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Chronic periodontitis is an infectious disease caused by periodontopathic bacteria in subgingival plaque. One major pathogen of this disease, Treponema denticola, has several virulence factors, including a major surface protein (Msp) and the surface protease dentilisin. The cytopathic effects of periodontopathic bacteria on epithelial cells disrupt the integrity of the barrier junction, resulting in the inflammation of periodontal tissue. The aim of this study was to investigate the effect of T. denticola virulence factors dentilisin and Msp on epithelial cells. The effects of T. denticola wild-type, Msp-mutant, and dentilisin-mutant strains on the contact junction in Madin-Darby canine kidney epithelial cells was evaluated based on ohmic values. Cultured oral carcinoma epithelial cells were scratched and exposed to the selected T. denticola strains and cell migration determined. Subsequent degradation of adherence proteins and proteins in the contact junctions was evaluated. Dissociation of cell contact junctions was detected in cells infected with wild-type T. denticola approximately 30 min after infection, but not in those exposed to the mutants. Inhibition of migration was observed in the wild-type and Msp-deficient mutants. The adherent proteins focal adhesion kinase, ZO-1, and paxillin were hydrolyzed by infection with the wild-type and Msp mutants. These results indicate that T. denticola disrupts the function of epithelial cells by hydrolyzing proteins at the intercellular junction and inhibiting healing of epithelial cells via hydrolyzed proteins associated with focal adhesion; Msp was also associated with these effects.

Capnocytophaga ochracea possesses a type-IX secretion system that exports proteins which have a gliding motility-associated C-terminal (CTD) domain. This system is found in several species of the Bacteroidetes phylum. Hyalin, a large protein encoded by Coch_0033 in C. ochracea ATCC 27872, has a CTD domain and is posited to be involved in quorum sensing according to the database of the Kyoto Encyclopedia of Genes and Genomes. This suggests that it plays a role in biofilm formation via interbacterial communication. The aim of this study was to investigate the potential role of the hyalin-like protein coded by the Coch_0033 gene in gliding and biofilm formation of C. ochracea. A hyalin-like protein-deficient mutant strain of C. ochracea, designated mutant WR-1, was constructed through insertion of the ermF-ermAM cassette into the target gene. The spreading feature at the edge of the colony was lost in the mutant strain. Crystal violet and confocal laser scanning microscopy revealed no difference between the quantity of biofilm organized by the mutant and that organized by the wild-type strain. These data suggest that the hyalin-like protein encoded by the Coch_0033 gene is indeed involved in C. ochracea gliding activity.

Candida albicans is the primary candidiasis-causing fungal pathogen in humans, and one of its most important virulence factors is the ability to form biofilms. Moreover, these biofilms are often resistant to antifungal agents, so there is a need to develop alternative elimination strategies and therapeutic agents for such infections. The antifungal activity of resveratrol, a phytoalexin polyphenolic compound, impairs the morphological transition of C. albicans under various hypha-inducing conditions and inhibits growth of the yeast-form and mycelia. The purpose of this study was to investigate the effect of resveratrol against C. albicans biofilm formation. The developmental, sustained, and mature stages of biofilm formation were affected or inhibited by resveratrol. Exposure to resveratrol at the developmental stage inhibited growth of C. albicans in a dose-dependent manner. A >30% reduction was observed in sustained biofilm growth in the presence of 200 μg/ml resveratrol in comparison with in its absence. In terms of disruption of matured biofilm, 6.25-100 μg/ml resveratrol significantly reduced cell viability of C. albicans compared with in a control sample (p<0.05). The present results indicate that resveratrol has the potential to serve as an anti-Candida treatment and preventive tool which functions by inhibiting existing or under-forming C. albicans biofilms.

Phenolic compounds in fruits such as cranberries have been shown to promote a number of biological activities. The purpose of this study was to investigate the effects of polyphenolic compound-containing lingonberry extract on oral streptococci and compare them with the known anti-cariogenic activity of cranberries. Water-soluble and polyphenol-rich fractions (Fractions I and II, respectively) were isolated from cranberries and lingonberries. The effects of those fractions on the biofilm formation ability and bioactivity of Streptococcus mutans MT8148R, Streptococcus sobrinus 6715, and Streptococcus sanguinis ATCC 10556 were then evaluated. Cranberry or lingonberry Fraction II (at 0.5-1 mg/ml) significantly reduced biofilm formation by S. mutans, S. sobrinus, and S. sanguinis. In contrast, cranberry or lingonberry Fraction I (at 0.5-2 mg/ml) increased biofilm formation by S. mutans and S. sobrinus, but not by S. sanguinis. Fractions I and II (at 1-2 mg/ml) also reduced the bioactivity of S. mutans, while Fraction II (at 0.5 mg/ml) enhanced the bioactivity of all tested strains. The results revealed that lingonberries contained a larger amount of polyphenol than cranberries and that they showed almost the same level of activity against the biofilm formation ability and bioactivity of oral streptococci. This indicates that polyphenol-rich lingonberry fraction offers a promising natural food derivative for prevention of dental caries.

Treponema denticola, an anaerobic spirochete found mainly in the oral cavity, is associated with periodontal disease and has a variety of virulence factors. Although in vitro studies have shown that T. denticola is able to penetrate epithelial cell monolayers, its effect on the epithelial barrier junction is not known. Human gingival epithelial cells are closely associated with adjacent membranes, forming barriers in the presence of tight junction proteins, including zonula occludens-1 (ZO-1), claudin-1, and occludin. Tight junction proteins are also expressed by Madin-Darby canine kidney (MDCK) cells in culture. In this study, the MDCK cell profile was investigated following infection with T. denticola (ATCC 35405) wild-type, as well as with its dentilisin-deficient mutant, K1. Basolateral exposure of MDCK cell monolayers to T. denticola at a multiplicity of infection (MOI) of 10 4 resulted in a decrease in transepithelial electrical resistance (TER). Transepithelial electrical resistance in MDCK cell monolayers also decreased following apical exposure to T. denticola (MOI=10 4 ), although this took longer with basolateral exposure. The effect on the TER was time-dependent and required the presence of live bacteria. Meanwhile, MDCK cell viability showed a decrease with either basolateral or apical exposure. Immunofluorescence analysis demonstrated decreases in the amounts of immunoreactive ZO-1 and claudin-1 in association with disruption of cell-cell junctions in MDCK cells exposed apically or basolaterally to T. denticola. Western blot analysis demonstrated degradation of ZO-1 and claudin-1 in culture lysates derived from T. denticola-exposed MDCK cells, suggesting a bacteria-induced protease capable of cleaving these tight junction proteins.

Periapical periodontitis usually results from microbial infection, with these microorganisms occasionally migrating to the root canal, which can lead to further, potentially life-threatening, complications. Here, the susceptibility of 27 bacterial strains to various antimicrobial agents was evaluated. These strains comprised 13 species; 16 of the strains were clinical isolates from periapical lesions. Each strain was inoculated onto blood agar plates containing one of the antimicrobial agents. The plates were incubated anaerobically at 37°C for 96 hr and the minimal inhibitory concentrations (MICs) determined. Ten strains required an MIC of 32 μg/ml or greater for amoxicillin, 6 for cefmetazole, and 5 for cefcapene among β-lactam antibiotics; 8 strains required an MIC of 32 μg/ml or greater for clindamycin, 4 for azithromycin, and 11 for clarithromycin among macrolide antibiotics; 3 strains required an MIC of 32 μg/ml or greater for ciprofloxacin and 2 for ofloxacin among fluoroquinolones. The effect of cefcapene on 5 strains was evaluated after biofilm formation to investigate the relationship between biofilm formation and susceptibility. All strains showed a decrease in susceptibility after biofilm formation. The results revealed that several antimicrobial agents commonly used in a clinical setting, including amoxicillin, cefmetazole, and clindamycin, are potentially effective in the treatment of orofacial odontogenic infections. The development of resistant strains, however, means that this can no longer be guaranteed. In addition, azithromycin, ciprofloxacin, and ofloxacin were more effective than the 3 β-lactam antibiotics tested. These results suggest that sensitivity testing is needed if odontogenic infections are to be treated safely and effectively.

Biofilm formation in dental unit water systems (DUWSs) can contaminate water from three-in-one syringes, air rotors, and low-speed handpieces. This may serve as a potential source of infection for dentists, dental staff, and patients, so these systems must be sterilized. Because slightly acidic electrolyzed water (SAEW) is often used as a disinfectant for food, the aim of this study was to investigate the possibility of using SAEW as a DUWS disinfectant. Slightly acidic electrolyzed water was injected into a dental unit and its effects evaluated. Chemical properties such as chlorine ion and potential hydrogen in the SAEW were measured. Detection of both ordinary and heterotrophic bacteria from the DUWS was performed by culture, and biofilm formation of the bacteria in the DUWS evaluated. Polymerase chain reaction (PCR) was used to detected contamination by nosocomial pathogens. Almost all the chlorine ions in the SAEW were exhausted during the two-day trials, and the pH value of the SAEW fell from 5 to 4. No viable cells were detected in the SAEW collected. Biofilm formation in the water from the DUWS with SAEW was almost at a baseline level, whereas that without SAEW was 4 times higher. The PCR analysis showed that no nosocomial infecting pathogens were detected in the SAEW. The present study demonstrated the antiseptic effect of SAEW in DUWS.

Porphyromonas gingivalis, a pathogen involved in the development of chronic periodontitis, has a number of major virulence factors, among which are its surface cysteine protease gingipains. The purpose of this study was to investigate the feasibility of inducing protective antibodies against P. gingivalis by means of immunization with recombinant Lactococcus lactis expressing the 44-kDa gingipain adhesion/hemagglutinin domain (Hgp44). Part of the Hgp44 sequence encoding the first 314 amino acid residues, residues 188-251, and residues 354-393 was amplified and inserted into shuttle plasmid pSGANC332, with the resulting chimeric plasmids designated as pISTY210, pCOL, and pSHGRP44A, respectively. After confirming the clone sequences, expression of recombinant proteins was investigated by immunoblot. The results revealed that while pISTY210 and pCOL both expressed the Hgp44 antigen on the surface of L. lactis, the level of expression was quite low. To enhance expression of the protein on the surface of the cells, cysteine residues were changed to serine residues by site-directed mutagenesis. Replacement of 3 out of 5 cysteine residues (pISTY213) significantly increased expression of the recombinant protein on the surface of the bacteria. Interestingly, replacement of the 4th cysteine residue (pISTY215) reduced antigenicity of the recombinant protein. These results indicate that expression of Hgp44 on the surface of L. lactis cells requires the replacement of several key cysteine residues, and that L. lactis expressing this antigen could be a promising candidate for immunization against P. gingivalis-induced periodontitis.

Periodontopathic bacteria such as Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Tannerella forsythia, Campylobacter rectus and Treponema denticola play an important role in the initiation and progression of periodontitis. The aim of this investigation was to evaluate the relationship between periodontal clinical parameters and the subgingival profile of periodontopathic bacteria. Twenty-six periodontitis patients (23-62 years of age; mean age, 40.2±13.2) with no systemic disease agreed to participate in the study. Periodontal clinical parameters, including probing depth (PD) and bleeding on probing (BOP) were recorded. Subgingival plaque samples were obtained from deep (PD≥4 mm) and shallow (PD≤3 mm) pockets in each patient for detection of P. gingivalis, A. actinomycetemcomitans, T. forsythia, C. rectus and T. denticola by polymerase chain reaction technique. The relationship between the periodontal pathogens and clinical parameters was determined with the Fisher exact test, and a statistically significant association was found between detection of P. gingivalis, T. forsythia, C. rectus and T. denticola and PD or BOP. T. denticola was the most prevalent pathogen in both shallow PD and deep PD sites. No statistically significant association was found between detection of A. actinomycetemcomitans and the clinical parameters examined. A statistically significant association was found between detection of the red complex bacteria and the clinical parameters. These results suggest that the red complex pathogens and C. rectus play an important role in the initiation and progression of periodontitis.

Aggregatibactor actinomycetemcomitans colonizes human periodontal lesions and is implicated in both aggressive periodontitis and chronic periodontitis. Clinical isolated colonies of A. actinomycetemcomitans were rough type. The rough type has a remarkable ability to adhere tenaciously to solid surfaces and colonize firmly. Rough type colonies change into smooth type colonies during the course of repeated inoculation and biofilm-forming activity ceases. Adherence by A. actinomycetemcomitans is mediated by the tight-adherence (tad) gene locus, which includes flp, rcpA and rcpB. In this study, we investigated the relationship between its biofilm-forming ability and expression of the flp, rcpA and rcpB genes associated with fimbriae protein production. First, we changed rough type strain organized biofilm on glass into smooth type and confirmed it by observation of biofilm on glass surfaces. Then, we carried out Real-Time PCR and found that expression of the rcpA and rcpB genes was clearly reduced in smooth type colonies. This suggests that expression of rcpA and rcpB plays a key role in biofilm formation by A. actinomycetemcomitans strains and the establishment of persistent infections in periodontal lesions.

Aggregatibacter actinomycetemcomitans is a pathogen associated with chronic and aggressive periodontitis and extra-oral infections. Fresh isolates of A. actinomycetemcomitans are fimbriated, forming small, rough-phenotype colonies on agar plates and also form biofilms. Recently, it has been reported that amyloid fibers are abundant in natural biofilms, and Escherichia coli and Salmonella spp. produce amyloid fibers that contribute to biofilm formation. This has yet to be reported, however, in A. actinomycetemcomitans. Amyloid binds the Congo red (CR) dye. In this study, therefore, we investigated amyloid formation in A. actinomycetemcomitans using a detection of CR-binding colonies on CR agar plates and CR-binding assay. All rough-phenotype strains formed dark red colonies and smooth-phenotype strains formed white or opaque red colonies on CR agar plates. Compared with smooth-phenotype strains, rough-phenotype strains showed higher CR-binding activity. CR-binding of rough-phenotype strain AKR was not affected by protease digestion or heating, whereas smooth-phenotype strain 29523 showed a marked reduction in CR-binding after both types of treatment. AKR showed amyloid-positive staining with CR to produce yellow green birefringence under polarized light, whereas 29523 showed amyloid-negative staining. These findings indicate that the CR-binding component of rough-phenotype A. actinomycetemcomitans is an amyloid-like fiber.

In order to find informative salivary biomarkers specific to oral cancer we examined expression of 4 kinds of cytokine in saliva. Levels of interleukins (IL-1beta, -6, -8) and osteopontin were measured by ELISA using whole saliva samples collected from 19 patients with oral cancer (9 men, 10 women; mean age, 60.9 years) and 20 healthy persons (15 men, 5 women; mean age, 32 years). Expression of the 4 cytokines was higher in patients with oral cancer than in healthy controls. The difference was significant in IL-6, in particular. The results suggest that saliva offers a potential target for a screening test aimed at detection of precancerous lesions.

The purpose of this study was to evaluate age-related differences in expression of vascular endothelial growth factor (VEGF) by periodontal ligament (PDL) cells. PDL cells were obtained from Wistar male rats weighing approximately 150 g each in the young group and 350 g each in the old group. PDL cells derived from upper and lower incisors were seeded in 35-mm culture dishes after primary culture. For cell proliferation assays, cells were detached and counted at 1, 3, 5, 7, 11 and 14 days after culture. VEGF mRNA expression was analyzed with TaqMan. The number of cells in both groups increased day by day, but the rate of increase in the young group was higher than that in the old group. VEGF mRNA expression in the young group increased from 3 to 14 days, but in the old group increased only slightly over the same time period. Expression ratios in the young group were higher than those in the old group, and there were significant differences between the young and old groups at 7 and 14 days of culture. In conclusion, the data revealed that PDL cells varied with age, and suggest that in view of such changes in cell proliferation and VEGF mRNA expression, age should be taken into consideration in periodontal treatment.

Defensins are known to play an important role in defense against bacteria. It is also known that immunity against infection is compromised with age. The purpose of this study was to evaluate the localization of human beta-defensin (HBD)-2 in human gingival epithelia according to age. Gingival epithelia in maxillary premolar buccal normal regions was immunohistochemically stained for HBD-2. Specimens were divided into two groups: 6 cases in a young group (<20 years old) and 7 cases in an elderly group (>50 years old). Expression of HBD-2 in gingival epithelium in young subjects was mostly detected in the superficial layer of the parakeratinized layer, while some areas of the spinous cell layer in elderly subjects were positive for HBD-2, as was the superficial layer. Two cases in the young group and 5 cases in the elderly group immunoreacted with HBD-2 in the spinous cell layer. Furthermore, immunoreaction was stronger in the elderly group. The results revealed HBD-2 positive cells in spinous cells in the elderly group and in the parakeratinized layer in the young group.

The purpose of this study was to investigate oral flora in independent persons aged over 80 years with more than 20 remaining teeth. The subjects were 22 participants of the 8020 campaign (6 males and 16 females) with a mean age of 81.3+/-1.6 years and an average of 24.7 teeth (Independent 8020 group). This group was compared with a group of 38 elderly people residing in nursing homes (10 males and 28 females) who had a mean age of 81.3+/-8.5 years and an average of 4.2 teeth (Nursing group with fewer teeth). Saliva samples were collected from the vestibular areas of the maxilla and mandible using cotton swabs. Cell numbers of microorganisms were expressed as colony forming units/ml (CFUs/ml) and compared between the two groups. The average number of Staphylococcus species was 65.2+/-74.4 CFUs/ml in the Independent 8020 group and 400.3+/-352.1 CFUs/ml in the group with fewer teeth (p<0.01); that of Candida albicans was 18.0+/-37.7 CFUs/ml in the Independent 8020 group and 152.9+/-211.9 CFUs/ml in the Nursing group with fewer teeth (p<0.05). Both species showed statistically significant differences between the two groups. This suggests that the Independent 8020 achiever group had better oral hygiene and that the presence of many teeth may be associated with an increased awareness of dental health.

Colonization by periodontopathic bacteria is a risk factor for peri-implantitis. The purpose of this study was to investigate the colonization by black-pigmented anaerobic bacteria that occurs between the time before fixture installation and 6 months after inserting superstructures in implant treatment in partial edentulous cases. Dental plaque was serially collected from around the natural teeth and implants in 12 patients in whom a dental implant was indicated, and Porphyromonas gingivalis and Prevotella intermedia were detected using polymerase chain reaction (PCR). One month after connecting the abutment, the detection rate of P. gingivalis per site from around the implants was 63.7% and that of P. intermedia was 50.8%. Six months after superstructure setting, the detection rate per site of P. gingivalis from around the implants was 56.8% and that of P. intermedia was 41.1%. When chromosomal DNA segmentation patterns in the isolated P. gingivalis and P. intermedia were compared using pulsed field gel electrophoresis (PFGE), the patterns in the natural teeth were in accordance with those in the implants in 3 of 4 cases (75.0%) in P. gingivalis and all cases in P. intermedia. This finding suggested that bacterial colonization around implants occurred early after the implant region was exposed to the intraoral cavity and that the bacteria were transmitted from the area around the natural teeth.

Recent studies have shown that invasive and non-invasive strains of Porphyromonas gingivalis can both be isolated from patients with periodontitis. We examined the interaction between an invasive 16-1 P. gingivalis strain and phagocytes obtained from human peripheral blood and guinea pig peritoneal cavity. Phagocytes from human peripheral blood, mainly polymorphonuclear leukocytes (PMNs) isolated by centrifugation in Ficoll Hypaque, and macrophages collected from the peritoneal cavity of guinea pigs, were exposed to P. gingivalis cells. After this exposure, greater numbers of the non-invasive P. gingivalis ATCC 33277 were observed in human PMNs and guinea pig macrophages compared with the invasive P. gingivalis 16-1. Electron microscopic observations showed that invasive 16-1 within phagosomes in human PMNs and guinea pig macrophages retained their surface fibrous structures as well as their outer membranes. Electron microscopic examination showed that destruction and damage to the cell membranes and inner structures were clear in human PMNs and guinea pig macrophages after exposure to invasive 16-1 for 6 and 24 hours; this was a clear difference from exposure to the non-invasive ATCC 33277. Release of lactate dehydrogenase (LDH) activities into the culture supernatant of PMNs after exposure to the invasive 16-1 for 4 and 6 hours was significantly greater than that after exposure to the non-invasive ATCC 33277 (p<0.05). On the other hand, the LDH activity after exposure for 21 hours to the invasive 16-1 was significantly lower than that of untreated cells and cells after exposure to the non-invasive ATCC 33277 strain (p<0.05). The PMN viabilities after exposure to cells of the invasive 16-1 for 3, 4, and 6 hours as evaluated by trypan blue staining were similar to those after exposure to cells of the non-invasive ATCC 33277, but that after exposure to the invasive 16-1 strain for 21 hours was significantly lower than that after exposure to cells of the non-invasive ATCC 33277 strain.

Porphyromonas gingivalis and Treponema denticola have been found together in lesions of human periodontitis. We examined the ability of a mixed infection by both bacteria to synergistically form abscesses and disturb immune responses in mice. Absorbance of an invasive P. gingivalis 16-1 strain grown in tryptic soy broth and T. denticola ATCC 33520 strain grown in TYGVS medium were adjusted. BALB/c mice were injected with 200 microliters of the cell suspension at a site on the lateral dorsal area. The sizes of the subsequent subcutaneous abscesses were measured with a caliper gauge, and the area was expressed in square mm. Mixed infections by P. gingivalis and T.denticola produced larger abscesses than those formed after mono-infections by either P. gingivalis or T.denticola. The abscesses caused by mixed infection reached their maxima on the 6th day and maintained that size for the subsequent 5 days. The delayed type hypersensitivities against extracted antigens of P.gingivalis in mixed infection mice were significantly lower than those in the mono-infected mice. However, the IgG response to sonicated antigen of P.gingivalis did not differ between the two groups. The sizes of the abscesses caused by mixed infections in mice immunized with whole cells of P.gingivalis 16-1 were compared to those caused in sham-immunized mice. The average size of the abscess caused by mixed infection in immunized mice did not differ from that in sham-immunized mice, but many of the abscesses in immunized mice ruptured on the 4th or 5th day, followed by recovery in two weeks. These results suggest that mixed infection with P.gingivalis and T.denticola attenuates protective immune responses.

Pustulosis palmaris et plantaris (PPP) is a chronic relapsing skin disease characterized by sterile intraepidermal pustules and, usually, by scaly erythematous skin on the palm, soles, or both. To determine the primary pathogenic factors involved in the onset of PPP, we analyzed the implications of heat shock proteins (HSPs), which are highly conserved immunogenic proteins, chronic microbial infections including marginal and periapical periodontitis, and delayed type hypersensitivity (DTH) against dental metals examined by patch tests. We found that the titers of IgG against HSPs of Escherichia coli GroEL and Actinobacillus actinomycetemcomitans DnaJ in the sera from PPP patients were higher than those in the control group (GroEL; p < 0.05). Two patients with PPP had more than 1,000 ng/ml serum IgE level. We found that the average serum IgG antibody level against human cytomegalovirus (HCMV) in the PPP patient group was higher than that of the healthy group (p < 0.05). DTH against dental metal was found in eight of 22 PPP patients. Six of eight patients reacted against more than two metals. Replacement of dental metal with resin in these patients resulted in the disappearance or remission of PPP. These findings suggest that the immune responses to HSPs produced by oral bacteria, HCMV infection, dental metal allergy, and their combinations are etiological factors for PPP. We concluded that various kinds of examinations, including oral cavity conditions and chronic infections, are essential to determine the major etiologic factors of PPP.

It has been suggested that infection by some mycoplasma species can act as possible cofactors in the acceleration of immunodeficiency in HIV-infected patients. The present study was designed to examine infections by oral mycoplasma species in HIV-seropositive (HIV(+)) patients. Mycoplasma salivarium and Mycoplasma orale were isolated from 59.5% and 16.7% of 42 HIV(+) patients, respectively. Non-M. salivarium and non-M. orale species were isolated from 40.5% of saliva samples from the HIV(+) group and 20.8% of those from 24 HIV-seronegative (HIV(-)) subjects, respectively. Although the production of superantigen by human peripheral lymphocytes in the isolated mycoplasma species from HIV(+) and HIV(-) subjects was evaluated, none of the examined mycoplasma strains, including ATCC strains of M. salivarium, M. orale, Mycoplasma buccae and Mycoplasma penetrans, were found to produce superantigen. Production of heat shock proteins (HSPs) by isolated mycoplasma strains was examined by immunoblotting using monoclonal antibodies against Helicobacter pylori HSP60. It was found that all the strains of M. salivarium, M. orale, and unidentified mycoplasma species isolated from HIV(+) and HIV(-) groups produced heat shock proteins. HSP production by oral mycoplasma may play a role in the immunomodulation of HIV(+) patients.

This study examined the effects of storage conditions such as time course, temperature, fluorescent light, and darkness on the components and antibacterial activity of formalin guaiacol (FG) used in endodontic treatment. We measured the quantities of formaldehyde and guaiacol in FG and antibacterial activities against Staphylococcus aureus, Porphyromonas gingivalis, and Porphyromonas endodontalis. The components and antibacterial activity of FG in the brown or transparent tightly sealed containers were not affected by temperature or fluorescent light throughout the 4 week test. However, in the loosely sealed containers, formaldehyde and guaiacol in FG sample decreased remarkably within one week, not only in a temperature- and time-dependent manner, but also under fluorescent light at 20 degrees C. Furthermore, the antibacterial activities in the FG sample were significantly attenuated in parallel with the decrease in formaldehyde levels. Fluorescent light caused color changes and crystallization of FG samples in the transparent containers. These results suggest that it is important to replace fresh FG every 5 to 7 days for endodontic treatment and that, in the dental office, it is advisable to store fresh FG in tightly sealed containers every 2 weeks to maintain its efficacy.