Invasion of dentinal tubules by root canal bacteria

Bacterial invasion of dentinal tubules and the clinical consequences have been recognized for over a century. However, while many components of the infected dentinal tubule microflora have been identified, it is likely that there are etiological agents involved in endodontic infections that have not yet been recognized. Bacterial invasion of coronal dentinal tubules occurs when the dentine is exposed to the oral environment and of radicular dentinal tubules subsequent to infection of the root canal system or as a consequence of periodontal disease. The content and architecture of a dentinal tubule can influence bacterial invasion, with tubule patency being important. This can account for regional variations in bacterial invasion and is particularly seen with dentinal sclerosis, where more advanced sclerotic changes in apical radicular tubules, especially in elderly individuals, limit bacterial invasion in this area. While several hundred bacterial species are known to inhabit the oral cavity, a relatively small and select group of bacteria are involved in invasion of dentinal tubules. Gram‐positive organisms dominate the tubule microflora in both carious and non‐carious dentine. The relatively high numbers of obligate anaerobes present, such as Eubacterium spp., Propionibacterium spp., Bifidobacterium spp., Peptostreptococcus micros, and Veillonella spp., suggests that the environment favors the growth of these bacteria. Gram‐negative obligate anaerobic rods, e.g. Porphyromonas spp., are less frequently recovered; however, with time, fastidious obligately anaerobic bacteria become established as principal components of the microflora and can be found within the deep dentine layers. In the early stages of infection, Gram‐positive bacteria dominate the microflora. The identification of adhesins that mediate these initial interactions of bacteria with dentine is important for understanding the development of tubule infection and in designing adhesion‐blocking compounds. Recent evidence suggests that streptococci and enterococci may recognize components present within dentinal tubules, such as collagen type I, which stimulate bacterial adhesion and intra‐tubular growth. Specific interactions of other oral bacteria with invading streptococci may then facilitate invasion of dentine by select bacteria. It is important therefore that the mechanisms of invasion and inter‐bacterial adhesion are understood to assist development of novel control strategies.     

Dentinal tubule invasion and adherence by Enterococcus faecalis

Aim To investigate dentinal tubule invasion and the predilection of Enterococcus faecalis for dentinal tubule walls. Methodology The invasion of dentinal tubules in extracted human teeth by E. faecalis was measured ex vivo after 8 weeks of incubation. The canal walls of 16 root sections were either intact or instrumented with or without smear layer present. Extent and maximum depth of tubule invasion were assessed histologically and compared between groups. In the adherence study, 44 vertically split root samples were prepared to expose longitudinally aligned dentinal tubules and fractured orthodentine (OD). Surfaces were exposed to E. faecalis (erythromycin resistant strain, JH2‐2 carrying plasmid pGh9:ISS1) and incubated aerobically for 2 h. Samples were processed for analysis using scanning electron microscopy. Bacterial adhesion to tubule walls versus fractured OD was calculated as number of cells per 100 μm². Results The strain of E. faecalis used in this study showed moderate to heavy tubule invasion after 8 weeks. In the adhesion studies, significantly more bacteria adhered to fractured OD than to dentinal tubule walls (anova , P < 0.001). With respect to the tubule wall, adherence was greater in inner versus outer dentine (P = 0.02) and greater when bacterial adhesion was tested in chemically defined medium than in phosphate‐buffered saline (anova , P < 0.001). Conclusions Although E. faecalis readily invaded tubules, it did not adhere preferentially to tubule walls. Initial colonization of dentinal tubules by E. faecalis may depend primarily on other factors.     

Penetration of smeared or nonsmeared dentine by Streptococcus gordonii

The purpose of this study was to investigate the penetration of smeared and nonsmeared dentine by Streptococcus gordonii. Prepared human roots, grouped as either nonsmeared or smeared, were immersed in a suspension of S. gordonii cells for 3 weeks. The roots were then prepared for scanning electron microscopy and histological analysis. Dentine discs prepared from coronal dentine were grouped similarly. Using a fluid filtration apparatus, the hydraulic conductance (Lp) of each disc was determined before and after incubation with bacterial suspension. Scanning electron microscopy evaluation of the roots following infection with bacteria showed no change in the smear layer (P<0.0001). Histological sections revealed that bacterial penetration of all the nonsmeared samples had occurred, while nine out of 10 smeared samples showed no bacterial penetration (P<0.0001). The Lp of the nonsmeared discs was significantly reduced by 42% (P<0.0001) after bacterial penetration. However, the smeared samples revealed a 1% reduction in Lp which was not significant (P > 0.05). The results suggest that dentinal smear layers are an effective barrier to dentinal tubule invasion by S. gordonii.     

Qualitative comparison of sonic or laser energisation of 4% sodium hypochlorite on an Enterococcus faecalis biofilm grown in vitro

The effectiveness of sonic activation, laser activation and syringe irrigation of 4% sodium hypochlorite in removing an Enterococcus faecalis biofilm was compared. Biofilms were grown in extracted human single rooted teeth using a flow cell apparatus. After 4 weeks' growth, teeth were subjected to each treatment using 4% sodium hypochlorite and radicular dentinal surfaces of the root canals were analysed by scanning electron microscopy. Results showed that sonic activation and syringe irrigation with sodium hypochlorite showed reduced numbers of bacterial cells on the radicular dentine but were not effective in eliminating E. faecalis in the dentinal tubules. Laser activation of sodium hypochlorite resulted in clean dentine walls and undetectable levels of bacteria within dentinal tubules. Qualitatively, sonic or laser activation of 4% NaOCl resulted in greater bacterial reduction compared with syringe irrigation, with laser activation producing the greatest overall reduction.     

Effect of conditioning films and a novel anti-adherent agent on bacterial adherence to dentine

Aim Adherence of bacteria to dentine is a prerequisite to infection of the root canal system, yet adherence of root canal bacteria to dentine is poorly understood. The aim of this study was to evaluate the effect of conditioning films and anti‐adherent compounds on bacterial adherence to dentine. Methodology Freshly extracted molar teeth were prepared and sectioned to give 225 discs of predetermined dimensions. The discs were allocated to two groups. Group 1 (n = 189) was divided into three subgroups (n = 63) and coated with one of three conditioning agents (artificial saliva, serum, or distilled water) prior to bacterial inoculation. Group 2 discs (n = 36) were treated with either a novel anti‐adherent agent (PC1036, Biocompatibles) (n = 18) or distilled water (n = 18) prior to conditioning with artificial saliva. Monospecies bacterial biofilms were generated on the dentine discs by incubating them in brain heart infusion broth (37 gL^?1) containing Streptococcus intermedius (Si), Enterococcus faecalis (Ef) or Lactobacillus fermentum (Lf) (originally isolated from infected root canals). The number of bacteria adhering to the discs in each of the groups was determined using standard serial dilution protocols. Additional discs were prepared under all conditions for scanning electron microscopy. Where appropriate, statistical analysis by one way anova , post hoc Bonferroni, and independent t‐test were used. Results Si adhered significantly better to dentine when conditioned with serum compared with artificial saliva (P = 0.005) or distilled water (P = 0.009). Conversely, Ef adhered significantly better to the control discs (distilled water) compared with serum conditioned discs (P = 0.016). The conditioning films had no effect on the adherence of Lf, which adhered to the dentine discs significantly less (P = 0.001) than either Si or Ef. The anti‐adherent coating significantly reduced the number of Si adhering to the dentine compared with the control (P = 0.012). Conclusion Given the importance of adherence in root canal infection it is conceivable that an anti‐adherent compound, could be used to prevent bacterial recontamination of cavities or the root canal system.     

Comparison of the number and diameter of dentinal tubules in human and bovine dentine by scanning electron microscopic investigation

Detailed information on dentine structure is essential for interpreting data from investigations on dentine-adhesive materials. The purpose here was to compare the number and diameter of dentinal tubules at similarly prepared surfaces of bovine permanent central incisors and human deciduous and third molars. In bovine teeth, crowns and roots were used; in human samples only the crowns were investigated. Tubule density in the middle layer was higher in bovine root (BR) dentine (number of tubules per mm(2)+/-SD: 23, 760+/-2453) than in human deciduous (HD) (18,243+/-3845), human permanent (HP) (18,781+/-5855), and bovine coronal (BC) (17, 310+/-2140) dentine. The corresponding values for the deep layer were 23,738+/-4457 (BR), 24,162+/-5338 (HD), 21,343+/-7290 (HP), and 20,980+/-4198 (BC). No significant differences were found for the number of dentinal tubules in bovine coronal dentine compared to the dentine of human deciduous and permanent molars. The mean diameter of bovine dentinal tubules was slightly, but not significantly, higher than in human dentine (middle layer/deep layer+/-SD): BC, 2. 85 microm+/-0.18/3.50 microm+/-0.33; BR, 3.10 microm+/-0.33/3.23 microm+/-0.30; HD, 2.55 microm+/-0.16/2.82 microm+/-0.28; HP, 2.65 microm+/-0.19/2.90 microm+/-0.22. These findings demonstrate that corresponding coronal dentine layers of human deciduous and permanent molars, and of bovine central incisors, are not significantly different in their number of tubules per mm(2) and their tubule diameter, whereas tubule density in bovine root dentine is significantly higher. These results suggest that provided standardized preparations are used, bovine incisor crown dentine is a suitable substitute for human molar dentine in adhesion studies.     

The permeability of dentine from bovine incisors in vitro

The permeability of coronal dentine was investigated by measuring the hydraulic conductance of dentine discs. Reductions in dentine thickness from the enamel side of disc resulted in a greater increase in permeability than reductions from the pulpal side. Scanning electron microscopy revealed fewer dentinal tubules with smaller diameters in superficial dentine than in deep dentine. The permeability of coronal incisor bovine dentine is six to eight times less than that of unerupted coronal human third molar dentine but similar to that of human root dentine.     

Efficacy of chlorhexidine in disinfecting dentinal tubules in vitro

Abstract Solutions of 0.2% and 2% chlorhexidine, 0.2% and 2% sodium hypochlorite (NaOCl) and normal saline were tested for their efficacy in disinfecting dentinal tubules following root canal irrigation in vitro. Freshly extracted bovine incisor roots were prepared as cylindrical shapes, 4 mm high and 5 mm wide with a lumen 2.1 mm wide. After mechanical removal of the cementum and elimination of the smear layer on the dentine surface with EDTA and NaOCl, the root sections were autoclaved and the dentinal tubules infected with E. faecalis (NCTC 775) by incubating in yeast extract glucose broth for 1 week. The root canals were irrigated with 20 ml of an irrigant solution using a syringe. Each irrigant was used in six specimens. Dentine was removed from the canal wall by sterile burs of increasing diameter to give samples 100, 100–300 and 300–500 μm deep. The dentine samples were then cultured to determine the presence and quantity of remaining micro-organisms. The results indicated that chlorhexidine and NaOCl were equally effective antibacterial agents at similar concentrations against the test microorganism. They significantly reduced the bacterial counts in the first 100 μm of dentinal tubules, however up to 50% of dentine samples remained infected following use of both agents.     

The cervical wedge-shaped lesion in teeth: a light and electron microscopic study

Background:  The cervical non-carious wedged-shaped lesion is controversial in that its aetiology may involve attrition, erosion, abrasion and stress-corrosion (abfraction). This study examined the histopathology of anterior teeth with cervical wedge-shaped lesions by light and electron microscopy to elucidate their pathogenesis.
Methods:  Ten undecalcified human teeth with cervical lesions were available for investigation. Patency of the dentine tubules was tested using red dye penetration from the pulp chamber. The morphology of normal and sclerotic dentine adjacent to the cervical wedge-shaped lesions was investigated by scanning electron microscopy. The numbers and diameters of dentinal tubules were measured at different levels beneath the surfaces of the lesions.
Results:  The gross and microscopic features of the worn teeth were described. Red dye penetration tests showed white tracts of sclerotic tubules contrasted with red tracts of patent tubules. Numbers of tubules per square area and diameters of patent and sclerotic tubules varied at different levels within the dentine due to deposits of intratubular dentine.
Conclusions:  The cervical wedge is shaped by interactions between acid wear, abrasion and dentinal sclerosis. No histopathological evidence of abfraction was found. Clinical diagnosis, conservation and restoration of non-carious cervical lesions need to take into account the extent of sclerotic dentine beneath wedge-shaped lesions.     

Dentine hypersensitivity

Based on the hydrodynamic theory for stimulus transmission across dentine, it would be logical to conclude that teeth exhibiting the clinical symptoms referred to as dentine hypersensitivity should have dentinal tubules open at the root surface and patent to the pulp. With the exception of studies on cut dentine, there is little direct evidence to support this conclusion. In this study, caries-free teeth with exposed cervical root areas scheduled for extraction which were classified as non-sensitive or hypersensitive after suitable stimulation were examined by scanning electron microscopy. Hypersensitive teeth showed highly significantly increased numbers of tubules per unit area (approximately 8 X) compared with non-sensitive teeth. Tubule diameters were significantly wider (approximately 2 X) in hypersensitive compared to non-sensitive teeth. The number of teeth showing the penetration of methylene blue through the zone of exposed cervical dentine was larger and the depth of penetration greater in hypersensitive teeth compared to non-sensitive teeth. The results provide further evidence that stimulus transmission across dentine in hypersensitive teeth is mediated by a hydrodynamic mechanism. An understanding of factors which open dentinal tubules would seem important if attempts to prevent or treat dentine hypersensitivity are to be successful.     

The effect of lathyrism on dentin structure of the rat incisors: a morphometric and scanning electron microscopic investigation

J Oral Pathol Med (2010) 39 : 424–430 Background: The present study was designed to study the effect of β‐aminopropionitrile (β‐APN), present in Lathyrus sativus grass pea consumed in drought prone areas, on dentin of the continuously erupting rat incisors. Methods: Eighteen adult male rats were used. In the experimental group (18 rats), lathyrism was induced by a once daily subcutaneous administration of β‐APN for 40 days. The maxillary and mandibular incisors were examined ultrastructurally and morphometrically. Results: The mean number of patent tubules, the mean area, perimeter and the area percent of the tubules were analyzed. Ultrastructurally, the dentinal tubules of both coronal and radicular dentin in the lathyritic group were narrower or even obliterated compared with those in the control. The coronal and radicular dentin of the lathyritic group exhibited an irregular lattice of non‐mineralized small branching collagen fibrils obliterating the dentinal tubules. The mean number of patent tubules in the control and lathyritic groups revealed an insignificant difference. The mean area of the tubules showed a statistically significant difference in lathyritic radicular dentin (P = 0.0353). The percentage of the total surface area of the dentinal tubules significantly decreased in the radicular dentin of the lathyritic group (P = 0.024). Conclusions: These findings indicated a deleterious effect of lathyrism on dentin, with a possible negative impact on developing teeth integrity.     

Dentine tubule infection and endodontic therapy implications

A critical review of the literature suggests that the microenvironment of dentinal tubules appears to favour the selection of relatively few bacterial types irrespective of the aetiology of the infection process; coronal dental caries or pulpar necrosis. These bacteria may constitute an important reservoir from which root canal infection and reinfection may occur following pulp necrosis or during and after endodontic treatment. Previous studies of this microflora have utilized microbiological culture techniques which need to be supplemented by those that allow In situ demonstration as well as identification of the bacteria. Newer treatment strategies that are designed to eliminate this microflora must include agents that can penetrate the dentinal tubules and destroy these microorganisms, since they are located in an area beyond the host defence mechanisms where they cannot be reached by systemically administered antimicrobial agents.     

A laboratory study of the effect of calcium hydroxide mixed with iodine or electrophoretically activated copper on bacterial viability in dentinal tubules

AIM: The aim of this laboratory study was to evaluate the ability of calcium hydroxide (CH), CH/iodine-potassium iodide (IKI) and electrophoretically activated copper to kill bacteria in dentinal tubules. METHODOLOGY: In an in vitro model of dentinal tubule infection, 42 cylindrical root specimens, prepared from freshly extracted bovine teeth were used. After removal of the smear layer, intracanal dentinal tubules were infected with Enterococcus faecalis for 3 weeks. CH alone or preparations of CH with copper or IKI were placed in the root canal for 1 week. In specimens containing copper/CH, an electrophoretic current(5 mA/5 min) was applied using two electrodes follow-ing placement of the medicament in the canal. Powder dentine samples obtained from the canal wall using ISO sizes: 025, 027, 029, 031 and 033 burs were examined for the presence of viable bacteria by inoculating agar plates and counting colony forming units (cfu). RESULTS: A significant difference was found between the experimental groups and the positive control group. CH and CH/IKI significantly (P < 0.001)reduced bacterial viability in dentinal tubules to a depth of 200 microm. Specimens with CH/IKI had significantly fewer viable bacteria than CH alone in tubules between the depths of 200-500 microm. Treatment with CH/copper and electrophoresis was most effective: specimens showed no viable bacteria in dentinal tubules to a depth of 500 microm from the root-canal space. CONCLUSIONS: IKI or electrophoretically activated copper additives can significantly improve the antibacterial properties of CH in dentinal tubules.     

Density of dentinal tubules affects the tensile strength of root dentin.

OBJECTIVES: The aim of this study is to count the dentinal tubules in the coronal and middle-apical third of root dentin of teeth extracted due to the progression of periodontal disease, and to compare the Ultimate Tensile Strength (UTS) of the same areas. The research hypothesis was that root dentin areas with different densities of dentinal tubules would also show different UTS values. METHODS: From 10 caries free maxillary central, lateral incisors and canines, extracted for periodontal reasons from three patients, cylindrical specimens approximately 10 mm long were prepared parallel to the long axis of the root and then divided into two parts using a low speed diamond saw one from the coronal third of the root, (Group 1) and one from the middle-apical third of the root (Group 2). The density of the dentinal tubules of the specimens of the two groups was measured by means of a scanning electron microscope and the UTS of the specimens was measured by a microtensile test. One way ANOVA was used to assess the effect of specimen location (coronal specimens vs. middle-apical specimens) on UTS. The differences in the density of dentinal tubules between coronal and middle-apical specimens were also subjected to statistical analysis using one-way ANOVA. RESULTS: UTS values of middle-apical specimens were found to be significantly (p < 0.05) higher than those of coronal specimens. The results of the one-way analysis of variance showed that the number of dentinal tubules of the samples from the coronal part of the root groups was significantly higher than that of samples from the middle-apical part (p < 0.05). SIGNIFICANCE: These results suggest that high values of tensile strength of the dentin are associated with low densities of dentinal tubules and that apical areas of root dentin are more resistant to tension than coronal ones.     

A morphometric analysis of the cross-sectional area of dentine occupied by dentinal tubules in human third molar teeth

The number and the mean percentage tubular cross-sectional area of dentinal tubules per square millimetre were calculated in specimens of coronal dentine of 13 intact human third molar teeth from patients 18 to 28 years of age. The dentine was fractured at various known distances from the dentino-enamel junction. Near the dentino-enamel junction the number of tubules per square millimetre was 22 000 and the mean tubular cross-sectional area was 3.6%. Midway between the pulpal wall and the dentino-enamel junction the number of tubules was 37000 mm^?2 and the mean tubular cross-sectional area was 6.2%. Close to the pulp the number of dentinai tubules was 48000 mm^?2 and the mean cross sectional area of tubules was 10.2 percent. The number of tubules per square millimetre more than doubled and the area occupied by tubules increased threefold from the dentine close to the dentine -enamel junction, to that close to the pulp. These differences in tubular patterns at different depths in dentine are clinically significant in dentine permeability, the treatment of traumatized teeth, and pain transmission in dentine.     

Bacterial eradication from root dentine by ultrasonic irrigation with sodium hypochlorite

The study aimed to evaluate intracanal irrigation procedures in eradicating bacteria from surface, shallow and deep layers of root dentine using extracted human teeth. Artificial bacterial smear layer was successfully produced by rubbing a mixture of dental plaque and artificially decalcified dentine or carious dentine on root canal walls. The reservoir holes were 3.5 mm in depth, 1 mm in diameter prepared 1.5 mm apart and parallel to the root canals on the decrowned planes, in which five separate bacterial species were placed ( Actinomyces israelii, Fusobacterium nucleatum, Propionibacterium acnes, Streptococcus mutans and Streptococcus sanguis ). Bacterial eradication after irrigation of the prepared canals was determined by bacterial recovery (i) from the root canal surfaces and shallow layers where bacteria were smeared artificially and (ii) from deeper layers of root canal dentine reservoir holes.
Ultrasonic irrigation with 5.5% and 12% NaOCl eradicated bacteria from artificial smear layer ( P <0.0001), whilst 12% NaOCl irrigation with a syringe was insufficient. Ultrasonic irrigation with water or 15% EDTA failed to eradicate bacteria from smeared surfaces. Ultrasonic irrigation with 12% NaOCl killed A. israelii, F. nucleatum, P. acnes, S. mutans , and S. sanguis placed in reservoir channels, although for F. nucleatum , a very small number of bacteria remained in five samples out of 12. Ultrasonic irrigation with less concentrated NaOCl failed to eliminate bacteria completely from reservoir channels in most samples. Ultrasonic irrigation with 12% NaOCl appeared to eliminate bacteria efficiently from surface, shallow and deep layers of root dentine.     

Human root caries: histopathology of arrested lesions.

The histopathology of arrested root caries lesions was examined in extracted human teeth. The main structural characteristics of arrested lesions were the completely mineralized surface area and the formation of a distinct sclerosis of the dentinal tubules. Intertubular dentin was, with the exception of the dentinal tubules, fully mineralized up to the surface. Dentinal tubules near the surface were either filled with ghosts of microorganisms or with crystals of different shapes. Sclerosis of the dentinal tubules was characterized by the presence of three different patterns of intratubular mineralization that occur in distinct regions of the zone of tubular sclerosis. The patterns were distinguishable by the type of crystals and their association with organic structures such as collagenous fibrils or odontoblast processes. It is suggested that arrested lesions are based on (1) the formation of an inner barrier that interrupts the diffusion of substrata from the pulp to invaded bacteria, (2) the formation of an outer barrier by a compact, highly mineralized surface region which blocks the diffusion of products of bacterial metabolism into dentin, and (3) an area of mineralization which extends from the outer barrier toward the root canal within demineralized dentin. The present study demonstrates the considerable potential of caries lesions in dentin to become arrested, and subsequently partially remineralized. These phenomena seem to depend on the severity of an active lesion and its location on the root surface. This should be taken into account when diagnosing root caries lesions. The potential of root caries lesions to become arrested indicates that the treatment concept of active root caries lesions should be reconsidered.     

Effect of electrophoretically activated calcium hydroxide on bacterial viability in dentinal tubules –in vitro

Abstract –  To evaluate the ability of electrophoretically activated calcium hydroxide (CH) to eliminate bacteria in dentinal tubules. In an in vitro model of dentinal tubule infection, 18 cylindrical root specimens prepared from freshly extracted bovine teeth were used. After removal of the smear layer, intracanal dentinal tubules were infected with Enterococcus faecalis for 21 days. In 12 specimens, CH paste was placed in the root canals for 7 days. In six of these, an electrophoretic current (10 mA per 10 min), using two electrodes, was applied after placing the medicament in the canal. Powder dentin samples obtained from within the canal lumina using ISO 025, 027, 029, 031 and 033 burs were examined for the presence of vital bacteria by inoculating agar plates and counting colony forming units. anova with repeated measures was used to analyze results. A significant difference was found between experimental groups and the positive control group. CH and electrophoretically activated CH significantly ( P  < 0.001) reduced bacterial viabilities in dentinal tubules to a depth of 200  μ m. Treatment with electrophoresis was significantly ( P  < 0.001) more effective than pure CH in depths of 200–500  μ m. Specimens treated with electrophoretically activated CH showed no viable bacteria in dentinal tubules to a depth of 500  μ m from the root canal space within 7 days. The time required for treatment of pulpal infection root resorption may be decreased, thus minimizing the risk of coronal fractures in young patients with traumatized teeth.     

Penetration of bacteria in bovine root dentine in vitro

AIM: The aim of this study was to develop a test model to quantify the penetration of bacteria into dentinal tubules. METHODOLOGY: The model consisted of two compartments separated by a bovine dentine specimen with a thickness of 1.5-3.1 mm. The root cementum was removed from the root surface and the specimens were oriented in the model with the pulpal side facing the inoculated chamber of the test model. One compartment contained the test organism and the other was filled with sterile broth that was evaluated for growth of the test organism. The depth of bacterial penetration was measured in the dentine with or without a smear layer using both a histological and a quantitative recovering grinding technique, after 6 weeks of exposure to the microorganisms. RESULTS: E. faecalis penetrated dentine significantly deeper than A. israelii (P < 0.001). After removal of the smear layer with EDTA, E. faecalis penetrated significantly deeper than in dentine pretreated with saline only (P < 0.01) or with a combination of saline and sodium hypochlorite (P < 0.01). Microorganisms were found in 89% of the cultured specimens and in 80% of the specimens that were evaluated with light microscopy. Total penetration through the dentine specimen and infection of the broth in the test compartment of the model occurred in only two out of 72 specimens. CONCLUSION: Collection and immediate culturing of infected dentine dust and counting colony forming units (CFU) allowed an overview of the number of bacteria per sample and was more sensitive than microscopy. Removal of the smear layer enhanced bacterial penetration.