria per unit weight of fuchsin-stained carious dentin cornpared with contiguous dentin that was not fuchsinstainable.

Clinical

aspect: Part 1

After administration of a local anesthetic, a rubber dam was applied to isolate the test tooth, The rubber dam was rinsed clean with water from the air-water syringe, and the cavity was prepared usingsterile burs in a high-speed handpiece with air-water cooling. The preparation was extended until a visually and tactually sound dentinoenamel junction (DEJ) was established. At that point, softenedcarious dentin was excavated with a sharp sterile spoon excavator. The samplewas placed on a sterile 2 X 2 inch gauze pad and divided approximately in half. Each half was quickly weighed in a preweighed aluminum pan on a scale (Federal Pacific Electric Co., Northboro, Mass.) accurate to 0.05 mg, Half of the samplewas stained with a 0.5% solution of basic fuchsin in propylene glycol for 10 seconds.This was adequate to completely stain the dentin asjudged by visual examination. Each half was submerged in a separatetest tube containing 10 ml of reduced transport fluid (RTF) with EDTA for transport to the bacteriology laboratory.?’

Clinical

aspect: Part 2

Freshly extracted carious teeth were obtained from the Oral Surgery Department of the University of Michigan, School of Dentistry. The teeth were placed immediately on extraction in a solution of RTF with EDTA. With minimal delay, the teeth were prepared using a sterile bur and a slow-speedhandpiece without air-water spray. The teeth were kept moist with fresh RTF with EDTA, and generation of excessiveheat was avoided by the use of the slow-speedhandpiece. When the outline form was established,a drop of sterile 0.5% basic fuchsin was applied to the prepared tooth for 10 seconds.The excessdye was flushed from the preparation with sterile saline solution from a 10 cc syringe through a blunted l&gauge needle. The stained dentin was excavated from the preparation with a sharp sterile spoon excavator until none remained. The tooth was restained with fuchsin dye for 10 seconds.If any newly exposed dentin absorbedthe stain, the previously excaJULY

1985

VOLUME

54

NUMBER

I

CARIES-DISCLOSING

DYE

Table II. Data from part 2* Sample IS

Location Pulpal

wall

Buccal

and

Weight

NS 2s

lingual

walls

of proximal

portion

Pulpal

wall

Buccal

and lingual

walls

of proximal

portion

NS 3s NS 45 NS 5s

DEJ, gingival

wall

DEJ, gingival

wall

NS 6s NS 7s NS 8S NS 9s NS 10 s NS

DEJ, buccal

and gingival

walls

of proximal

portion

DEJ, buccal

and gingival

walls

of proximal

portion

walls

of proximal

portion

DEJ, gingival DEJ, buccal

wall and gingival

2.40 0.50 2.10 0.65 1.05 0.65 0.95 0.40 1.05 0.50 0.80 0.60 0.75 0.45 1.35 0.35 0.90 0.65 0.35 0.45

(mg)

Log 10 CFU/mg 6.2253 3.9845 7.3766 4.1038 6.5855 3.7251 6.4346 2.5185t 6.5340 2.51851 5.8621 3.4579 5.6425 2.5185t 5.7882 2.5185t 6.6665 2.5185? 5.5302 2.5185t

S = Fuchsin stained; NS = fuchsin nonsrained. *Mean: S = 6.2688; NS = 3.0380; I = 11.40; ,!I = 0.0001. tNo growth detected.

vated stained dentin was discarded and the newly stained material was excavated. This procedure was repeated until no further absorption of the fuchsin dye could be demonstrated. At this point, the current sample of excavated fuchsin-stained dentin was weighed and submerged in 10 ml of RTF with EDTA. An additional dentin sample was then obtained from the first nonstained layer, weighed, and placed in 10 ml of RTF with EDTA.

Laboratory

procedure

The dentin samples in RTF with EDTA were dispersed with .a Tekmar homogenizer (Teckmar Co., Cincinnati, Ohio) for 20 seconds and sonified (Sonifier Cell Disruptor, Heat Systems Ultrasonics, Planeview, N.Y.) for 10 seconds.‘8 Each sample was thoroughly mixed in a Vortex mixer (Scientific Industries, Bohemia, N.Y.), and 1 ml of the sample RTF with EDTA solution was drawn off and placed in 9 ml of RTF with EDTA, thereby creating a loo-fold dilution. After thorough mixing of the loo-fold dilution in the Vortex mixer, 1 ml of the dilution was placed in 9 ml of RTF with EDTA to create a lOOO-fold dilution (Fig. 1). A spiral plater (Spiral Systems, Cincinnati, Ohio) was used to plate 0.03 ml of each sample dilution on plates of MM10 culture media.25 After plating, the samples were placed in an anaerobic chamber at 37” C and incubated for 5 days. The plates were counted using a dissecting microscope; the number of CFU was recorded and converted to CFU/mg of dentin. THE

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DENTISTRY

RESULTS Part 1 Five sample sets of carious dentin were examined. Table I provides the data. The data were analyzed by the two-sample t test, which yielded a value oft = .08, giving a significance of p = .93. Therefore, no significant difference between the CFU/mg of fuchsin-stained and unstained samples could be demonstrated. Testing was discontinued at five sample sets because the data reflected no significant differences. Since the basic fuchsin/propylene glycol stain did not significantly affect the growth of the bacteria indigenous to carious dentin, part 2 of the study was initiated.

Part 2 Ten teeth provided 20 samples (10 fuchsin-stained and 10 nonstained). The data are presented in Table II. To more nearly satisfy the normality assumption of the two-sample t test, the total counts of CFU/mg were converted to CFU log base 10 values. If a no growth condition was found, a value of 330 CFU/mg (or log,, 2.5185) was used for statistical purposes, because this was the minimal sensitivity of the testing procedure. Statistical analysis by two-sample t test demonstrated that the fuchsin-stained dentin contained more CFU/mg than the nonstained dentin (t = 11.4; p < .OOOl). The fuchsin-stained dentin always contained demonstrable bacteria, while six of the 10 nonstained samples showed no growth. When the means of the CFU/mg of the fuchsin-stained and nonstained dentin samples were 53

ANDERSON,

compared, it was found that the average fuchsin-stained sample contained 1300 times more CFU/mg than the adjacent nonstained sample. There was a strong tendency for the fuchsin-nonstained samples taken at the DEJ to be below minimal detectable CFU/mg. Sample 6 was the only exception. Samples taken from the pulpal wall and the buccal and lingual walls of the proximal portion demonstrated a significant number of CFU/mg in fuchsin-nonstained areas.

DISCUSSION Caries removal during cavity preparation, although it is biologically desirable, appears to be a clinical uncertainty from a bacteriologically necessary standpoint. 14,I’. ?‘,” The long-term significance of bacteria remaining within dentin is unknown. Some authors have used quantitative culturing procedures and reported significant reductions in CFU/mg counts when medicaments such as zinc oxide and eugenol and calcium hydroxide were applied. “. 2’,‘2 In teeth in which medicaments were not used, the residual flora was capable of doubling during the month following insertion of a restoration.” Other authors have used qualitative culturing procedures and shown continued viability of some organisms after prolonged periods beneath a restoration.“,” It appears that survival or growth of residual dentinal organisms is predicated on their ability to fulfill their nutritional requirements. If nutrients are available, either through marginal leakage or diffusion from the pulp, increased numbers of organisms can occur and possibly lead to renewal of the carious process. The location of the residual caries organisms within a cavity preparation may be an important determinant of recurrent caries. If the bacteria are located on the pulpal or axial wall of the preparation where medicaments can be applied and retained, and if nutrient sources from microleakage are unavailable, then most of the bacteria will die.18,19. 22 In such teeth, there is little evidence of caries progression. ‘j, I6 However, if the bacteria reside in areas near the cavosurface margins of restorations that exhibit microleakage, they have access to a relative abundance of intraoral nutrients. These conditions appear to prevail at the DEJ under metallic restorations. A recent study demonstrated that fuchsin-stainable dentin was present at the DEJ in 59% of the teeth after caries removal in cavity preparation.26 The present study showed that similarly stained dentin contained significantly more CFU/mg than nonstained dentin. This suggests that removal of all fuchsin-stainable dentin could greatly decrease the chances of viable bacteria remaining at or near the cavosurface boundary. This finding is supported by the early work with fuchsin of Fusayama and Terachima* and Fusayama and Kurosaki”’ but contradicts later findings that the fuchsin dye preceded bacterial invasion.” 54

LOESCH,

ANI)

CHARRENIiAl’

This study also demonstrated that dentin containing less than 10,000 CFU/ m g was normally not disclosed 1~ the fuchsin dye, whereas counts of greater than 550,OO~~ CFU/mg dentin were readily stained. This suggests :h,~i the dye can be used to approximate the bacterial load (ii the dentinal surface because a certain bacterial tn,ts~, apparently needs to be present before the dve is absorberf by the dentin. The clinical sequela of viable bacteria remaining a~.01 near the DEJ is not known. It is hypothesized that ;: significant amount of so-called recurrent caries is in Iat:r residual caries, or caries that was not excavated :?I rhe initial preparation. Use of a basic fuchsin/propylen(-, glycol or other diagnostic dye could limit the amount oi infected dentin that remains at the time of caviry preparation. A reduction of the residual bacterial concentration by a factor of 1300 was demonstrated by using fuchsin dye as a diagnostic guide in this study. The dye technique should be viewed as an adjunctive clinic*ai procedure to aid the dentist in treatment. of carious teeth. The investment of time is minimal, and the removal oI cultivable bacteria is more assured with the USC of’ 3 dye.

CONCLUSIONS Within the limitations of this study, the following conclusionsare made. 1. A 0.5% solution of basic fuchsin in propylene glycol showed no statistically significant effect on the viability of the bacteria indigenous to carious dentin. 2. Cariously affected dentin, as delineated by a 0.5% solution of basic fuchsin in propylene glycol, contained 1300 times more CFU/mg than dentin that did not take up the dye. 3. There was a strong tendency for the DE J to be free of demonstrable bacteria if all fuchsin-stained dentin was excavated. 4. The deepestportion of a eariouslesion can contain low numbers of bacteria in advance of fuchsin-stainable dentin. REFERENCES I. 2.

3.

4. 5. 6. 7

CARIES-DISCLOSING

8. 9. 10. 11. 12. 13.

14. IS. 16. 17. 18.

19.

DYE

Fusayama ‘r, Terachima S: DifTerentiatiob of two layers of carious dentin by staining. J Dent Res 51:866, 1972. Franc0 SJ, Kelsey WP: Caries removal with and without a disclosing solution of basic fuchsin. Oper Dent 6~46, 1981. Fusayama ‘T, Kurosaki N: Structure and removal of rarious dentin. Int Dent J 22:401, 1972. Ohgushi K. Fusayama T: Electron microscopic structure of the two layers of carious dentin. J Dent Res 54~1019, 1975. Sato Y. Fuaayama T: Removal of dentin by furhsin staining. J Dent Res 55:678, 1976. Shimizu C, Yamashita T, Fusayama T: Carious change of dentin observed on longspan ultrathin sections. J Dent Res 60~1826, 1981. Znnder H.\: Bacteria in the dentin after cavity preparation. Illinois Dent J 9:207, 1940. Seltzer S: The bacteriologic status of dentin after cavity preparation. J Am Dent Assoc 27:1799. 1940. Besic FC: The fate of bacteria sealed in dental cavities. J Dent Res 22~349, 1943. Schouboe ‘I‘, Macdonald JB: Prolonged viability of organisms sealed in dentinal caries. Arch Oral Biol 7:52S, 1962. Lrung RI,. Loesche WJ- Charbeneau GT: EHect of Dycal on bacteria in deep carious lesions. J Am Dent Assoc 100:193, 1980. King JB, (Irawtord JJ, Lindahl RL: Indirect pulp capping: A

Structural effects of bleaching vital rat teeth

20. 21. 22. 23.

24. 25. 26.

bacteriologic study of deep carious dentin in human teeth. Oral Surg 20:663, 1965. Fisher FJ: The viability of micro-organisms in carious dentine beneath amalgam restorations. Br Dent J 121:413, 1966. Fisher FJ: The effect of a calcium hydroxide/water paste on micro-organisms in carious dentine. Br Dent J 133:19, 1972. Fisher FJ: The effect of three proprietary lining materials on micro-organisms in carious dentinr. Br Dent J 143:231, 1977. Fairbourn DR, Charbeneau GT. I,oesche WJ. Ellcct of improved Dycal and IRM on bacteria in deep various lesions. J Am Dent Assoc 100~547. 1980. Syed SA, Loesche WJ: Survival of human dental plaque flora in various transport media. Appl Mwobiol 24:6X, 1977. Loesche WJ, Syed SA: The predominant cultivabit Ilora ot plaque and carious dentin. Caries Res 7:X1, 1973. Anderson MH: A Comparison of Digital and Optical (Criteria Used for Detecting Sound Dentin. blaster‘s Thesis. 1982, Llniversity of hlichigan, School ot Dcntistr\

on tetracycline-stained

William Pamela

R. Ledoux, D.D.S.,* R. B. Malloy, D.D.S., Ph.D.,** R. V. V. Hurst, D.D.S.,**+ McInnes-Ledoux, B.D.S., M.Sc. (Dent.),**** and Roger Weinberg, Ph.D.*****

Louisiana

State University, School of Dentistry, New Orleans, La.

S

taining of permanent teeth by tetracycline is a frequently encountered problem. Tetracycline is a broad-spectrum antibiotic used prophylactically and therapeutically to treat infections of the respiratory, gastrointestinal, and urinary tracts. Although tetracycline’s ability to stain developing teeth has been known since 1956,’ it is still the drug of choice for somemedical conditions, for example, cystic fibrosis.2-4 Tooth discoloration ranges from gray or brown to black and causesgreat anguish for children in a society that places a high value on appearance.’ Methods for Presented at the International Association for Dental Research, Orleans, La. *Clinical Assistant Professor, Department of Orthodontics. **Private practice, Lafayette, La. ***Private practice, New Orleans, La. ****Assistant Professor, Department of Biomaterials. *****Professor. Department of Biometry. THE

JOURNAL

OF PROSTHETIC

DENTISTRY

New

restoring original tooth color have included (1) complete crown coverage, (2) laminate veneers, (3) removal of surface enamel with subsequentdirect bonding of composite resin veneers, and (4) bleaching. Complete crown coverage offers good esthetics but destroys large amounts of tooth tissue. In addition, this technique requires maintaining young patients for the rest of their lives with complete crowns. Laminate veneersrequire considerablelaboratory and operatory time, as well as the addition of possible unacceptable bulk to the tooth. In addition, marginal refinement is difficult.6 Compositeresin veneers,a new technique, show great promise. However, patient cooperation is essentialfor the elimination of gingivitis before application, and maintenance of good oral hygiene after treatment. In addition, the dentist must create acceptabletooth color without the aid of accurate shadeguides. 55