This study was carried out to investigate the presence and types of bacteria invading the deep layers (0.5-2.0 mm from the surface of the root canal wall) of infected dentine of human root canals by sampling with an anaerobic glove box system the split surfaces of eight freshly extracted teeth. More bacteria were recovered after incubation in an anaerobic glove box than after aerobic incubation in air with 30 per cent CO2. Out of 256 predominant bacterial isolated, 205 isolates (80 per cent) were obligate anaerobes. These findings suggest that the environment of deep layers of endodontic dentinal lesions is anaerobic and favours the growth of anaerobes. Among the obligate anaerobic isolates, strains belonging to Lactobacillus (30 per cent) and Streptococcus (13 per cent) were predominant, followed by Propionibacterium (9 per cent). No strains of obligate anaerobic Gram-negative rods were isolated. The microflora of deep layers of infected root dentine is somewhat similar to that of the deep layers of carious lesions in coronal dentine.

Anaerobic procedures were adopted to demonstrate the early bacterial invasion of non-exposed dental pulps, and to isolate and identify the bacteria. Of 19 freshly extracted teeth which originally exhibited deep dentinal lesions, clinical examination and electric pulp testing showed that nine of them had no pulpal exposure. Thus the pulps of these teeth were covered by clinically sound dentine beneath the carious lesion. Bacteria were found to have invaded the pulps of six of these nine teeth. The predominant bacteria were obligate anaerobes belonging to the genera Eubacterium, Propionibacterium and Actinomyces. Other obligate anaerobes were Lactobacillus, Peptostreptococcus, Veillonella and Streptococcus. The bacterial composition resembled that of the deep layers of dentinal lesions described previously, suggesting that the bacteria isolated in this study had passed through some individual dentinal tubules, to invade the dental pulp.

The bactericidal efficacy of metronidazole against bacteria in carious dentin was clarified by measuring (1) the difference between bacterial recovery from suspensions of carious dentin on metronidazole-containing BHI-Blood agar plates (10 μg/ml) and control plates and (2) the difference between bacterial recovery from carious lesions of freshly extracted teeth, covered by α-tricalcium phosphate (TCP) cement containing metronidazole (5%) for 1–3 days and that covered by TCP only. More than 10³ bacteria per milligram sample were recovered from carious dentin. More than 99% of the bacteria were, however, not recovered when samples were inoculated on metronidazole-containing BHI-Blood agar plates or when the lesions were covered by TCP cement containing metronidazole, indicating that metronidazole effectively disinfected the carious dentin.

The bactericidal efficacy of metronidazole (MN) against bacteria of carious dentin was estimated by measuring bacterial recovery with or without application of MN to dentinal lesions in vivo. More than 10³ colony-forming units of bacteria per milligram of sample were recovered from the samples of carious dentin. However, none were recovered from the samples of dentinal lesions which had been covered by Α-tricalcium phosphate (TCP) cement containing MN in situ for 1 day, 1 month, 1 year and/or 2 years in vivo. Placebo experiments in which carious dentin was covered by TCP cement without MN showed that TCP itself was not bactericidal. Bactericidal efficacy of MN in situ is clearly demonstrated, indicating that MN is a valuable material in caries treatment to disinfect carious dentin.

Abstract:For better bacterial recovery from endodontic lesions, several bacterial culture conditions were studied. Samples taken from endodontic lesions of 26 freshly extracted teeth, and the bacterial growth was determined in 11 kinds of growth media under aerobic (in air with or without addition of 5% CO2) or anaerobic conditions (in an anaerobic glove box).Among the media tested, bacteria of endodontic lesions were most efficiently recovered in brain heart infusion medium containing 4% sheep blood (BHI-Blood) under anaerobic conditions. For routine dental practice, serum can be substituted for blood which makes the media opaque, resulting in clear media, and thus bacterial growth can be judged more easily.